Download Single Molecule Optical Magnetic Tweezers Microscopy Studies of

SINGLE MOLECULE OPTICAL MAGNETIC TWEEZERS MICROSCOPY STUDIES OF
PROTEIN DYNAMICS
Qing Guo
A Dissertation
Submitted to the Graduate College of Bowling Green
State University in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
August 2015
Committee:
H. Peter Lu, Advisor
John Farver
Graduate Faculty Representative
Neocles Leontis
John Cable
© 2015
Qing Guo
All Rights Reserved
iii
ABSTRACT
H. Peter Lu, Advisor
This dissertation presents our research work aiming at conformational manipulation of
single enzyme protein molecules, performed by single molecule magnetic tweezers correlated
with optical fluorescence spectroscopy. To experimentally investigate the enzyme-substrate
interactions and the related conformational fluctuations, we have developed a new approach to
manipulate the enzymatic conformation and enzyme-substrate interaction at the single-molecule
level by using a combined magnetic tweezers and simultaneous fluorescence resonance energy
transfer (FRET) spectroscopic microscopy. By a repetitive pulling-releasing manipulation of a
Cy3-Cy5 dye labeled 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) molecule
under the conditions with and without enzymatic substrates, we have probed and analyzed the
enzymatic conformational dynamics. Our results indicate that the enzymatic conformational
flexibility can be regulated by enzyme-substrate interactions: (1) the enzyme at its conformationperturbed state has less flexibility when binding substrates, and (2) substrate binding to the
enzyme significantly changes the enzyme conformational flexibility, experimental evidence of so
called entropy trapping in and enzyme-substrate reactive transition state. Furthermore, our
results provide significant experimental analysis of folding-binding interactions of the enzymesubstrate interactions, and reveal the dynamic nature of the enzyme-substrate interactions. We
also find supportive results from Steered Molecular Dynamics (SMD) Simulation, showing that
in our studies, conformational manipulation by magnetic tweezers is able to distort the active
domain of the enzyme molecules to an extent that significantly beyond thermal conformational
fluctuations.
iv
Furthermore, we have also revealed the impact of partially unfolding the enzyme
molecules on their activity by using single-molecule TIRF-magnetic tweezers spectroscopy to
manipulate conformation of the enzyme molecules to a partially unfolded, yet not fully
denatured condition. By conformationally distorting horseradish peroxidase (HRP) molecules via
magnetic tweezers at the single molecule level, we successfully manipulated and examined the
activity changes of the HRP catalyzed H2O2-Amplex Red reaction. We have observed
significant tolerance of the enzyme activity to the enzyme conformation in its deformed or
partially-unfolded states. We have identified that (1) enzymatic activity can be manipulated by
our TIRF-magnetic tweezers at single molecule level; and (2) enzyme molecules in partially
unfolded conformation are still capable of showing significant activity, although at a lower but
measurable level, due to the enzymatic active site conformational fluctuation and substrate
binding induced folding-binding conformational changes. We further provide our understanding
of the enzyme behavior based on enzymatic conformational fluctuation, enzyme-substrate
interactions, enzyme-substrate active complex formation, and protein folding-binding
interactions.
v
To The Memory of My Dear Grandfather
vi
ACKNOWLEDGMENTS
In my journey of becoming a Doctor of Philosophy at Bowling Green State University, I
have received help and guide from many wise and friendly people. I am deeply grateful to them,
and I appreciate their help and friendship.
During the past six years of graduate study, if I have made any progress, either as a
scientific researcher, or as a people in the society, I would like to attribute all of them to Dr. H.
Peter Lu. I will be forever feeling in debt to him, for the countless support, patience, guidance,
encouragement that Dr. Lu gave me. To me, Dr. Lu is the paragon in both being a successful
scientist and being a socially welcomed people. He not only guides me in my work, but also
offers me a lot of help in my life. It is him who improved my understanding of science. I would
by no means to become Doctor without his chronic guidance and help.
I am thankful to all my other committee members: Dr. Neocles Leontis, Dr. John Cable
and Dr. John Farver for their precious time. I also want to acknowledge all the group members,
both current members and past members from Dr. H. Peter Lu’s group, for setting a
communication-free and hard-working environment. Especially, I want to thank Dr. Yufan He
for his teaching and guidance to me these years. I also want to thank Jin Cao, Desheng Zheng,
Yuanmin Wang and Zijian Wang for their friendship.
I also want to say thank you to many faculty and staff at the Center for Photochemical
Sciences and the Department of Chemistry: Nora Cassidy, Alita Frater, Charles Codding, and
Doug Martin, Hilda Miranda, for their help.
I would also like to thank my family for their love and support in these years. Without
their understanding and support, I would never be able to enjoy working on science for so many
years.
vi
TABLE OF CONTENTS
Page
CHAPTER I. INTRODUCTION ..........................................................................................
1
1.1
Introduction of Single Molecule Spectroscopy..............................................
1
1.2
Introduction of Single Molecule Protein Conformational Dynamics ............
4
1.3
Introduction of Single Molecule Studies of Enzyme .....................................
10
1.4
Introduction of Magnetic Tweezers ...............................................................
12
1.5
Research Objective and Specific Aims, and Dissertation Overview .............
17
1.6
References ......................................................................................................
18
CHAPTER II. EXPERIMENT .............................................................................................
25
2.1
2.2
Principles of Experimental Techniques .........................................................
25
2.1.1
Principles of Confocal Microscopy ...................................................
25
2.1.2
Principles of Forster Energy Transfer (FRET) .................................
28
2.1.3
Principles of Total Internal Reflection Microscopy (TIRFM) ..........
33
2.1.4
Signal Detection techniques: Introduction to APD and EMCCD......
40
2.1.5
Basics of Magnetic Tweezers: Force Calibration ..............................
45
Experiment Details.........................................................................................
48
2.2.1 Experimental Setup of Single Molecule FRET Correlated with Magnetic
Tweezers ........................................................................................................
48
2.2.2 Experimental Setup of Single Molecule TIRFM Correlated with Magnetic
2.3
Tweezers ........................................................................................................
50
2.2.3
Steered Molecular Dynamics (SMD) Simulation ..............................
51
Materials and Sample Preparation .................................................................
52
vii
2.4
Selection of Magnetic Beads .........................................................................
54
2.5
References ......................................................................................................
56
CHAPTER III. MANIPULATING AND PROBING ENZYMATIC CONFORMATIONAL
FLUCTUATIONS AND ENZYME-SUBSTRATE INTERACTIONS BY SINGLEMOLECULE
FRET-MAGNETIC TWEEZERS MICROSCOPY ..............................................................
58
3.1
Introduction ...................................................................................................
58
3.2
Materials and Methods ...................................................................................
60
3.2.1
HPPK Protein .....................................................................................
60
3.2.2
Sample Preparation ............................................................................
62
3.2.3
Experimental System .........................................................................
65
3.2.4
Force Calibration ...............................................................................
66
Results and Discussion ..................................................................................
67
3.3.1
FRET Measurement ...........................................................................
67
3.3.2
Repetitive Conformational Manipulation of Single HPPK Molecule
3.3
Observed by FRET Spectroscopy ..................................................................
3.3.3
Probing Conformational Flexibility of Single HPPK Protein Molecule by
Single Molecule FRET-Magnetic Tweezers Spectroscopy ...........................
3.3.4
69
71
Conformational Dynamics Manipulation by Single Molecule FRET-
Magnetic Tweezers Spectroscopy..................................................................
76
3.4
Conclusion .....................................................................................................
78
3.5
References ......................................................................................................
79
CHAPTER IV. INTERROGATING THE ACTIVITIES OF CONFORMATIONAL
DEFORMED ENZYME B BY SINGLE MOLECULE TIRF-MAGNETIC TWEEZERS
viii
MICROSCOPY
............................................................................................................
89
4.1
Introduction ....................................................................................................
89
4.2
Materials and Methods ...................................................................................
89
4.2.1
Materials ............................................................................................
92
4.2.2
TIRF Measurement ............................................................................
93
4.2.3
Sample Preparation ............................................................................
94
Results ............................................................................................................
96
4.3.1
Single-Molecule TIRF Imaging Measurement of HRP Activity .......
96
4.3.2
Analysis of Single-Molecule Activity Trajectories Measured under Force
4.3
Pulling and Releasing Conditions ..................................................................
4.3.3
97
Repetitive Force Pulling-Releasing Manipulation of Enzyme
Conformation for Impacting Enzymatic Activity .......................................... 102
4.4
Discussion ...................................................................................................... 104
4.5
Conclusion ..................................................................................................... 107
4.6
References ...................................................................................................... 108
CHAPTER V. STEERED MOLECULAR DYNAMICS SIMULATION STUDIES OF THE
CONFORMATIONALLY DEFORMED ENZYMES MANIPULATED BY SINGLE
MOLECULE MAGNETIC TWEEZERS .............................................................................. 120
5.1
Introduction .................................................................................................... 120
5.2
Estimating Conformational Stretching Extent from HPPK Simulation ........ 121
5.3
SMD Simulation Study of HRP Protein Molecule ........................................ 125
5.3.1 SMD Simulation of HRP Protein Molecule in One Tethering
Condition........................................................................................................ 125
ix
5.3.2
SMD Study on All Possible Stretching Type of HRP Protein
Molecule ........................................................................................................ 129
5.3.3
5.4
Distortion in Unfolding Simulation ................................................... 131
References ...................................................................................................... 132
CHAPTER VI. DESIGN AND IMPLEMENTATION OF A QUADRUPOLE
MAGNETIC TWEEZERS..................................................................................................... 133
6.1
History of Instrumental Design for Magnetic Tweezers................................ 133
6.2
The Multi-Channel Magnetic Tweezers ........................................................ 136
6.2.1
An Introduction of the Multi-Dimensional Magnetic Tweezers Setup in
Our Lab .......................................................................................................... 136
6.2.2
An Improvement: Developing the New Generation Magnetic
Tweezers ........................................................................................................ 140
6.3
References ...................................................................................................... 142
x
LIST OF FIGURES
Figure
Page
1.1
Free Energy Diagram of Enzymatic Reaction ...........................................................
8
1.2
A Conceptual Scheme of the Sample System ............................................................
14
2.1
Conceptual Figure of Confocal Microscopy ..............................................................
27
2.2
Principle of Förster Resonance Energy Transfer .......................................................
30
2.3
The Distance-FRET Efficiency Relationship ............................................................
32
2.4
Spectrum Profile of Chromophore Molecule Cy3 and Cy5.......................................
33
2.5
A Conceptual Figure of Total Internal Reflection Phenomenon ...............................
35
2.6
A Conceptual Scheme of Evanescent Wave ..............................................................
37
2.7
Conceptual scheme of total internal reflection microscopy (TIRFM) .......................
39
2.8
Principle of Photodiode Module used in our experiment ..........................................
42
2.9
Principle of Electron Multiplied Charge Coupled Device (EMCCD) .......................
44
2.10 Magnetic field-Distance curve of the magnet used in experiment ............................
47
2.11 Experimental setup of single molecule FRET-Magnetic Tweezers spectroscopy.....
49
2.12 Experimental setup of single molecule TIRF-Magnetic Tweezers spectroscopy ......
50
2.13 A conceptual scheme of single molecule protein immobilization method ................
53
2.14 Force-velocity response of magnetic beads in different size .....................................
55
3.1
Structural information of HPPK molecule. ................................................................
61
3.2
Preparation of single molecule HPPK sample. ..........................................................
63
3.3
A conceptual scheme of the experimental system .....................................................
65
3.4
Single-molecule FRET data of a Cy3-Cy5 labeled HPPK in experiment .................
68
3.5
Repetitive force pulling and releasing manipulation of Single HPPK molecules .....
70
xi
3.6
Perturbing and characterizing enzyme-substrate binding interaction by single-molecule
FRET magnetic tweezers microscopy .......................................................................
3.6
73
Conformational fluctuation rate distributions calculated from autocorrelation analysis of
HPPK with substrate ATP and HP added. .................................................................
75
4.1
A conceptual scheme of our experimental system.....................................................
92
4.2
Preparation of single molecule HRP sample ......................................................
95
4.3
Single-turnover detection of HRP enzyme catalysis .................................................
97
4.4
Histogram results of turnover events from 30 individual HRP molecules ................
98
4.5
Analysis of the relationship between turnover event, mean waiting time and product burst
of single HRP molecules ............................................................................................ 101
4.6
Response of HRP enzymatic activity to repetitive magnetic pulling force ............... 103
4.7
Conceptual scheme of conformational fluctuation of single enzyme protein when being
deformed by external force ........................................................................................ 107
5.1
SMD simulation of HPPK molecule pulling by magnetic tweezers. ......................... 124
5.2
SMD simulation results show the scheme of the distortion of active site when the protein
is pulling by magnetic tweezers ................................................................................. 127
5.3
Three residue pairs to illustrate distortion on the active domain when an HRP molecule is
stretched by magnetic tweezers ................................................................................. 130
5.4
Active site conformational distortion in larger unfolding situation ........................... 131
6.1
A conceptual scheme of the quadrupole magnetic tweezers setup ............................ 137
6.2
Conceptual scheme of an experiment testing the function electromagnet poles ....... 138
6.3
A testing experiment examination the electromagnet functions. ............................. 139
6.4
A circuit diagram of the new controlling system for the quadrupole electromagnet. 141
1
CHAPTER I. INTRODUCTION
This chapter is dedicated to the introduction of single molecule studies of protein
conformational dynamics and a brief introduction of single molecule magnetic tweezers
(MTW).
1.1 Introduction of Single Molecule Spectroscopy
The capability of probing and manipulating molecules at single molecule level and
even to manipulate chemistry reactions by probing chemical bonds has been a chronic
dream by chemists. With the development of lasers and microscope techniques, it started
to become possible in experiment in late 20th century. Single molecule spectroscopy
originates from the studies on solid state science, which used fluorescence spectroscopy
to detect a single molecule in crystalline or amorphous solids as a probe of local structure
and dynamics in solids. The first successful single molecule experiment has been
achieved by Moerner’s group back to 1989, using sensitive doubly modulated absorption
approach to detect a single molecule in solids at low temperature.1 By 1990, a single
molecule observation in liquid has been achieved;2 in 1993, Betzig and Chichester have
firstly observed immobilized single molecule at room temperature by near-field scanning
microscope.3 Afterward, single molecule spectroscopy has already been regarded as a
potentially rich field and powerful approach for biophysics research, although the
experimentally technical difficulties of near field scanning method still limit the
application of single molecule methods. From 1994 to 1997, imaging single molecule at
room temperature using far field microscopy and Raman spectroscopy have been
achieved.4,5 In 1998, Xie and Lu firstly applied single molecule room temperature
2
fluorescence spectroscopy in enzymatic dynamics study, which finally started a new era
using single molecule approaches in biophysics research.6
Afterward, single molecule techniques have been widely used in extensive studies on
biophysics field. Depending on research focus, those works since then can be sorted into
two large directions. One of them is focused on imaging and spectra analysis, such as
Surface Enhanced Raman Spectroscopy (SERS),4 Förster Resonance Energy Transfer
(FRET) spectroscopy,7 and more recent studies on super-resolution imaging methods for
biological systems such as Stimulated Emission Depletion (STED) Microscopy,
Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction
Microscopy (STORM).8‐10 In the other direction, scientists are more interested in
mechanical characters of biomolecules, and hence developing a lot of techniques to apply
force on target sample at single molecule level, such as Atomic Force Microscopy
(AFM),11‐12 Optical Tweezers developed by Chu, Bustamante and Block,13‐15 Magnetic
Tweezers developed by Bustamante and Bensimon16,17, Anti-Brownian Electrokinetic
Trap developed by Moerner18 Biomembrane Force Probe developed by Evans,19 etc. A
lot of theoretical studies also spring out to enhance the single molecule field, such as
protein folding-unfolding theory developed by Wolynes and Onuchic,20, 21 and single
molecule level enzymatic reaction theory developed by Xie and Lu, etc. 4,22
Single molecule approaches can provide new information that cannot be obtained
from traditional ensemble level experiments in chemical and biological research. In
ensemble level measurements, which means that individual behavior of molecules are
indistinguishable, experimentally observed parameters are usually average characteristics.
By removing ensemble averaging effects, single molecule techniques are scientifically
3
valuable mainly in three aspects. Firstly, single molecule measurements generate
frequency histogram of the distribution of experimentally observed values. Statistical
indicators of these distributions will bring more information, i.e. multi-peak histogram
which reflects multiple intermediate states; the shape of the histogram which reflects
fluctuation flexibility of respective parameters. Such characters are especially important
when the sample systems are heterogeneous. Secondly, single molecule methods make
post-synchronization of time-dependent processes with many molecules involved
unnecessary. For a process such as enzymatic reaction or protein-protein interaction
which involves multiple different states, when we measure them at ensemble level, at any
given time, the specific states of molecules in our detection signal will be averaged out
and cause loss of information from observation of molecule intermediate states.
However, if we can monitor one molecule at a time with proper time resolution, at any
given time, its specific states will be recorded without any averaging out effect, and
hence give us more information of the sample states on time domain. As a result, more
dynamical information can be obtained in single molecule measurements. For example,
in single molecule detections, since all the signals are from one same molecule, we can
apply correlation analysis to analyze the fluctuation rate of certain states of the molecule,
or we can even analyze time-involved disorder of parameters of one single molecule.23
Thirdly, in single molecule observation, new effects can be discovered, or even become
diagnostics of molecule system. For example, absorption spectra frequency of single
molecule has been found to be an indicator of molecular local spatial environment;24, 25
Fluctuation rate from autocorrelation analysis can be a diagnostic approach to reveal
process which involves multiple intermediate states.
4
Those advantages make single molecule spectroscopy become especially
powerful for biophysics studies. On one hand, biological process such as protein foldingunfolding or enzyme catalysis process at molecule level are more dynamic rather than
static, which means time-dependent fluctuation plays a key role in those processes. On
the other hand, single molecule techniques also provide us detailed structure detecting
methods and even manipulation methods of biological systems such as DNA or protein
molecules, membrane systems. Moreover, many biophysical processes such as DNA
translation, gene expression, or transportation occurring on membranes involve very few
molecules or even single molecule.26 In later chapters, our discussion will mainly focused
on protein conformational dynamics and structure-function relationship of enzyme
molecules at single molecule level.
1.2 Introduction of Single Molecule Protein Conformational Dynamics
Protein are participants in various types of processes in living organisms such as
metabolic reactions, gene expression, response to external stimulation, etc. and hence
constitute an active research area in life science. As a result, understanding the structure
and dynamics of protein molecules and the mechanism of their functions has been
intriguing scientists’ interest for a long time. The prototype of modern idea of protein
structure has been developed since 1950s.27 The first protein having its three-dimensional
structure resolved is myoglobin, achieved by Kendrew in 1958 using X-Ray
Crystallography. In 1965, Blake resolved the structure of lysozyme, making it the first
enzyme and the second protein molecule having its structure revealed. Since then, by the
help of several techniques such as X-Ray Crystallography, Nuclear Magnetic Resonance
(NMR) and Cryo-Electron Microscopy (Cryo-EM), more than 35,000 distinct protein
5
sequences have been resolved. Nowadays, crystal structures of many complex protein
molecules such as NMDA receptors can be decoded in experiments.28
In biophysics and biochemistry studies today, a major concerned topic is the
structure-function relationship. The structure of protein molecules are heterogeneous
biopolymer which is formed by folding a particular linear sequence consisting of 20
naturally occurring amino acids. Once the sequence is determined, the amino-acids chain
will be regulated by many different types of non-covalent force, including hydrogen
bonding, hydrophobic forces, electrostatic forces, van der Waals forces, etc. and therefore
folded into a 3-D structure spontaneously. Such 3-D structures, although recently found
to be fluctuating in natural condition, are called stable conformation of the protein
molecules. For protein molecules, it has been found out that structure and function of
protein molecules are intimately related. For example, specific conformational
recognition processes are involved in antibody-antigen binding interaction; enzymesubstrate conformational docking is the key step in enzymatic catalytic function;
neurotransmitters rely on conformation-specific binding of receptors that ties on
membrane protein of the target cell to achieve neuro-signal transportation, etc.
Protein conformation is also dynamic rather than static. In real biological
environment, conformations of protein molecules are not limited in one certain
configuration, but fluctuate all the time. For example, internal motions of enzyme
molecules have been found fluctuating at different time scales ranging from pico-seconds
to seconds.
Furthermore, in biological processes such as protein-ligand interactions or
enzyme-substrate interactions, protein molecules undergo a lot of intermediate states with
different conformations, while time dependent study on conformation facilitate us to
6
understand mechanisms of those processes. Such dynamic characteristics of protein
conformation allow us to reveal rich information from time-resolved single molecule
spectroscopy. For example, in protein folding-unfolding studies, each conformation of a
given protein molecule reflects different potential energy; the landscape consisting of
those different energies as a function of different conformation reveals the folding
probability of protein molecules.20 In single molecule enzymology studies, the
conformational fluctuation rate of enzyme molecules which can be obtained from
autocorrelation analysis of experimental time trajectories can even reveal the existence of
multiple intermediate states which enzyme molecules undergo during enzymatic
reactions.29
Based on previous knowledge of crystal structures of protein molecules, extensive
single-molecule studies of time-dependent information of protein conformation have
come out. In recent decades, both theoretical and experimental research works have been
done to study dynamic character of protein conformation in many different processes.
For example, theoretical models based on the concept of energy landscape describing
protein folding-unfolding process have been developed by Wolynes and Onuchic,20,21
experimentally observations of protein folding-unfolding and folding-binding process
have been studied by many groups,30,31 intermediate states of enzyme protein during its
catalytic process have been revealed by Lu since 2004, etc.32 Recently there is even a
research field named independent disordered protein which is specifically focused on
conformational fluctuation regulation of protein molecules.
One type of proteins that are especially important is enzymes. Enzymes are
protein molecules that can speed up chemical reactions by thousands or even millions of
7
times by changing the energy barrier, or so-called activation energy to accelerate the
formation of intermediate states of given reactions. Typically, such processes are
accomplished via specific binding process between substrate molecules and active site on
enzyme molecules. The term ‘active site’ refers to a part of an enzyme molecule, taking
charge of binding with substrate to catalyze chemical reactions. The active site on an
enzyme molecule typically consists of a few amino acid residues which directly
participate in the recognition of substrate molecules to initiate the catalytic reaction
mechanism. The catalysis process in a chemical reaction is usually triggered by a
collision between a substrate and the active site, which then evolved into a specific
binding process between the substrate and the active site. Some enzymes have their
active sites accessible in their 3-D conformations; some enzymes have their active sites
buried inside their 3-D structure, requiring conformational change to allow the substrate
to access the active site.
The ability of enzyme to increase chemical reaction or biological reaction rate and
the specificity of enzyme function intrigues extensive interests of chemists for a long
time. From ensemble level experiments, kinetic model describing enzymatic reaction has
been established a hundred years ago.33,34 Models describing the kinetics of an enzymatic
reaction can be shown in equation 1.1.
k
k3
kcat
1
ZZZ
X
E + S YZZ
→ EP ⎯⎯→
E+P Z ES ⎯⎯
k
2
(1.1) In equation 1.1, E stands for enzyme, while S and P are short for substrate and products
respectively. Rate constant of each steps are noted as k1 to k3 and kcat. An enzymatic
reaction is a process that including at least three steps: the first step is the enzyme 8
substrate compleex formation
n, while the second
s
step iis chemical rreaction by bbreaking andd
form
ming chemicaal bonds, and
d the final step will be prroduct releassing. The ennergy change
du
uring an enzy
ymatic reacttion is descriibed in figurre 1.1.
Figurre 1.1. Free energy diagrram showing
g that enzym
me increase cchemical reaaction by
lowerring activatio
on free energ
gy barrier. The
T blue currve indicatess an enzymattic reaction,
whilee the black curve indicates the same chemical reaaction withoout enzyme ccatalysis.
Acron
nyms stand for: E for en
nzyme; S forr substrate; P for productt; ES for enzzymesubsttrate complex
x; EP for enzyme-produ
uct complex. ES* stands ffor the transsition state.
ΔG*caat and ΔG*uncat are activaation free eneergy of the eenzymatic reeaction with and without
enzym
me respectiv
vely.
9
Theory to describe the overall rate of enzymatic reaction has also been established
by Michaelis and Menton since 1913, which can be summarized in equation 1.2.34
v = kcat [ E ]0
[S ]
K M + [S ]
(1.2)
In equation 1.2, v stands for the reaction rate, [E]0 is the enzyme concentration, kcat is the
maximum number of substrate molecules that being converted into product during the
reaction per enzyme molecule per second. KM indicates the substrate concentration when
the reaction rate reaches its half-maximum value. [E] and [S] stands for concentration of
enzyme and substrate respectively.
Despite kinetic model has been well-developed to describe mechanism of
enzymatic reactions at ensemble level, structure-function question for enzyme molecules
remains as a challenge. Since late 1990s, with the development of single molecule
techniques, research on enzymatic reaction has also entered a new level. Fluorescencebased optical observation methods have been developed, which allow us to monitor timedependent structure information of single enzyme protein molecule or reaction turnover
events; theoretical models for enzyme at single molecule level has been developed in
recent years too.22 Although some conventional approaches such as NMR and XRD can
resolve enzyme protein conformation in crystal structure form, it has to rely on recently
developed single molecule methods to probe the impact of enzyme conformation to its
activity during reactions, which will be discussed in the following section.
10
1.3 Introduction of Single Molecule Studies of Enzyme
Using single molecule approaches, such as single molecule fluorescence
spectroscopy and single molecule force spectroscopy, we are able to interrogate
conformational dynamics and the associated functions of enzyme molecules at single
molecule level. The advantages of single molecule techniques which have been
discussed in section 1.1 make recent research of enzymatic reactions rely on single
molecule approaches to answer the structure-function questions of enzyme molecules and
to reveal their dynamic characteristics: For a given enzymatic reaction, what is happening
to enzyme molecules when they perform catalytic function? How do substrate ligands
affect or even regulate enzyme-substrate binding events? What does the conformation of
enzyme molecules at intermediate states look like? Is there one single intermediate or
multiple intermediate states of enzyme-substrate complex during a reaction? What is the
impact from conformational fluctuation of enzyme molecules on their functions, and can
we manipulate those conformational fluctuations? Compared to traditional ensemble
level experiments studying enzymatic reactions, single molecule level experiments allow
us to directly probe the formation of enzyme–substrate reactive complex or even the
conformation of active sites on enzyme protein molecules, and to answer those questions.
In this dissertation, our research works are focused on studying conformational
dynamics of enzyme protein molecules at single molecule level. Enzyme-substrate
interactions determine the formation of enzyme-substrate complex which is the initial
triggering step of an enzymatic reaction. Understanding the enzyme-substrate interaction
will be helpful in characterizing formation of enzymatic transition states that defines the
reaction pathway, energetics, and the dynamics. A critical factor determining the rate of
11
enzyme–substrate interactions is enzymatic conformational change. Protein
conformation has been found to be a critical impact factor in determining binding affinity
toward ligands.35 As a result, conformational regulation can significantly affect active
site-substrate binding process. For example, conformational stability of tertiary structure
has been found to have important influence on enzymatic activity;36,37 by manipulating
conformation of enzyme molecules, even controlling enzyme-substrate interaction is
possible.38
To understand the role of conformational change of enzyme molecules in catalytic
process, many different models have been developed. Back in 1894, Fisher proposed a
lock-key hypothesis, qualitatively describing the high selectivity of enzyme-substrate
interactions using a metaphor which is ‘the specificity of the combination of a key and
respective lock’.39 In 1958, Koshland firstly suggested an ‘induced-fit’ theory, claiming
that the binding interaction between an enzyme protein and substrate molecule induces
conformational change to the enzyme protein. By mid 1960s, a ‘conformational selection’
paradigm was proposed, stating that conformations of enzyme molecules are fluctuating
all the time, and substrate ligands will select those conformations that are compatible
with enzyme-substrate binding, resulting in a conformational distribution shift to those
reaction-favored ones.40,41
In our studies in this dissertation, we also try to interrogate the impact of enzyme
conformation to their catalytic function. We developed magnetic tweezers correlated
with single molecule fluorescence spectroscopy for the purpose of probing the enzyme
protein conformational dynamics and the associated enzymatic function. Firstly, we
successfully achieved repetitive conformational manipulation of single enzyme protein
12
molecule. Later, using this conformational manipulation method, we try to interrogate the conformational selection mechanism by probing the conformation flexibility under
different conditions aiming at revealing the influence of enzyme-substrate interactions on
conformational dynamics of enzyme molecules, and to discover dynamic nature of the
enzymatic active transition state formation process.
We further make the attempt to interrogate structure-function relationship at
molecule level in enzymatic reactions. Traditional enzymatic stability studies focused on
ensemble level activity of enzyme at different physical conditions or chemical
environment without probing corresponding change in conformation of enzyme
molecules. Hence, the impact to enzymatic activity from partial conformational change,
which is the condition that enzyme molecules are not unfold or denatured, but only with
some stretching or distortion on its conformation remains unclear. Using single molecule
magnetic tweezers as conformational probe to manipulate enzyme activity at single
molecule level with simultaneous optical observation of turnover events, we are trying to
answer this question.
1.4 Introduction of Magnetic Tweezers
Force is a key parameter which is involved in all types of biological processes.
For example, the motions of motor proteins generate biological force;42 Ligand-receptor
recognition processes involve binding force;43 Protein folding-unfolding events can be
easily affected by external force.44 There are also some force-driven processes, such as
gene expression or cellular motions.45 There are even many proteins having mechanical
functions , such as cytoskeletal proteins or muscle proteins.46 Hence, the capability to
13
measure and to apply force onto single molecules in order to probe those fundamental
processes becomes a major concern in biophysical and biochemistry studies.
Applying molecular level force to manipulate molecules that involved in
biological processes typically require mechanical force at 10-12 Newton (pico-newton, or
pN) scale. Meanwhile, such manipulation approaches also need to achieve a high spatial
precision at 10-9 meter (nm) scale. In the last 30 years, there are plenty of techniques
developed for this purpose, the most successful and thus commonly used techniques are
Atomic Force Microscopy (AFM), Optical Tweezers, Magnetic Tweezers, Biomembrane
Force probe (BFP) and Microneedles.11‐19,47 In our studies, we use magnetic tweezers as
the manipulation approach to apply force on single protein molecules.
Magnetic force has been used in biological and medical field for a long time. For
example, magnetic resonance imaging (MRI) has been widely used as a clinical
diagnostic method; magnetic force for drug targeting has also been developed since
2006.48 The concept of magnetic tweezers is to employ magnetic field to apply force
through magnetic nanoparticles as force sensor conductor onto target molecules. A
classical way of doing single molecule magnetic tweezers experiment is shown in figure
1.2. A target bio-molecule sample, which can be either single DNA or protein molecule
or even biological specimen, is immobilized on the glass cover slip tethered by covalent
bond. And in the other end of the sample molecule, a paramagnetic nanoparticle is linked
via specific covalent bond. This paramagnetic particle will serve as the force sensor,
conducting force applied by external magnetic field to apply force onto the sample
molecule. The applied force can be regulated by controlling the magnetic field. To apply
magnetic field, either permanent magnet or electromagnet can be used, while the field
114
can be
b tuned by either
e
spatiallly controllin
ng the positioon of magneet or controllling the
current applied on
n electromag
gnet.
Figurre 1.2. A conceptual sch
heme of the sample
s
systeem in magneetic tweezerss experimentts.
In briief, we tether a single prrotein molecu
ule to a moddified glass ccoverslip at oone end by
TESP
PA-DMS lin
nkers and bou
und to a super-paramagnnetic bead att the other ennd via biotinn-
15
streptavidin specific binding. When external magnetic field is applied, the protein
molecule will be pulled via sensing the force through the magnetic bead.
The earliest single molecule work applying magnetic tweezers to manipulate biomolecules can be traced back to 1992, which is the study by Bustamante using magnetic
beads to measure the elasticity of single DNA molecules. The term ‘magnetic tweezers’
has not been widely used until 1996, when Bensimon used magnetic force to wring out
DNA molecules. Since then, magnetic tweezers have been widely applied in many
different biophysical research works as a molecular force manipulation approach. There
are extensive works improving the setup of magnetic tweezers;49 many different DNA
molecules have been characterized in their mechanical parameters using magnetic
tweezers;50 Some researchers try to use magnetic tweezers as a force calibration approach
or a force clamp technique;51 Some studies trying to use magnetic force for cellular
nanoparticle transportation have also been achieved.52
Why do we need to develop magnetic tweezers? The answer is that as a molecule
level force probe, magnetic tweezers have some significant advantages compared to other
alternative methods such as AFM and optical tweezers. Firstly, it can apply pulling force
either small as sub-picoNewton49 or large as close to nanoNewton. In contrast, AFM can
only generate pulling force not smaller than 5 pN until 2012, with a complicated
modified experiment setup and micro-machine modified cantilever. The reason lies in
different working mechanism of these approaches. In experiments, AFM needs real-time
response of the physical position of AFM cantilever to calibrate real-time applied force.
Hence, the spring constant of the AFM cantilever will become the limiting factor of how
small the force can be achieved. On the other hand, magnetic tweezers do not require any
16
corresponding physical feedback observation to provide force information in
experimental measurements, although force calibrations need to done independently.
Details of force calibration in magnetic tweezers experiment will be discussed in Chapter
II.
The second advantage is that direct physical contact or chemical contact to target
molecules is not necessary in magnetic tweezers experiments. Magnetic tweezers indeed
require a magnetic nanoparticle, or named magnetic beads to direct tethered onto sample
molecules as a force sensor to conduct applied force by magnetic field. However, in
experimental measurements, it still allow a remote control, or ‘controlling via field’ type
force to be applied. As a result, in magnetic tweezers experiments, any inaccuracy from
direct physical contact of force applying part in instrument such as AFM cantilever
vibration or displacement can be avoided. Also remote control significantly facilitates
the experiment design and reduces complexity for any correlated measurement.
The third character of magnetic tweezers as a single molecule level force
manipulation method is that any photon-damage to the sample or a photon induced
background noise to a correlated simultaneous single-molecule spectroscopic
measurement can be avoided. Compared with optical tweezers, which always require
lasers at infrared region and high power to create optical potential field trap to manipulate
protein spatial position, magnetic tweezers is beneficial in this aspect not only by avoid
of inducing photon damage or photon noise, but also has the advantage by not to induce
any heat as side effect of high power lasers into experimental systems.
17
Finally, magnetic tweezers allows manipulating large number of molecules
simultaneously as long as those molecules have paramagnetic micro beads tethered on
them. In recent years, using an improved modified technique, AFM can also apply force
on multiple protein molecules at the same time.53 However, considering the instrumental
complexity and force scale limit of AFM, magnetic tweezers is still a unique technique to
apply force on multiple molecules. These specificities make the magnetic tweezers
approach promising for protein conformational dynamics studies, especially for our
research purpose in this dissertation.
1.5. Research Objective and Specific Aims, and Dissertation Overview
Previously, research works using magnetic tweezers to study single bio-molecules
majorly focused on manipulating DNA topological structure, or resolving unfolding
dynamics of poly-protein molecules. Manipulating single protein conformation and to
further probe the impact of structure change of protein to its function using magnetic
tweezers remains unchallenged. The objective of our research is using magnetic tweezers
correlated with single molecule fluorescence optical spectroscopy to manipulate
conformation of single protein molecule, and to probe structure-function relationship of
enzyme protein at single molecule level.
This dissertation consists of six chapters. Chapter I discussed about scientific
motivation of the research work in this thesis. Chapter II described instrumental setups in
our experiments and some necessary background knowledge including principles of some
experimental techniques used in our studies. Chapter III presents our work using a
combined magnetic tweezers and simultaneous fluorescence resonance energy transfer
18
(FRET) spectroscopic microscopy to manipulate the conformation of a Cy3-Cy5 dye
labeled 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) molecule and
enzyme-substrate interaction at single-molecule level. Chapter IV shows that by
applying conformational distortion of horseradish peroxidase (HRP) molecules via
magnetic tweezers at single molecule level, we successfully manipulated and examined
the activity changes of HRP catalyzed H2O2-Amplex Red reaction. We made further
theoretical studies using Steered Molecular Dynamics (SMD) simulation method to have
a detailed understanding about the impact on enzyme active domain when external force
is applied via magnetic tweezers in chapter V. In chapter VI, we discussed a newly
developed technical improvement for higher controlling ability for next generation of
magnetic tweezers.
1.6. References
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24 Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L.T.; Itzkan, I.; Dasari, R.R.; Feld, M.S.
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25 Lu, H.P.; Xie, X.S. Single-Molecule Spectral Fluctuations at Room Temperature.
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26 Tamarat, Ph.; Maali, A.; Lounis, B.; Orrit, M. Ten Years of Single-Molecule
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28 Karakas, E.; Furukawa, H. Crystal Structure of a Heterotetrameric NMDA Receptor
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37 Iyer, P.A.; Ananthanarayan, L. Enzyme Stability and Stabilization—Aqueous and
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25
CHAPTER II. EXPERIMENTS
This chapter is dedicated to the description of experimental techniques in our
single molecule magnetic tweezers (MTW) studies and the sample preparation
procedures in our experiments.
2.1 Principles of Experimental Techniques
2.1.1 Principles of Confocal Microscopy
The prototype of the concept of ‘confocal’ can be traced back in 1940, when Hans
Goldmann developed a slip lamp system for eye examination.1 The so-called confocal
microscope came out firstly at 1950s, developed by Marvin Minsky, who was a postdoc
scientist at Harvard, trying to image biological events in his brain tissue study in 1955.2
By 1971, laser was induced as light source for the first time in confocal microscope.3‐4 In
mid 1980s, confocal microscope has been evolved into its modern form, typically a beam
scanning confocal microscope.5 A conceptual figure of confocal microscopy is shown in
figure 2.1. The central idea of confocal microscope is to use two pinhole apertures at
both ends of the optical path: one aperture is put in front of the laser light source, and
another aperture is set in front of the photon detector module. Once these two pinhole
apertures form optical conjugate plane, fluorescence signals from unwanted part of
specimen which are out of focus can be blocked.6
In traditional wide field fluorescence microscope, the incident light excites the
whole sample specimen. As a result, it is difficult to use such illuminating method to
pinpoint a specific small volume of specimen, since fluorescence emitted from all parts of
the specimen always arrive through the same optical path to the photon detector
26
simultaneously, forming unwanted background noise, lowering down signal-noise ratio.
By using the confocal illuminating approach, we can block out lights from all parts other
than the focal volume on the specimen, only allowing emission light from a very close
region around the focal point on sample place to be detected and hence to achieve a high
spatial resolution. In practical, the best vertical imaging resolution of confocal
microscope can be 0.5µm, while the best horizontal imaging resolution can be 0.2 µm,
making the spatial imaging volume close to the diffraction limit.
In experiments, two additional points are noteworthy. Because of the
fluorescence signal from out-of-focus part on the specimen will be blocked by the
conjugate pinhole apertures, the overall signal will only be fluorescence from the focused
volume in the specimen. Hence a low signal level is expected, although with a higher
signal-noise ratio compared to traditional wide field microscopy. To have a practical
detection, we will need photomultiplier tube (PMT) or Avalanche Photodiode (APD) as
detector module. We use APD in our confocal microscopy setup as detector. Another
point worth mentioning is that since the imaging capability of confocal microscope is
limited in the focal volume of the specimen, it will require a scanning process to have an
image of a large area of the specimen, i.e. a 5µm × 5µm region. For one single protein
molecule having a few hundred of amino acid residues, such as 6-hydroxymethyl-7,8dihydropterin pyrophosphokinase (HPPK) or horseradish peroxidase (HRP) which has
been used in our experiment, the overall size of the molecule is limited within 10 nm.
Hence it will be an ideal imaging approach using confocal microscopy to detect protein
molecules. In our studies, we selectively labeled two dye molecules Cy3 and Cy5 on a
227
singlee HPPK prottein moleculle while usin
ng confocal m
microscopy to study connformationall
dynam
mics of HPP
PK moleculee, which willl be discusseed in Chapterr 3.
Figure 2.1. A concep
ptual figure of
o confocal m
microscopy.. Green liness indicate
incident laser sou
urce light, wh
hile red lines stand for eemission fluoorescent lighht after
excitaation of sam
mple specimeen.
28
2.1.2 Principles of Forster Energy Transfer (FRET)
Förster resonance energy transfer (FRET) spectroscopy, or fluorescence
resonance energy transfer spectroscopy as an alternative term sometimes, has been
regarded as one of the remarkable cornerstones in biophysical study field. The concept
of FRET originates around 1948, named by Theodor Förster, a German Physical
Chemist.7 The central idea is outlined below. When two different chromophore
molecules are close to each other in a few tens of angstrom and getting excited by
incident light, excitation energy can be transferred between the two dye molecules by
dipole-dipole resonance interactions, as shown in figure 2.2. In this energy transfer, the
chromophore sending out energy is named donor, and the chromophore that receiving
energy is named acceptor. When a donor molecule is excited from its ground state (S0 in
figure 2.1) to its excited state (S1 in figure 2.1) by incident light, the excited donor will
disperse its energy in two processes that competing with each other: coming back to its
ground state while emitting photon to form ‘fluorescence from donor’, or nonradioactively transfer this energy to the acceptor molecule, forming ‘fluorescence from
acceptor’ by long-range inter molecular dipole-dipole coupling, which is named Förster
energy transfer. The rate of Förster energy transfer can be calculated by equation 2.1.8
kT =
1
τD
×(
R 6
) R0
(2.1)
In equation 2.1, kT is the energy transition rate constant, τD is the fluorescence
lifetime of donor chromophore, R is the distance between donor molecule and acceptor
molecule, R0 is a distance parameter named Förster distance, which depending on various
factors: specific spectroscopic and mutual orientations of different chromophore
29
molecules, refractive index of solvent, and the overlap of emission spectrum of donor and
absorption spectrum of acceptor.9 The parameter we observe in experiment is the
quantum yield of Förster energy transfer, named FRET efficiency, described by equation
2.2.
E= (Energy transferred from Donor to Acceptor)/(Energy absorbed by donor)
(2.2)
The relationship of FRET efficiency E and FRET rate constant kT can be
described as equation 2.3.
E=
k ET
k f + k ET + ∑ ki
(2.3)
In equation 2.3, kET is the FRET rate constant; kf stands for the radioactive decay
rate of donor, while ki indicates all other processes in which donor molecule decay from
its excited state.
330
Figurre 2.2. A con
nceptual figu
ure of the prrinciple of reesonance eneergy transferr between
two different
d
chro
omophore molecules.
m
Experimeental measureement of thee FRET efficciency reliess on the relattionship
show
wn in equation 2.4. The directly
d
obseerved parameeters are steaady state fluuorescence
intensity of donorr (FD) and accceptor (FA). FDA indicattes the fluoreescence intensity of
donorr with the ex
xistence of acceptor,
a
whiile FD indicaates the fluorrescence inteensity of
donorr when only donor moleecules exist in sample sollution.
E=
FD − FDA
FD
(2.4
4)
31
It has been discovered that the rate constant of such energy transfer is
proportional to the inverse sixth power of the distance between donor and acceptor, as
shown in equation 2.5.
E FRET =
1
1 + ( r / R0 ) 6
(2.5)
In equation 2.5, r is the distance between donor molecule and acceptor molecule,
R0 is the Förster distance of the donor-acceptor pair. Hence, Förster resonance energy
transfer efficiency can be an indicator of the distance of two different chromophore
molecules, as long as the distance between the two chromophore molecules is in the scale
of a few nanometers. FRET spectroscopy takes advantage of this phenomenon, using
two or even more different dye molecules as donor and acceptor, labeling those dye
molecules on certain positions of biological specimen, to monitor the distance between
those positions, and hence to obtain structural information of biological specimen.
The relationship between donor-acceptor distance and FRET efficiency make
FRET spectroscopy become a ‘molecular scale ruler’ especially for biophysical studies.
The effective distance of FRET spectroscopy is 10 to 100Å.10 On the other hand, when
the distance between donor and acceptor is close to the Förster distance R0, FRET
efficiency is most sensitive to the change of donor-acceptor distance, as shown in figure
2.3. Hence, in practical, FRET is widely used as a precise molecular level ruler to
measure distance around 40Å to 60Å in biological specimens.
332
Figurre 2.3. The distance-FRE
d
ET efficienccy relationshhip.
Successfu
ul FRET meaasurement allso requires that approprriate chromoophore shoulld
be ch
hosen. The Förster
F
reson
nance energy
y transfer reqquires overlaap between eemission
specttrum of dono
or and absorp
ption spectru
um of accepttor, which m
means that thhe energy
releassed from don
nor moleculees can be efffectively recceived by accceptor moleccules. In ouur
studiees, we use Cyanine
C
3 (C
Cy3) as donorr and Cyaninne 5 (Cy5) aas acceptor, w
while their
specttrum profile is shown in figure 2.4. The
T Forster distance of ddye pair Cy33 and Cy5 iss
5.4nm
m. 11‐12
333
Figurre 2.4. Specttrum profile of Chromop
phore moleccule Cy3 andd Cy5.
2.1.3 Principles of Total Intternal Reflection Microoscopy (TIR
RFM)
on is a comm
mon physicaal phenomennon that happpening whenn
Total inteernal reflectio
h a boundaryy of differentt medium. S
Since light iss
a beaam propagatiing wave passses through
also one
o type of electromagn
e
et propagatin
ng wave, tottal internal rreflection is iinvolved as a
charaacter of lightt. In brief, on
nce a beam of
o light is paassing througgh a boundaary of two
differrent optical medium,
m
wh
hose refractiv
ve index are n1 and n2 reespectively, w
while n1>n2,
partiaal of the ligh
ht will go thrrough the meedium; partiaal of the lighht will be refflected back
to thee same side of
o the incideent light, as shown
s
in figu
gure 2.5. However, whenn the angle
formeed by incident light and the normal direction
d
to tthe boundaryy surface is larger than a
certaiin angle, notted as θ in figure 2.5, thee incident ligght will be 100% reflecteed back
34
internally to the incidence side. This phenomenon is called total internal reflection, and
the angle θ is named critical angle, as shown in figure 2.5. 13
One point need to be specially mentioned is that if a beam of light is shedding
from on medium, hitting to another which has a lower refractive index, i.e., if n1<n2 in
figure 2.5, total internal reflection phenomena will not occur in any angle. There is no
concept as ‘critical angle’ anymore in this condition, and the term ‘total internal
reflection’ does not refer to this situation. 14‐15
The physical principle of total internal phenomena can be explained by Snell’s
law. When a beam of light is propagating from one medium with refractive index n1 to a
different refractive index n2, the angles formed by the optical beam to the normal
direction to the boundary surface are noted as θ1 and θ2 respectively, as shown in figure
2.5. Then the relationship between these two different refractive index and angles can be
described by equation 2.6, which is called Snell’s law.
n1 sin θ1 = n2 sin θ 2 (2.6)
Hence, when n1 is larger than n2, sinθ1 will be smaller than sinθ2. That means a
‘bending’ of the light path, and a larger θ2 compared to θ1. As a result, when θ2 reaches
90o, θ1 is still at a sharp angle that between 0o and 90o. For any larger θ1 since then, the
incident light will always be total internally reflected back to the original medium. The
θ1 at that point is the critical angle, as shown in figure 2.5.
335
Figurre 2.5. A co
onceptual fig
gure of total internal refleection phenoomenon.
There is an
a interesting
g and importtant side effeect of total innternal refleection
pheno
omenon, nam
med evanesccent wave. When
W
a beam
m of light is propagatingg in a medium
m
with refractive in
ndex n1, hittin
ng to the bou
undary of annother mediuum with smaaller
refracctive index n2 in an angle that is larg
ger than criti cal angle annd hence youu get total
intern
nally reflecteed to the orig
ginal medium
m, there is sttill some waave ‘penetratting’ to the
otherr medium, traavel along th
he boundary of the two m
mediums. A
As shown in ffigure 2.6,
evaneescent wave is a near-fieeld wave evaanescent wavve traveling along the x direction,
and decay
d
along z direction.
36
The physical principle of evanescent wave is as below. First, after a beam of
planar wave is hitting a boundary of two different medium, the transmitted part of the
wave can be described by equation 2.7.
k T = kT sin(θT )x + kT cos(θT )z (2.7)
In equation 2.7, kT is the wave vector of transmitted wave, θT is the virtual angle
of the transmitted wave to the normal direction of the boundary, x and z is direction unit
vector shown in figure 2.6. From equation 2.6 we have
sin(θT ) =
n1
cos(θ1 ) n2
(2.8)
The angle θ1 is the angle between incident wave and normal direction of the
boundary surface, as shown in figure 2.6. Hence, when n1>n2, sinθT is larger than 1,
resulting in a complex value cosθT:
cos(θT ) = i sin 2 (θT ) − 1 (2.9)
On the other hand, from electrodynamics, we have description of transmitted
plane wave as below:
E T = E 0 e i ( k T •r −ω t ) (2.10)
Combined equation 2.7, 2.9 and 2.10, we have
ET = E0e − kT
sin 2 (θT ) −1z i ( kT sin(θT ) x −ωt )
e
(2.11)
337
Equation 2.11 directly
y shows the character off evanescent wave: traveeling throughh
x direection, decay
y along z dirrection. In brief,
b
evanesccent wave w
will only be iintense withiin
one th
hird of the wavelength
w
starting
s
from
m the boundaary surface. In our experriments, we
use 532 nm lasers as excitatio
on incident light,
l
hence tthe generateed evanescennt wave is
nce approxim
mately 170 nnm starting frrom the
consiidered to be effective witthin a distan
samp
ple plane.
Figurre 2.6. A co
onceptual sch
heme of evaanescent wavve when totaal internal refflection of
optical beam occu
urs.
38
An alternative representation of equation 2.10 is as below: 16
z
I = I 0 exp(− ) d
(2.11)
In equation 2.11, I is the intensity of evanescent wave, z is the perpendicular
distance to the sample surface, d is characteristic exponential decay depth, which can be
described as below:
d=
1
−
λ sin 2 θ I
( 2 − 1) 2 4π n2 sin θ
(2.12)
In equation 2.12, λ is wavelength of incident light, n2 is refractive index of the
transmitted substrate shown as figure 2.6; θ is the critical angle, while θI is incident angle
which is the angle formed by incident light and normal direction to the sample plane.
Total internal reflection microscope (TIRFM) takes advantage of evanescent
wave to form a molecular-level fluorescence signal detection method. A conceptual
graph of TIRFM is shown in figure 2.7. A beam of incident lasers is hitting through
objective to the top surface of glass coverslip, and get entirely reflected back by tuning
incident angle larger than critical angle. Evanescent wave will be generated close to the
top surface of the coverslip, where the specimen is put. Immersed oil is always used to
ensure that refractive index value is identical during the optical path of incident light
before it hits the top surface of glass coverslip. Details of TIRFM in my experiments will
be discussed in the experiment setup section.
339
Figurre 2.7. Concceptual scheeme of total internal
i
refleection microoscopy (TIRF
FM). Greenn
arrow
w lines indicaate incident light source, while red liines indicatee signal beam
m light
emittted by samplle specimen after being excited
e
by evvanescent w
wave.
40
2.1.4 Signal Detection techniques: Introduction to APD and EMCCD
Introduction to Avalanche Photodiode (APD)
In the magnetic tweezers-confocal FRET measurement which is the experiment
performed in chapter III, we use avalanche photodiode (APD) as optical signal detection
approach. In the magnetic tweezers-TIRFM measurement which is the experiment
performed in chapter IV, we use electron multiplied charge coupled device (EMCCD) as
optical signal detection method. In this section, we will briefly introduce the basic
principles of these two fluorescence signal detection techniques.
Avalanche photodiode (APD) is a type of semiconductor electronic device that
based on photoelectric effect, converting light to electric signals with high sensitivity.
The working principles of semiconductor photodiode as photodetector is shown in figure
2.8A and 2.8B. The core part is a PIN diode, which has one a p-type semiconductor and
one n-type semiconductor at two ends, with a wide semiconductor region in middle. The
p-type part and n-type part are usually heavily doped while the semiconductor region is
undoped. When one photon comes in as external light signal, an electron with proper
excited and raised from the valance band to the conduction band to generate an electronhole pair will be formed in the semiconductor region. External bias voltage will let these
two electrons to drift quickly away from the junction region. Once there are light hitting
the detector with multiple photons, such electron movement will form a current flow
whose intensity is proportional to the incident light. In this way, light signals can be
converted to electronic signals to be detected precisely and quantitatively.
41
However, in traditional PIN diode, only one electron-hole pair will be triggered
by one incoming photon. For single molecule experiments, the light signal will always
be weak, which means there may not to be enough photons to trigger electric signals with
satisfied signal-noise ratio. In some single molecule experiments, there are even photons
arriving to the detector one by one.17 APD will be used instead to discern signals when
only a few photons are collected. As shown in figure 2.8C, the core part of APD is the
built-in signal amplification region named depletion layer, typically a silicon photodiode
structure, where electron multiplication occurs. When one photon arrived from the n-side,
an electron-hole pair is formed. The n-side is negatively doped, while the p-side is
positively doped, and a thin p-layer is coated on the n-side. An external reverse bias
voltage is applied, which means p-side is connected to cathode, and n-side is connected to
anode. The electron-hole pair in the depletion layer will then move towards respectively
to the PN junctions due to external reverse bias voltage: electron will run back toward
original n-side, and the ‘hole’ runs toward p-side. Once the external reverse bias voltage
is high enough, i.e. higher than 105 V/m, the electron will collide to the thin p-layer on
the n-side, resulting in ionization generating more electron-hole pairs. In this way, one
single incident photon is capable to trigger multiple electron-hole pairs. Such process is
named impact ionization, or avalanche effect. As a result of avalanche effect, the signal
level become detectable, named current gain effect. The gain will be around 100 when
reverse bias voltage is around 100-200 V. By appropriate doping techniques, the gain
can even be high as 1000.
442
Figurre 2.8. Princciple of Phottodiode Mod
dule used in oour experim
ment. (A) Traaditional PIN
N
photo
odiode when
n there is no incident ligh
ht. (B) Workking mechannism of tradittional PIN
photo
odiode when
n light is inco
oming. (C) A conceptuaal figure of aavalanche phhotodiode
configuration.
43
Introduction to Electron Multiplied Charged Coupled Device (EMCCD)
In the magnetic tweezers-TIRFM measurement which is the experiment
performed in chapter IV, we use electron multiplied charge coupled device (EMCCD) to
monitor fluorescence signal of released products from single molecule enzyme catalysis
events.
The core part of traditional CCD is a photoactive layer consisting of a silicon
capacitor matrix array to sense photons from external light. Nowadays it is a common
technique used in digital cameras or optical scanners. For single molecule level signal
detection, similar to traditional photodiode, traditional CCD also cannot provide
sufficient signal-noise ratio due to lower photon level in experiments. Hence, EMCCD is
used instead. The principle of EMCCD is as described in figure 2.9. In brief, EMCCD
also utilized the phenomenon of impact ionization to amplify the electron signal from
active pixels hit by incident light. The core part of EMCCD is a gain register placed
between the output amplifier and the shift register, as shown in figure 2.9. The gain
probability at every stage in the gain register is typically small as 2%. However, in one
EMCCD, there will be hundreds of stages in the gain register, and resulting in overall
high gain. When an incoming image signal hits on the active pixels, the information of
the image will be stored as converted electron signal in the image storage area. Then, the
stored information can be either read out row by row from the serial shift register to
generate a normal CCD image, or to pass through the electron multiply register, amplify
signal via impact ionization, to generate an electron multiplied image. One point needs to
be mentioned is that time for signal readout process in both CCD image and EMCCD
444
image cannot be neglected. It
I may requirres tens of m
millisecond ttime for one frame of
image which con
nsisting of 51
12 times 512
2 pixels. Figurre 2.9. Princciple of Electtron Multipllied Charge C
Coupled Devvice (EMCC
CD).
45
2.1.5 Basics of Magnetic Tweezers: Force Calibration
An introduction of principle, history and application of single molecule magnetic
tweezers has already been discussed in chapter 1.4. In this section, we will go through
some experimental details of magnetic tweezers.
In practical, when setting an experiment using magnetic tweezers as single
molecule level force manipulation approach, two major aspects need to be deliberately
considered. First of all, an appropriate observation approach needs to be correlated with
magnetic tweezers. This is because unlike other single molecule level manipulation
technique such as AFM or optical tweezers, magnetic tweezers does not have a way to
directly obtain the sample conformation by its own. In physical essential aspect, the core
part of magnetic tweezers will be a piece of magnet, despite sometimes it can be designed
into many different complicated forms. Hence, to carry out a single molecule level
magnetic tweezers experiment, at least one type of single molecule optical observation
setup needs to be set to provide information of target sample molecules during the
experiment. Secondly, appropriate combination of magnetic field and magnetic beads
need to be chosen to convey the magnetic force onto target sample molecules. In this
section, our discussion will focused on force calibration for magnetic tweezers. And
details of correlating magnetic tweezers setup with correlated single molecule optical
observation methods will be discussed in section 2.2.
Mechanical force from external magnetic field is applied on a targeted protein
through a paramagnetic bead linked covalently to the single protein molecule. To
quantitatively understand the force that applied on protein molecules by the magnetic
46
tweezers, we note that there are a number of specific approaches to estimate the
mechanical forces applied though a magnetic field on a paramagnetic bead: (1)
Measuring and model analyzing the Brownian motions of a tethered paramagnetic bead;18
(2) Monitoring the dragging motion of a small number of magnetic beads in liquid
environment with known viscosity;19,20 (3) Observing the displacement of a micropipette
with a magnetic bead attached at its end, etc.19 Different methods for measuring torque
on magnetic beads have also been developed.21 Nevertheless, each of the estimation
approaches bears specific merit of estimation with certain error bars. We have applied a
model analysis based on the measured magnetic field strength curve (Figure 2.8) as a
function of the distance between the magnetic tip and the sample surface.
We calibrate the applied force by estimating the magnetic field gradient to get the
magnetic moment of the beads tethered on the single protein molecule. For a magnetic
bead in an externally-produced magnetic field B, noting its magnetic moment as m, then
the potential energy U is:
(2.13)
U = -m × B
For a given magnetic bead, its magnetic moment m is the product of the volume
magnetization M and volume V of the bead. Therefore, the force F that is applied on the
magnetic bead can be calculated:
F = -∇U = -∇(-m ⋅ B ) = m ⋅∇B = MV ⋅∇B = MV
∂B
∂z
(2.14)
In our experiments, the magnetic field applied is approximately 1100 Gauss. As
an approximation, we only consider the magnetic field gradient in one direction
47
perpendicular to the sample plane. Thus the field gradient can be estimated from the
curve shown in Figure 2.10. In this way the value of field gradient is calculated to be
55±15 T/m. When calculating the field gradient, position error of up to 1mm is taken
into consideration as uncertainty of distance between the magnet and the sample plane.
The volume V of paramagnetic bead is 0.6×10-18m3, and the volume magnetization M is
43×103 A/m.22 In our calculation, we have considered the factor that M here is the
saturation magnetization, an approximation that may bring error less than 25%. Hence
the force is calculated 1.4±0.4 pN from equation 2.14. The typical force applied to the
targeted single-molecule HPPK proteins is roughly 1-3 pico-Newton that is weaker than a
typical hydrogen bonding force of 6-9 pico-Newton.
Figure 2.10. Magnetic field-Distance curve of the magnet used in experiment. Each
data point is repetitively measured for five times. The blue point indicates the position of
the magnet in the experiment which is set 4 mm above the sample plane to generate a
magnetic field with approximately 1100 Gauss strength.
48
2.2 Experimental Details
2.2.1 Experimental Setup of Single Molecule FRET Correlated with Magnetic
Tweezers
In chapter III, the measurement was carried out by a correlated setup combining a
two-channel laser scanning microscope with magnetic tweezers, to take single-molecule
FRET imaging as simultaneous optical observation of protein conformation while
mechanical manipulation of the same protein molecule by magnetic tweezers is
performing.
The setup configuration is shown in figure 2.11. In brief, we use the singlemolecule photon stamping approach to record the single- molecule FRET fluctuation
time trajectories photon by photon for both the donor and acceptor simultaneously. The
fluorescence images and photon-counting trajectories are acquired with an inverted
confocal microscope (Axiovert 200, Zeiss). The excitation laser (532 nm continuouswave (CW) Crystal laser) confocal beam is reflected by a dichroic beam splitter (z532rdc,
Chroma Technology) and focused by a high-numerical aperture objective (1.3 NA, 100×,
Zeiss) on the sample at a diffraction limited spot of ~300 nm diameter. In order to obtain
the fluorescence images and intensity trajectories, the emission signal is split by using a
dichroic beam splitter (640dcxr) into two color beams c entered at 570 nm and 670 nm
representing Cy3 and Cy5 emissions, respectively. The signals from two channels are
detected by a pair of Si avalanche photodiode single photon counting modules (SPCMAQR-16, Perkin Elmer Optoelectronics). Typical images (10 μ m × 10 μ m) are acquired
by continuously raster-scanning the sample over the laser focus with a scanning speed of
449
4ms/p
pixel, with each
e
image of
o 100 pixelss × 100 pixells. The fluorescence inttensity
trajecctories of thee donor (Cy 3) and accep
pter (Cy5) arre recorded bby a two-chaannel
Picoh
harp 300 (PiccoQuant) ph
hoton-stampiing set-up. A permanentt magnet is cconnected onn
an ind
dependent z-axis stage to control its distance to the sample gglass coversllip, while X-Y dirrection in-plaane movemeent is controllled by the tuuning the tw
wo-layer sam
mple stage byy
comp
puter. Figurre 2.11. Exp
perimental setup of single molecule FRET-Magnnetic Tweezzers
specttroscopy.
550
2.2.2 Experimental Setup off Single Mole
lecule TIRFM
FM Correlateed with Maggnetic
Tweeezers
In chapterr IV, the meaasurement was
w carried oout by a correelated setup combining a
inverrted laser scaanning micro
oscope with magnetic tw
weezers, to taake single-m
molecule
TIRF
F imaging as simultaneou
us optical ob
bservation off enzymatic activity whiile
mech
hanical manipulation of the
t same enzzyme molecuule by magnnetic tweezerrs is
perfo
orming.
The setup
p configuration is shown in figure 2.12. In brief, we carried oout the TIRF
F
measurements by
y using an in
nverted confo
ocal microsccope (Olymppus IX 71 wiith 60 x
W laser (Crysstalaser) gennerating evannescent wavee for total
objecctive) with a 532 nm CW
intern
nal excitation
n. Emitted signal
s
are filltered with oone beam spllitterfilter (C
Chroma
Techn
nology, z532
2rdc), one 54
45 nm long--pass filter annd then beinng collected bby an
Electtron Multiply
ying Charge Coupled Deevice (EMCC
CD: ProEM 512B, PI coo.).
51
Figure 2.12. Experimental setup of single molecule TIRF-Magnetic Tweezers
spectroscopy.
2.2.3 Steered Molecular Dynamics (SMD) Simulation
In chapter V, we performed steered molecular dynamics (SMD) simulation to
have a single molecule level understanding of the impact of conformational manipulation
to the function of target protein molecule. The scene at molecule level in our studies is as
below. Firstly, we immobilized one single enzyme protein molecule at one given residue
position. A external pulling force is then applied to another given residue position on the
sample enzyme protein molecule. In chapter III, we achieved conformational
manipulation of HPPK protein molecule by magnetic tweezers at single molecule level.
In chapter IV, we studied HRP enzyme protein using the same conformational
manipulation approach to discover what impact such conformational manipulation can
have to protein function. Hence, Steered MD simulation can provide us additional
valuable information of a molecule level scene connecting the studies in chapter III and
chapter IV. There are several different software packages for the purpose of normal MD
simulation, such as GROMACS, CHARMM, AMBER, etc. However, the scene in our
studies that ‘pulling one single protein from two given residue position’ requires a special
type MD simulation named Steered MD (SMD), with NAMD as software package
respectively. NAMD was developed by the Theoretical and Computational Biophysics
Group in the Beckman Institute for Advanced Science and Technology at the University
of Illinois at Urbana-Champaign. More details of our SMD simulation will be discussed
in chapter V.
52
2.3 Materials and Sample Preparation
In our experiments, we prepared single molecule immobilized protein samples in
the way described in Figure 2.13.In brief, we tether a single protein molecule to a
modified glass coverslip at one end by TESPA-DMS linkers and bound to a superparamagnetic bead (Dynabeads® MyOne™ Streptavidin T1, 1.05-µm diameter,
Invitrogen Company) at the other end via biotin-streptavidin linkers. The glass coverslip
is modified as below. Firstly, a clean glass coverslip is immersed overnight in NaOHethanol solution. The coverslip was next washed by distilled water, blow-dried by air
flow, and incubated with a DMSO solution containing a mixture in 10% concentration
consists of TESPA and isobutyltrimethoxysilane in 1:10000 ratio overnight. The low
concentration of each solution was to make sure that the distribution of protein molecules
on cover glass is separate so that one bead does not attach to multiple protein molecules.
More details such as incubation time and buffer pH will be discussed in chapter III and
chapter IV specifically.
553
Figurre 2.13. A conceptual scheme
s
of siingle molecuule protein im
mmobilizatioon method.
A cleean cover glaass was treatted by 3-amiinopropyltrieethoxy-silanne (TESPA) m
mixed in
isobu
utyltrimethox
xysilane in 1:10000
1
ratio
o in DMSO ssolution. Prrotein molecuules are
tetherred onto the siliconized cover glass at
a one end bby Dimethyl suberimidatte-2HCl
(DMS
S-2HCl) which reacting with amine group on TE
ESPA, and aat the other eend to a
param
magnetic beaad by biotin--streptavidin
n bonding. P
Protein molecules use thee amine
group
p from its Ly
ysine residuees to form bo
ond at both eends. As a rresult, all Lyysine residuees
are acccessible to become the linking posiition of one ssingle proteiin molecule, leading to a
multiiple-possibility of protein
n tethering condition.
c
D
Details will bbe discussedd in chapter
III an
nd chapter IV
V.
54
2.4. Selection of Magnetic Beads
We chose the magnetic beads with 1 µm diameter for our experiment in order to
apply force at pN scale on protein molecules. In experiment, an easy way to have a quick
test of the applied force on magnetic beads is using the Stokes Formula Force=6πƞrv,
where ƞ is the viscosity of the solution; r is radius of the magnetic bead; and v is the
moving speed through the solution monitored from microscope. Although we use a
different approach to quantitatively estimate the applied force by our magnetic tweezers,
as shown in our previous publication, we still rely on watching velocities of free
magnetic beads in solution to have an estimation of the force scale we applied. We
assume our PBS buffer solution has similar viscosity as water, since the concentration of
the buffer solution is low as 50mM. The diameter of eyepiece on microscope is
approximately 3 cm, while the objective amplification is 60 and eyepiece amplification is
10. As a result, by observing through eyepiece we can monitor an area of 50 µm. As
shown in figure S5, we can see that magnetic beads with small diameter at 100 nm scale
need to be driven faster than 1 mm/s to generate even 1 pN force, indicating that it is not
a good choice to use beads at 100 nm scale for our experiment. On the other hand,
although based the Stokes Formula, larger beads always appears to be better, we also
need to consider that our experiment is observing protein molecules, with their size at 1
to 10 nm scale. Hence, we hope to limit the bead size as small as possible to decrease the
impact of the magnetic beads to local solution environment. As a compromise of these
two thoughts, we chose the 1µm size beads for our measurements.
55
Fig. 2.14. Force-velocity response of magnetic beads in different size.
56
2.5 References
1
Goldmann, H. Spaltlampenphotographie und –Photometrie. Ophthalmologica 1939, 98
(5/6): 257–270.
2
Minsky, M. Microscopy Apparatus: US 3,013,467.
3
Egger, M. D. (1971). Scanning Laser Microscope for Biological Investigations. Applied
optics. 1971, 10 (7): 1615–1619.
4
Amos, W.B.; White, J.G.: How the Confocal Laser Scanning Microscope Entered Biological
Research. In: Biology of the Cell / under the Auspices of the European Cell Biology
Organization. Band 95, Nummer 6, September 2003, S. 335–342.
5
Prasad, V.; Semwogerere, D.; Weeks, E.R. Confocal Microscopy of Colloids. J. Phys.: Cond.
Mat. 2007 19, 113102.
6
Förster, T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Annalen der Physik
1948, 437, 55-75.
7
Förster T. Fluorescence of Organic Compounds Gettingen: Vandenhoeck & Ruprecht:
1951:312.
8
Stryer, L.; Haugland, R. P. Energy Transfer: A Spectroscopic Ruler. Proc. Natl. Acad. Sci.
USA 1967, 58, 719-730.
9
Clegg, R.M. Fluorescence Resonance Energy Transfer. Curr. Opin. in Biotech. 1995, 6:103-l
10.
10 Ha, T.; Enderle, T.H.; Ogletree, D.F.; Chemla, D.S.; Selvein, P.R.; Weiss, S. Probing the
Interaction Between Two Single Molecules: Fluorescence Resonance Energy Transfer
Between a Single Donor and a Single Acceptor. Proc. Natl. Acad. Sci. USA 1996, 93, 62646268.
57
11 Deniz, A.A.; Dahan, M.; Grunwell, J.R.; Ha, T.; Faulhaber, A.E.; Chemla, D.S.; Weiss, S.;
Schultz, P.G. Single-Pair Fluorescence Resonance Energy Transfer on Freely Diffusing
Molecules: Observation of Förster Distance Dependence and Subpopulations Proc. Natl.
Acad. Sci. USA 1999, 96, 3670–3675.
12 Ambrose, E.J. A Surface Contact Microscope for the Study of Cell Movements. Nature 1956,
178 (4543): 1194.
13 Yanagida, T.; Sako, Y.; Minoghchi, S. Single-Molecule Imaging of EGFR Signalling on the
Surface of Living Cells. Nature Cell Biology 2000, 2 (3): 168–172.
14 Axelrod, D. Total Internal Reflection Fluorescence Microscopy in Cell Biology". Traffic
2001, 2 (11): 764–774.
15 Axelrod, D. Cell-Substrate Contacts Illuminated by Total Internal Reflection Fluorescence. J.
Cell Biol. 1981, 89, 141-145.
16 He, Y.; Lu, M.; Lu, H.P. Single-Molecule Photon Stamping FRET Spectroscopy Study of
Enzymatic Conformational Dynamics. Phys. Chem. Chem. Phys., 2013, 15, 770-775.
17 Smith, S. B.; Finzi, L.; Bustamante, C. Direct Mechanical Measurements of the Elasticity of
Single DNA Molecules by Using Magnetic Beads. Science 1992, 258, 1122-1126.
18 Haber, C.; Wirtz, D. Magnetic Tweezers for DNA Micromanipulation. Rev. Sci Instrum.
2000, 71, 4561-4570.
19 Kollmannsberger, P.; Fabry, B. High-Force Magnetic Tweezers with Force Feedback for
Biological Applications. Rev. Sci Instrum. 2007, 78, 114301.
20 Forth, D.; Sheinin, M.Y.; Inman, J.; Wang, M.D. Torque Measurement at the SingleMolecule Level. Annu. Rev. Biophys. 2013, 42, 583-604.
21 Note: the value is according to the product specification from Invitrogen Company.
58
CHAPTER III. MANIPULATING AND PROBING ENZYMATIC
CONFORMATIONAL FLUCTUATIONS AND ENZYME-SUBSTRATE
INTERACTIONS BY SINGLE-MOLECULE FRET-MAGNETIC TWEEZERS
MICROSCOPY
3.1 Introduction
Conformational change of protein molecules is often critical for the biological
functions, affecting the affinity and selectivity of protein-protein and protein-ligand
interactions, and further regulating the catalytic activity of enzymatic reactions.1-4 For
example, an enzyme can have different activities with different conformations.5-7 Thus,
manipulating protein conformations can be an effective approach to study the relationship
between protein conformation and function.8-30
One of the central questions in protein functions is the impact of ligand binding to
conformational fluctuation or conformational flexibility changes of protein molecules,
especially enzyme-substrate interaction.3 The answer to this question serves a critical
understanding of the enzyme-substrate interactions and the enzymatic active transition
state formation. In recent years, a number of novel single-molecule approaches
combining single-molecule optical spectroscopy with mechanical force manipulation
approaches have been developed to achieve protein conformational manipulation, such as
atomic force microscope (AFM), optical tweezers, and magnetic tweezers, etc.9-11,21-36
Here we report our newly developed approach using magnetic tweezers correlated with
single-molecule FRET spectroscopy to study ligand-binding impact on enzymatic
conformation by force manipulating single enzyme molecule conformation with
59
simultaneous optical observation of the enzyme conformational fluctuations under
different conditions of with and without enzymatic substrate.
Compared with other approaches for manipulating single protein molecules, such
as AFM or optical tweezers, magnetic tweezers has a number of desirable and
complimentary specificities: (1) magnetic tweezers can apply a pulling force either in a
fine scale as small as sub-picoNewton37 or in a relative large scale close to
nanoNewtons;38 (2) magnetic tweezers does not require physical contact or chemical
contact to target molecules; (3) magnetic tweezers does not induce either photo-damage
to the sample or a photon background noise to a correlated simultaneous single-molecule
spectroscopic measurement; (4) magnetic tweezers allows manipulating conformation of
a large number of molecules simultaneously as long as the molecules are tethered to
paramagnetic micro beads. These specificities make the magnetic tweezers approach
promising for protein conformational manipulation. Since 1990s, extensive studies on
manipulating single biological molecules by using magnetic tweezers have been
reported.39-45 The applications of magnetic tweezers manipulating biological molecules
have been extended from DNA wringing46,47 to polymer protein molecules pulling48, 49.
The correlated theoretical simulations have also been developed in recent years.48,49
Nevertheless, to our knowledge, conformational manipulation by magnetic tweezers and
correlated simultaneous single-molecule FRET spectroscopic analysis of a single protein
molecule has not been reported.
60
3.2 Materials and Methods
3.2.1 HPPK Protein
In our experiment, we chose fluorescence dye labeled 6-hydroxymethyl-7,8dihydropterin pyrophospho- kinase (HPPK) as a model system to measure the FRET and
magnetic tweezers manipulations in the solutions with and without enzymatic substrates.
HPPK is an 18 kDa 158-residue monomeric enzyme protein molecule with the biological
function to catalyze the transferring of pyrophosphate from ATP to 6-hydroxymethyl-7,8dihydropterin (HP), releasing adenosine monophosphate (AMP) and 6-hydroxymethyl7,8-dihydropterin pyrophosphate (HPPP) as products.50-53 The fluorescent dyes Cy3
(donor)/Cy5 (acceptor) was labeled to the mutated amino acid residue 48 on loop 2 and
residue 151 close to the active site of the enzyme, respectively, as shown in Figure 3.1.
HPPK molecules were bound to the glass cover slip at one end by 3triethoxysilylpropylamine-Dimethyl Suberimidate links and attached to a superparamagnetic bead at the other end by biotin-streptavidin links. The HPPK molecule was
labeled at residue position 48-151 with Cy3 and Cy5 dye molecules respectively. We
choose Cy3-Cy5 dye labeled HPPK as a model system to study the effect of external
force triggering on enzymatic conformational dynamics by using combined magnetic
tweezers manipulations and correlated FRET measurement in the solution with and
without enzymatic substrates.
61
Figure 3.1. Structuraal informatio
on of HPPK molecule. (A
A) Crystal sttructure of H
HPPK moleccule
from protein databan
nk (PDB ID: 1HKA). Th
he mutated dyye-labeled reesidue and L
Lysine residuue
has been pointed out (black spotss on the molecule). The HPPK moleecule was labbeled at resiidue
4
with
h Cy3 and Cyy5 dye moleccules which are the greeen and red sppot in the schheme
position 48-151
respectiv
vely. (B) An example of single moleccular image of the HPPK
K molecule oobserved by
confocal microscope. (C) Full seequence of HPPK
H
proteinn.
62
3.2.2 Sample Preparation
As shown in Figure 3.1, the fluorescent dyes, Cy3 and Cy5 as FRET donor and
acceptor, were labeled to the mutated amino acid residue 48 on loop 2 and residue 151
close to the active site of the enzyme, respectively. The Cy3/Cy5 fluorescent dye pair
was statistically labeled to the mutated enzyme with thiolation. HPPK molecules with
this type of dye labelling have been used to study conformational dynamics in previous
published work from our group.54 The HPPK molecules were bound to the glass cover
slip at one end by 3-aminopropyltriethoxy-silane (TESPA)-Dimethyl Suberimidate•2HCl
(DMS) linkers and linked to a super-paramagnetic bead (Dynabeads® MyOne™
Streptavidin T1, 1.05-µm diameter, Invitrogen Company) at the other end via biotinstreptavidin bond. Protein immobilization was carried out through a routine procedure
shown in figure 3.2. In brief, a clean glass coverslip was immersed overnight in NaOHethanol solution. The coverslip was next washed by distilled water, blow-dried by air
flow, and incubated with a DMSO solution containing a mixture in 10% concentration
consisting of TESPA and isobutyltrimethoxysilane in 1:10000 ratio overnight. The
coverslip was then washed by distilled water and consecutively transferred and incubated
for 4 hours in each system below: 15 mL PBS buffer solution pH=8.0, containing 10nM
Dimethyl Suberimidate•2HCl (DMS•2HCl); 15mL PBS buffer solution pH=7.4,
containing 1nM HPPK; 15 mL PBS buffer solution pH=7.4, containing 10 nM NHSPEO12-biotin; 15 ml PBS solution pH=7.4, containing 1µl magnetic beads stock solution
which is commercial available. The low concentration of each solution was to make sure
that the distribution of protein molecules on cover glass is adequately separated so that
one bead does not attach to multiple protein molecules. Meanwhile, low concentrations
63
of TESP
PA are used to ensure that immobiilized protein moleculees are distriibuted
separately enough from
f
each other
o
for obttaining sing
gle moleculle FRET im
mages.
Figure 3.2. Preparatiion of singlee molecule HPPK
H
samplee. We tethereed protein m
molecules at one
end to the coverslip by
b Dimethyll suberimidatte-2HCl (DM
MS-2HCl) annd at the othher end to a 1 µm
n-streptavidin
n bonding. F
Force was appplied by addding externaal
size paramagnetic beead by biotin
magneticc field and heence the mollecule could
d feel it throuugh the beadds.
In
n our experiments, we conducted the single-m
molecule FR
RET measu
urements wiith
simultan
neous magn
netic tweezeers pulling of
o HPPK en
nzyme moleecules in PB
BS buffer
solution under the conditions
c
with
w and wiithout the en
nzymatic reeaction subsstrates. Thee
me moleculees were imm
mersed in a solution co
ontaining 50
0 mM pH=7
7.4
immobillized enzym
PBS bufffer solution
n as imaging
g buffer and
d 1 mM 6-h
hydroxy-2,5
5,7,8-tetram
methylchrom
man-
64
2-carboxylic (Trolox) solution as oxygen scavenger to protect dye molecules from
photobleach.
We also have noted that either biotin or DMS can only be tethered to a HPPK
molecule via connection with lysine in the amino acid sequence, which leads to multiple
possible tethered condition of the protein molecule to coverslip or magnetic beads.
However, in each FRET measurement, we focused on a specific individual HPPK
molecule during our repetitive manipulation by magnetic tweezers. Consequently,
although we did not necessarily pinpoint that a pair of specific lysine residues tethered to
a specific protein molecule, our observation of the reproducible FRET changes under
periodically applied magnetic field demonstrates that successful single-molecule level
protein conformational manipulation is achieved.
All the attachment to HPPK molecule, either biotin or DMS, can only be fulfilled via
connection with lysine in the amino acid sequence. Hence there are five possible positions in an
HPPK molecule to allow attachment of magnetic particle via biotin or attachment to cover glass
via DMS. As a result, there are five possible positions on one HPPK molecule available to
attachment to either coverslip or magnetic bead. In the reaction two of these five positions will
be occupied by attachment to either coverslip or magnetic bead. The combination brings
multiple different possible types for the relative position of chromospheres and the lysine
attached to glass or bead on a single HPPK molecule. Although from the crystal structure we can
preclude some tethering conditions that are less possible: for example, in figure 3.1A we can see
that it is essentially impossible for two linkers to consecutively tether on residue 154 and residue
157, we still are not able to pinpoint one deterministic specific amine residue pair for protein
tethering. On the other hand, during our FRET measurement, we focused on one certain HPPK
65
moleculee, no matter what
w certain
n type it is. Iff we can obsserve its reprroducibly FR
RET change
under perriodically ap
pplied magneetic field, wee are able to say conform
mational mannipulation iss
achieved
d via magnetiic tweezers, although wee do not knoow which cerrtain two of the five lysinne
on the HP
PPK protein
n is attached.1-3
3.2.3 Ex
xperimentall System
The
T essentiaal componen
nt of our maagnetic tweeezers devicce is a homeemade coneeshape peermanent magnet
m
moun
nted on an independen
i
nt 3D translaational mov
vement stag
ge
that conttrols the mo
ovement of the magnett, as shown in Figure 3
3.3. Details of the setup
p
have alreeady been discussed
d
in
n Chapter 2.2.1.
Figure 3.3.
3 A conceptual scheeme of the experimenta
e
al system. (A
A) Cy3-Cy
y5 labeled
HPPK kinase
k
moleccules are tethered on a modified g
glass coversslip that waas positioned
d in
a buffer solution ch
hamber. Th
he inset paneel presents tthe conceptt of the con
nformationaal
66
manipulation of a single HPPK molecule by magnetic tweezers. The cover glass is
treated by 3-aminopropyltriethoxy-silane (TESPA) and isobutyltrimethoxysilane in
1:10000 ratio. Dimethyl Suberimidate•2HCl (DMS) is used as cross linker to immobilize
HPPK protein molecule on the treated cover glass. The immobilized HPPK molecules
are tethered through NHS-PEO12-biotinlinking the lysine residue of HPPK to the
streptavidin-coated magnetic beads. (B) Magnetic Field-Distance curve of the magnet
used in experiment. The blue data point indicates the position of the magnet in our
experiments: the magnet is set 4 mm above the sample plane to generate a magnetic field
with approximately 1100 Gauss.
3.2.4 Force Calibration
In our experiments, mechanical force from external magnetic field is applied on a
targeted protein through a paramagnetic bead linked covalently to the single protein
molecule. As the discussion in chapter 2.1.5, we calibrate the applied force by estimating
the magnetic field gradient to get the magnetic moment of the beads tethered on the single
protein molecule. For a magnetic bead in an externally-produced magnetic field B, noting
its magnetic moment as m, then the potential energy U is:
U
-m ⋅ B
(3.1)
For a given magnetic bead, its magnetic moment m is the product of the volume
magnetization M and volume V of the bead. Therefore, the force F that is applied on the
magnetic bead can be calculated:
F = -∇U = -∇(-m ⋅ B ) = m ⋅∇B = MV ⋅∇B = MV
∂B
∂z
(3.2)
In our experiments, the magnetic field applied is approximately 1100 Gauss. As
an approximation, we only consider the magnetic field gradient in one direction
67
perpendicular to the sample plane. Thus the field gradient can be estimated from the
curve shown in Figure 3.3B. In this way the value of field gradient is calculated to be
55±15 T/m. When calculating the field gradient, position error that up to 1mm is taken
into consideration as uncertainty of distance between the magnet and the sample plane.
The volume V of paramagnetic bead is 0.6×10-18m3, and the volume magnetization M is
43×103 A/m.55 In our calculation, we have considered the factor that M here is the
saturation magnetization, an approximation that may bring error less than 25%. Hence
the force is calculated 1.4±0.4 pN from equation 3.2. The typical force applied to the
targeted single-molecule HPPK proteins is roughly 1-3 pico-Newton that is weaker than a
typical hydrogen bonding force of 6-9 pico-Newton.
3.3 Results and Discussion
3.3.1 FRET Measurement
Figure 3.4A shows a pair of FRET donor-acceptor (D-A) fluorescence intensity
trajectories from a single Cy3-Cy5 labeled HPPK molecule under force manipulation by
magnetic tweezers. The FRET efficiency E is calculated from equation 3.3, in which ID
and IA stand for the emission intensity of donor and acceptor, respectively. Figure 3.4C,
the histogram of the FRET efficiency, shows the distribution of FRET efficiency.
E=IA/(ID+IA)
(3.3)
The FRET efficiency reflects the distance between the two dyes labeled on protein
molecules, described by equation 3.4, in which R is the distance between donor Cy3 and
acceptor Cy5 while R0 is a constant determined by the transition donor-acceptor dipole–
dipole interaction. In this experiment, when a pulling force is applied by the external
68
magneticc field, we are able to probe
p
the conformatio
onal changess from the ssimultaneou
us
single-m
molecule FR
RET efficien
ncy trajecto
ories.
EFRET=1/[1+(R/R0)6]
(3.4)
Figure 3.4.
3 Single-molecule FRET
F
data obtained
o
fro
om a Cy3-C
Cy5 labeled HPPK und
der
magneticc field man
nipulation. (A)
( A portion of a pairr of single-m
molecule flu
uorescence
intensity
y time trajecctories of FR
RET donor (green linee) and accep
ptor (red lin
ne). (B) The
FRET effficiency caalculated fro
om the pair of fluoresccence intenssity trajecto
ories of the
donor an
nd acceptor in A. (C) The
T FRET efficiency d
distribution
n deduced frrom B.
69
3.3.2. Repetitive Conformational Manipulation of Single HPPK Molecule Observed by
FRET Spectroscopy
Figure 3.5A shows the FRET efficiency distribution measured from a single HPPK
enzyme under the enzymatic reaction conditions with the substrate of ATP and HP added
in PBS buffer. With the magnetic field applied, the mean of the FRET efficiency is
significantly shifted from 0.5 to 0.3, which suggests that the single-molecule HPPK
enzyme is stretched out in conformation under the external pulling force. The result
shown in Figure 3.5 demonstrates that our combined technical approach of magnetic
tweezers correlated single-molecule FRET spectroscopy is sensitive and capable of
manipulating and measuring molecule conformational changes simultaneously. To
further demonstrate the reproducibility and effectiveness of the force manipulation of
enzyme conformations by the magnetic tweezers correlated single-molecule FRET
spectroscopy, we have conducted a repetitive force pulling and releasing manipulation of
single HPPK enzyme molecules. Figure 3.5B shows that the single-molecule FRET
efficiency toggles between 0.5 and 0.3 reflecting the enzyme conformational changes due
to the manipulation by the external force pulling and releasing, demonstrating high
reproducibility and feasibility of the force manipulation of the conformational changes of
the single-molecule enzymes. It is intriguing that the FRET distribution shows a bimodal
distribution pattern around efficiency value 0.2 when the enzyme molecule is pulled by
magnetic force, which is most likely due to the force perturbation of the molecule, and the
molecule still has significant conformational flexibility under the weak force
manipulation. Nevertheless, the focus of this control experiment is to demonstrate the
feasibility of the repetitive force manipulation of the overall enzyme conformational
70
changes and distributions whille the confo
ormation flu
uctuations o
of the enzym
me are still
allowed and measurrable.
Figure 3.5
3 Repetitiive force pu
ulling and reeleasing maanipulation of individu
ual kinase
enzyme molecules. (A) The FRET
F
efficieency distrib
butions of siingle HPPK
K moleculess
f
pullin
ng (Red) and releasing (Blue) man
nipulation. (B) The FR
RET efficiency
under a force
responsee of a singlee HPPK pro
otein molecu
ule being reepetitively ttoggled witth (Red) and
d
without (Blue) the external
e
maagnetic forcce. These F RET distrib
butions are obtained frrom
a series of
o continuo
ous single-m
molecule FR
RET measurrements witth the substtrate of ATP
P
and HP added
a
in the PBS buffe
fer solution.
71
3.3.3. Probing Conformational Flexibility of Single HPPK Protein Molecule by Single
Molecule FRET-Magnetic Tweezers Spectroscopy
In an enzymatic reaction, the enzyme-substrate interaction is the crucial step
determining the overall reaction dynamics as well as the reactivity and selectivity,
according to the Michaelis-Menton mechanism and recent experimental and theoretical
studies.56-66 The enzyme-substrate complex formation can regulate both static and
dynamic conformations of the enzyme, and the enzyme-substrate complex requires a
specific molecular conformation to form an active enzyme-substrate complex state ready
to react and convert the substrate to the product.
By probing the conformational fluctuations of single-molecule enzyme under the
conditions of with substrate and without substrate, we have observed a significant change
in conformational fluctuation distribution induced by the external force. Figure 3.6A and
3.6D show the enzymatic conformational distributions of HPPK in the buffer solution
without the substrate, under the conditions of without (Figure 3.6A) and with (Figure
3.6D) the external pulling force, respectively. Figure 3.6B and 3.6E show the enzymatic
conformational distributions in the buffer solution with the substrate of 100 μM ATP, 100
μM HP and under the conditions of without (Figure 3.6B) and with (Figure 3.6E) external
pulling force manipulation, respectively. Comparing the distributions in Figure 3.6A and
Figure 3.6B, measured under no external force manipulation, it is remarkable that the
enzyme-substrate interaction narrows the conformational fluctuation range significantly,
indicated by the smaller standard deviation of the FRET efficiency distribution (Figure
3.6B), which suggests that the enzyme-substrate interaction decreases the enzymatic
conformational flexibility and the overall spatial accessibility. It is known that the
72
enzyme-substrate interaction can narrow and limit the enzyme conformational flexibility
and accessible space, according the well demonstrated conformational selection
mechanism or induced fit mechanism.67-73 However, due to the external force
manipulations, the enzyme-substrate interaction is not able to cause a significant change
in standard deviation of conformational fluctuation distributions, as shown in Figure 3.6D
and Figure 3.6E, suggesting the external force manipulation provides a dominating impact
on the enzyme conformational flexibility and limits the impact of the enzyme-substrate
interaction on the enzyme conformational flexibility.
For more quantitative understanding of the impact of enzyme-substrate interaction
on enzymatic conformation fluctuation and the impact of external force manipulation on
the enzyme-substrate interaction, we further use the standard deviation of FRET
efficiency distribution to quantitatively characterize the broadness of the FRET efficiency
distributions as well as the conformational flexibility.74,75 More flexible enzymatic
conformation gives a wider enzymatic conformational fluctuation distribution in range,
and a larger standard deviation of the conformational distribution. The results (Figure
3.6C and 3.6F) suggest that (1) the enzyme molecules without substrate have more
flexible conformational fluctuations, which is indicated by the larger standard deviation in
the FRET efficiency distribution; (2) the enzyme molecules with substrate interaction
have more spatially confined conformational changes and less flexible conformational
fluctuations, which is indicated by the smaller standard deviation in the FRET efficiency
distribution; and (3) an external force pulling on an enzyme molecule decreases the
impact of selective binding-folding enzyme-substrate interaction at the enzymatic active
site. This attribution is further supported by the results measured under the conditions
73
with and
d without su
ubstrate presence: the enzymatic conformational distrib
butions undeer a
pulling force
f
perturrbation (Fig
gure 3.6D, 3.6E,
3
and 3. 6F) show leess differen
nce in the
standard
d deviation in
i FRET effficiency com
mparing to the same sttandard dev
viation
measureed in HPPK without thee pulling fo
orce perturb
bation (Figu
ure 3.6A, 3.6
6B, and 3.6
6C) .
Figure 3.6.
3 Perturb
bing and chaaracterizing
g enzyme-su
ubstrate bin
nding interaaction by sin
nglemoleculee FRET maagnetic tweeezers micro
oscopy. (A)) Distributio
on of FRET
T efficiency
y of a
single ap
po-HPPK molecule.
m
(B
B) Distribution of FRE
ET efficiency
y of HPPK in ATP and
d HP
substratee solution. (C) The sttandard dev
viation of FR
RET efficieency of sing
gle HPPK
moleculees measured
d under the conditions of with (so
olid line) an
nd without ((dashed linee)
74
substrate in buffer solution and without the force perturbation. (D) Distribution of FRET
efficiency of a single HPPK molecule under magnetic force pulling and without substrate
ATP and HP added. (E) Distribution of FRET efficiency of a single HPPK molecule
under magnetic force pulling in the solution with substrate ATP and HP added. (F) The
standard deviation of FRET efficiency of single HPPK molecules measured under the
conditions of with (solid line) and without (dashed line) substrate in buffer solution and
with the force perturbation. The error bar in both figure 3.6C and 3.6F are forth central
moment of FRET distribution. Evidently, under the force perturbation, the enzyme
conformation is less sensitive to enzyme-substrate interactions comparing to the results in
3.6C when the measurement is under no external force perturbation.
Our results demonstrate that the enzymatic conformational fluctuation accessible
space is strongly influenced by the enzyme-substrate interactions, which provides
experimental evidence showing the critical role of the protein-ligand interactions in a
possible conformation selection mechanism of enzyme-substrate complex formation.
Conformations of protein molecules undergo dynamical fluctuations under physiological
conditions, while the existence of ligands induces conformational regulation energetically
and spontaneously favor to those conformations involving in ligand-active sites binding
interactions.76-79 Consequentially, the conformational distribution narrows down to a
ligand-binding accessible conformational subset out of the broad conformational
distribution of the apo HPPK enzymes without involving in protein-ligand interactions, as
shown in Figure 3.6C and 3.6F.
75
Figure 3.7.
3 Conform
mational flu
uctuation raate distributtions calcullated from aautocorrelattion
analysis of HPPK with
w substraate ATP and
d HP added
d. (A) Autoccorrelation ffunctions frrom
ntensity trajectory meaasured underr the condittion of with
hout magnettic pulling
FRET in
force. Green line in
ndicates don
nor while reed for accep
ptor. (B) Au
utocorrelatio
on function
ns
RET intensitty trajectory
y measured under the ccondition off with magn
netic pulling
from FR
force app
plied. Green line indiccates donor while red fo
for acceptorr. (C and D) Fluctuatio
on
rate distrributions off FRET don
nor under co
onditions w
with and with
hout magneetic pulling
force. (E
E and F) Fluctuation raate distributtions of FR
RET accepto
or under con
nditions witth
and with
hout magnettic pulling force.
f
76
3.3.4. Conformational Dynamics Manipulation by Single Molecule FRET-Magnetic
Tweezers Spectroscopy
To further characterize the changes in conformational dynamics with respect to the
conformational flexibility and the accessibility of the conformations associated with the
enzyme-substrate interactions, we analyze the autocorrelation functions of fluorescence
fluctuation trajectories of our single molecule FRET measurements (Figure 3.7A and
3.7B), under the conditions of with and without the magnetic field, for both donor (Figure
3.7C and 3.7D) and acceptor (Figure 3.7E and 3.7F), while the HPPK molecule is with
the substrate of ATP and HP added in the PBS buffer solution. Conformational
fluctuation rate can be calculated from the exponential decay rate of autocorrelation
function, and the essentially same decay rates between the autocorrelation functions of
donor and acceptor in both A and B strongly indicate that the fluctuations are from the
same origin, the single-molecular FRET. We have studied 30 different timing on FRET
trajectories of one single molecule under both with and without magnetic pulling force
conditions to have the distributions of conformational fluctuation rate as shown in Figure
3.7C, 3.7D, 3.7E and 3.7F. Figure 3.7C and 3.7D show the distributions of
conformational fluctuation rate calculated from autocorrelation functions, and the
distributions show a remarkable broadening, under the condition of with the magnetic
pulling force, comparing to that of measured under without the magnetic pulling force.
This result indicates an increase in distribution range of conformational fluctuation rate
under the magnetic force pulling. Similar change triggered by magnetic tweezers pulling
force also occurs consistently in acceptor fluctuation rate distributions (Figure 3.7E and
3.7F).
77
Such broadenings in the conformational fluctuation rate distribution are consistent
with the results from the standard deviation analysis of FRET efficiency distributions.
According to Figure 3.6C and 3.6F, it is the substrate binding interaction that leads to less
flexible conformational fluctuations of HPPK protein molecule, while such interaction is
weakened by applied external pulling force. Consequently, weakened enzyme-substrate
interactions also result in an apparent broadening of conformational fluctuation rate
distribution. Enzyme-substrate interaction is highly sensitive to the protein
conformational perturbation by external pulling force. With the force pulling, such
enzyme-substrate interaction is perturbed and weakened, releasing the protein from being
constrained by ligand-binding interaction, resulting in a broader range of the enzyme
conformational fluctuation rate.
Our results show that the small external force of about 1.4±0.4 pN can apparently
impact the enzymatic conformational fluctuation distribution, and the enzymatic
conformational flexibility or conformational fluctuation distribution is critical for
enzyme-substrate interaction, thus the small external force can impact the interactions
between enzyme and substrate. This low external pulling force is not sufficient to rupture
the protein tertiary structures as the rupture force is at least 18 pN for HPPK55 or even not
sufficient to break hydrogen bonds as a typical hydrogen bonding force is about 4 pN and
higher.80 Therefore, the low external force we applied to an individual HPPK enzyme
molecule likely only causes a deformation of tertiary structure of the HPPK enzyme
molecule.
78
To illustrate the fact that the small external pulling force is capable of impacting
the enzymatic function, such as enzyme-substrate interaction, we note that the external
pulling force applied on an single enzyme molecule through the magnetic tweezers, even
if the force is at similar scale competing with the thermal fluctuation forces, is an onedirection constant force that capable of deviating the conformational fluctuation energy
landscape, leading to a deformation of the HPPK enzyme molecule. As an analogy, it is
simply like a random walk on a tilted energy landscape by an external and constant force
field. Evidently, the one-direction pulling force decreases enzymatic conformational
flexibility and affecting the enzyme-substrate interaction impacting enzymatic function.
Furthermore, we emphasize that this work only focuses on the understanding of the
enzyme-substrate interactions in forming the enzyme-substrate reactive complex, which is
the first step of an enzymatic reaction, and our future work will focus on identify and
characterizing the impact of the external force manipulation on the enzymatic reaction
turnover activities.
3.4. Conclusion
In summary, we have demonstrated that our correlated single-molecule FRETmagnetic tweezers microscopy is capable of manipulating the conformation of single
enzyme molecules, and in turn, manipulating the enzyme-substrate interactions, by
applying and controlling a pulling force on single kinase molecules. Technically, the
correlated magnetic tweezers single-molecule FRET spectroscopy is a potentially
powerful tool to interrogate the protein conformational dynamics and the associated
protein functions. Using our approach, we are able to interrogate the conformational
selection mechanism by exam the conformation flexibility and conformational fluctuation
79
accessible space when the enzyme is under interacting and not interacting with the
substrate molecules. We have observed that the enzyme-substrate interaction provides a
strong conformational selection effect through a folding-binding interacting process
shifting the conformational fluctuation to more confined spatial range; whereas, under the
force pulling, distorted enzyme conformation has a weaker interaction with the substrate,
leading to a weak conformational selection effect and folding-binding interacting
dynamics.
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89
CHAPTER IV. INTERROGATING THE ACTIVITIES OF CONFORMATIONAL
DEFORMED ENZYME B BY SINGLE MOLECULE TIRF-MAGNETIC
TWEEZERS MICROSCOPY
4.1 Introduction
One of the central focuses in protein study is the structure-function relationship, the
impact of different conformations to the properties of protein molecules. There has been
intensive research reported on that protein molecules with their tertiary structure perturbed or
even partially unfolded may be related to misfunction or causing diseases, because changing
protein conformations typically leads to significant differences in their affinity, selectivity, and
reactivity. 1-22 In modern enzymology, it has been extensively explored that the enzymatic
conformation-function relationship, especially in the dynamic rather than the static perspectives,
plays a critical role to understand enzyme mechanism at molecular level. 23-28 For example, in an
enzymatic reaction, forming enzyme–substrate reactive complex often involves significantly
enzymatic active site conformational changes, being a critical step in defining enzymatic reaction
potential surface, reaction transition state, and reaction pathways. Such enzyme–substrate
interaction process has been demonstrated to be significantly affected by enzyme conformation
deformations. 1,4 18-20
Traditional enzyme studies focused on enzyme molecules at enzymatic reaction
conditions while the enzymes are fully folded or in their natural states. Such research works on
enzymatic stability studies focused on ensemble level activity of enzyme at different physical
conditions or chemical environment without probing corresponding change in conformation of
enzyme molecules.29-37 In recent years, more and more researches have focused on studying
90
enzymes and their activities at their deformed, unfolded, or force-manipulated states.3,7,38-40
Typically, such experiments on enzymes are under non-physiological or non-enzymatic reaction
conditions, or even under denatured conditions under which the enzyme unfolds.41-44 On the
other hand, conformational manipulation on single protein molecule by applying mechanical
force has been achieved by several different approaches. For example, Atomic Force
Microscopy (AFM) have been widely used to study conformational dynamics of single protein
molecules;45-48 Optical tweezers has been applied to study folding pathway or even
conformational folding transition state of single protein molecules;49-51 Magnetic tweezers has
also been explored to study protein conformational dynamics.40,52
Recently, rupturing a single enzyme molecule and observing its recovery of activity
under enzymatic reaction conditions has been achieved using AFM.7 However, manipulating
conformation of a single enzyme molecule in its deformed and partially unfolded states under a
physiological enzymatic reaction condition with simultaneous observation of its activity remains
a challenge, exploring how critical the enzyme conformational stability as well as dynamically
fluctuating and externally force perturbed enzymatic states impact on the enzymatic activities. A
single molecule level observation of enzymatic reactivity in the contest of conformational
deformation of the enzyme protein molecules will provide us a fundamental understanding of the
dependence of enzymatic reactivity on the conformational changes and stability. The impact of
different conformation on enzyme function has also been the focus of theoretical studies.23-24, 5355
A range of key questions on how the enzymes work can be investigated. For example, does a
conformation-deformed or even partially unfolded enzyme molecule still have measurable
enzymatic reactivity? If so, how much activity will be left at various degree of external force
perturbation? And in terms of molecular folded conformations, how much can an enzyme
91
molecule tolerate such conformational deformation under an enzymatic reaction condition, i.e.,
under enzyme-substrate binding interaction conditions? Here we report our work towards
obtaining the answers for these questions.
In Chapter III, we have investigated that enzyme–substrate interaction induced enzymatic
active site fluctuation dynamics and flexibility associated with induced fit and folding-binding
mechanism by manipulating the enzyme conformations and fluctuations at single molecule level
using our home developed single molecule FRET magnetic tweezers which generating forces at
1-10 picoNewton scale.52 Here we report our new approach to manipulate single molecule
catalytic activity using magnetic tweezers to deform the conformation of horseradish peroxidase
(HRP) enzyme at single molecule level, correlated with total internal reflection (TIRF)
microscope to simultaneously observe the fluorogenic enzymatic turnovers, as shown in Figure
4.1. There are specific advantages of using magnetic tweezers to provide an external mechanical
force to manipulate single molecule enzyme, including, (1) wide force range from less than
hydrogen bonding force to protein rupture force; (2) no photo-damage and cross talk to single
molecule spectroscopic measurements of enzymatic activity and enzyme conformational
changes; and (3) capability to simultaneously applying force on a large number of single
molecules.38-40,56-59 Combined with TIRF microscopy as optical measurement, it provides us the
unique capability and opportunity to interrogate conformation-function relationship of enzyme
molecules under enzymatic reaction conditions, specifically studying the impact of deforming
protein conformation on protein function at single molecule level.
92
4.2 Mateerials and Methods
M
4.2.1. Ma
aterials.
Horseradish
H
peroxidase
p
(H
HRP) is a 34
4 kDa 306-reesidue monoomeric enzym
me. The HR
RPcatalyzed
d reaction co
onverts hydro
ogen peroxid
de (H2O2) annd non-fluorrescent probee substrate N
Nacetyl-3,7
7-dihydroxy
yphenoxazinee (APR) into
o fluorescentt resorufin. This enzym
matic reactionn is
fluorogen
nic, in which
h only the reeleased produ
uct moleculees emit fluorrescence thatt is detectablle by
total internal reflectio
on fluorescence microscopy. In our experiment,, we choose supernetic beads (Dynabeads®
(
® MyOne™
™ Streptavidiin T1 Invitroogen Companny) with 1.005
paramagn
µm diam
meter, covalen
ntly attachin
ng the beads to biotin co--factor linkinng to the HR
RP molecules
through a biotin-strep
ptavidin link
k. The HRP enzyme moolecules are iin substrate ssolution
consistin
ng of 50 mM PBS buffer solution (pH
H =7.4), 1000 nM N-acetyyl-3,7dihydrox
xyphenoxazin
ne (Amplex Red, APR) and 100 nM
M hydrogen pperoxide (H2O2).
93
Figure 4.1. A conceptual scheme of our experimental system. Horseradish peroxidase (HRP)
molecules are tethered at one end to a modified glass coverslip. The immobilized protein
molecules are tethered at the other end to magnetic beads through biotin-streptavidin linking.
4.2.2. TIRF Measurement.
TIRF measurements are carried out by using an inverted confocal microscope (Olympus
IX 71 with 60 x objective) with a 532 nm CW crystal laser generating evanescent wave for total
internal excitation. Emitted signal is filtered with a long-pass beam splitter and collected by an
Electron Multiplying Charge Coupled Device (EMCCD: ProEM 512B, PI co.). We conduct the
single molecule total internal reflection optical measurements and pulling manipulation via
magnetic tweezers simultaneously. Magnetic force was applied through those attached
superparamagnetic beads on HRP molecules. The essential component of our magnetic tweezers
device is a permanent magnet generating magnetic field, which was mounted on a specially
made stage enabling the magnetic probe to move along any direction and for any desired
distance. The sample chamber was put on an x-y stage capable of applying in-plane adjustment.
The distance between the magnet and the sample cover glass is 4 mm, implying an 1100 Gauss
magnetic field at the sample.
In our experiments, we applied mechanical force to single enzyme molecules by applying
external magnetic field and sensing through tethered magnetic bead on those single enzyme
molecules. Quantitative calculation of the force generated by our magnetic tweezers has been
discussed in Chapter 3.2.4. In brief, pulling force roughly at 1-3 pN can be applied on the
targeted single HRP protein molecules using our magnetic tweezers setup. Although the force is
94
weaker than hydrogen bonding force which is typically 6-9 pN, we have demonstrated that force
at this scale is capable to trigger conformational response in our previous publication.52
4.2.3. Sample Preparation.
As shown in Figure 4.1, the HRP molecules were bound to the glass cover slip at one
end by 3-aminopropyltriethoxy-silane (TESPA)-Dimethyl Suberimidate•2HCl (DMS) linkers
and linked to a streptavidin coated superparamagnetic bead (Dynabeads® MyOne™ Streptavidin
T1 Invitrogen Company), at the other end via biotin-streptavidin bond. Protein immobilization
was carried out through a routine procedure as shown in Figure 4.2. In brief, a clean glass
coverslip was firstly immersed overnight in NaOH-ethanol solution, and the coverslip was next
washed by distilled water, blow-dried by air flow, and incubated with a DMSO solution
containing a mixture in 10% concentration consisting of TESPA and isobutyltrimethoxysilane in
1:10000 ratio overnight. The coverslip was then washed by distilled water and consecutively
transferred and incubated for 4 hours in each system below: 15 mL PBS buffer solution pH=8.0,
containing 10nM Dimethyl Suberimidate•2HCl (DMS•2HCl); 15 mL PBS buffer solution
pH=7.4, containing 10 nM HRP; 15 mL PBS buffer solution pH=7.4, containing 10 nM NHSPEO12-biotin; 15 ml PBS solution pH=7.4, containing 1µl magnetic beads stock solution which
is commercial available. The low concentration of each solution was to make sure that the
distribution of the individual enzyme molecules on cover glass is adequately separated so that
one bead does not attach to multiple enzyme molecules. Meanwhile, low concentrations of
TESPA are used to ensure that immobilized protein molecules are distributed separately enough
from each other for obtaining single molecule TIRF images.
95
Figure 4.2.
4 Preparatiion of singlee molecule HRP
H sample. We tethereed protein moolecules at oone
end to the coverslip by
b Dimethyll suberimidatte-2HCl (DM
MS-2HCl) annd at the othher end to a 1 µm
size paramagnetic beead by biotin
n-streptavidin
n bonding. F
Force was im
mplied by adding externaal
magneticc field and heence the mollecule could
d feel it throuugh the beadds.
We
W also havee noted that either
e
biotin or DMS cann only be teth
thered to a H
HRP moleculle via
connectio
on with lysin
ne in the amiino acid sequ
uence, whichh leads to m
multiple possiible tetheredd
condition
n of the proteein moleculee to coverslip
p or magnetiic beads. Thhere are six llysine residuues
on one HRP
H moleculle: residue 65
5, 84, 149, 174,
1 232, 2411. In our expperiment, eaach HRP
moleculee are immobiilized on cov
verslip via DMS-lysine
D
ccovalent linkk at one out of these six
lysine ressidues, and being
b
tethereed to magnettic beads thrrough anotheer lysine resiidue out of thhe
96
rest five options. Therefore, in our sample preparation process, each HRP molecules can be
immobilized by using any two of the total six lysine residues to link toward DMS and biotin.
There are 15 possible lysine combinations in total, leading to 15 possible different tethering
conditions. Since we are not able to pinpoint that a pair of specific lysine residues tethered to a
specific protein molecule, we have performed Steered Molecular Dynamics (SMD) simulation
for all 15 possible tethering conditions for HRP molecule, showing that when being stretched by
external force, the active site of HRP molecule will be deformed beyond the scale of its normal
fluctuation in most of possible conditions. Details of the SMD simulation will be discussed in
chapter V.
4.3. Results
4.3.1. Single-Molecule TIRF Imaging Measurement of HRP Activity
Figure 4.3B shows the time trajectories of fluorescence intensity of a single HRP
molecule under a fluorogenic enzymatic assay condition. Time resolution of our TIRFM
imaging measurement is 20ms per frame, while each measurement lasts 60 seconds and
accumulates totally 3000 imaging frames with 10 ms data readout time for each frame. The
fluorescence signals we take into account as enzymatic turnover events are the photon count
spikes above the threshold of the trajectory. The threshold is set three times the standard
deviation larger than the distribution mean value of the histogram deduced from the whole
trajectory over time domain. In brief, we firstly fit the TIRF experimental real time trajectories
by Gaussian distribution. Then we set the threshold value of signal from real time trajectory as
‘higher than three times the standard deviation above the mean value of overall trajectory’ part.
97
In this way, we have the confiden
nce level at least
l
larger tthan 95% to discern signnals from
backgrou
und noise.
Figure 4.3.
4 Single-tu
urnover detecction of HRP
P enzyme caatalysis. (A) Exemplary fluorescencee
time trajeectory of sin
ngle HRP mo
olecule obserrved from TIRFM measurement. (B
B) Timedistributiion of the TIIRFM trajecttory. On tim
me domain, siignals abovee threshold aare taken intoo
account as
a turnover events.
e
(C) Segment of the TIRFM turnover trajjectory. τofff is the waitinng
time betw
ween sequen
ntial reaction
n events.
4.3.2. An
nalysis of Sin
ngle-Molecu
ule Activity Trajectories
T
s Measured u
under Forcee Pulling an
nd
Releasing
g Condition
ns.
We
W analyzed 30 individuaal moleculess under the eexternal forcce manipulation of pullinng
and releaasing, shown
n in Figure 4.4. The HRP
P enzyme moolecules are in 50 mM P
PBS buffer
solution (pH
( =7.4) with
w Amplex Red (100 nM
M) and H2O2 (100 nM) aas substratess. We apply
1100 Gau
uss magneticc field to gen
nerate appro
oximately 1.55pN pulling force for “pulling”, whiile
not apply
ying any field for “releassing”. Figurre 4.4A and 44.4B show thhat when beeing pulled bby
external magnetic
m
forrce, the num
mber of turno
over events ooccurred on tthose HRP m
molecules
evidently
y decrease, in
ndicating a decrease
d
of catalytic
c
actiivity of thosee HRP enzym
me moleculees.
98
Figure 4.4.
4 Histogram
m results off turnover events from 300 individual HRP molecuules. (A)
Turnoverr events histo
ogram when
n no force is applied on H
HRP molecuules, the “releasing” grouup of
HRP. (B)) Turnover events
e
histog
gram when th
he HRP mollecules are ppulled by appplied force frrom
magneticc tweezers, th
he “pulling”” group of HR
RP.
To
T further qu
uantitatively characterizee the impact of conformaational distorrtions on thee
enzymatiic activity off HRP moleccules by usin
ng the mechaanical force manipulatioon, we analyzze
both the distribution of the turnov
ver waiting time
t
and disstribution of detected phooton from
released products bassed on the siingle-molecu
ule fluorogennic trajectoriies recordedd, as shown iin
Figure 4..5. The turnover waiting
g time, τoff, is the time innterval betweeen two conssecutive deteected
fluorogen
nic turnover events, abov
ve-threshold
d fluorescencce signal inteensity jumpss. The turnoover
waiting time, the tim
me that needeed for actual events of caatalytic produucts formation, is negatiively
99
proportional to the enzymatic reactivity. In an enzymatic reaction, product formation rates are
determined by the rate of substrate diffusion and enzyme-substrate complex formation, besides
the reaction and releasing products. Although individual waiting time values are stochastic, the
mean waiting time, <τoff>, and its distributions are defined by enzymatic reaction rate. In our
experiment, we analyze the mean waiting time <τoff> over the turnover trajectories from each
HRP enzyme molecule. When under pulling force via magnetic tweezers, deformation of the
HRP enzyme occurs, the <τoff> accordingly increases due to the decrease of the time-averaged
single-molecule catalytic rates of HRP enzyme, as shown in Figure 4.5A.
We have further evaluated the enzymatic reaction activity changes under force pulling by
counting photon bursts from the product releasing events. We calculate the integral area of
fluorescence signal above the threshold by three times standard deviation higher than the mean
value of the whole time trajectory. Figure 4.5B shows the distribution of the total photon counts
from the product turnover events for each examined single molecule HRP under both force
pulling and non-force pulling conditions. The total photon counts of enzymatic turnover photon
burst events are calculated by counting all the photons above the threshold with and without
magnetic pulling force for each single molecule HRP examined. Under the force pulling, the
product burst counts decrease significantly in identical with the decreased number of turnover
events, presumably associated with significant enzyme conformation deformation. This result of
the enzymatic reaction activity decreases with the increase of the enzyme conformation
deformation by force manipulation is consistent with the results of <τoff> analysis.
To reveal that if the reduced activity is the result of change in substrate binding, we have
further studied the change in enzyme-substrate binding affinity by calculating the equilibrium
Dissociation Constant Kd, which is defined in equation 4.1 and equation 4.2.
100
koff
ZZZX
ES YZZ
ZE+S k
on
Kd =
koff
kon
=
τ off
τ on
(4.1) (4.2)
In equation 4.1, E and S stand for enzyme and substrate. Rate of binding and dissociation
of enzyme-substrate complex are characterized by kon and koff respectively. Waiting time τoff and
on-time τon are defined as shown in figure 4.5A. The result of Kd calculation is shown in Figure
4.5D, in which blue cubic spots stand for when the HRP enzyme molecules are released with no
pulling force applied on them, and red triangle spots indicate the condition that ‘pulling force is
applied by magnetic tweezers’ when the enzyme molecules are stretched and hence
conformational deformed by external pulling force. We can find that when being stretched by
external pulling force, the dissociation constant Kd become larger, indicating a weaker ligandsubstrate binding ability, which is as expected, since the enzyme conformation has been
deformed. Although a deformed enzyme molecule can rely on fluctuation to come back to its
active conformation, the stability will be affected to be less due to external force.
101
Figure 4.5.
4 Analysis of the relatiionship betw
ween turnoveer event, meaan waiting tiime and prodduct
burst of single
s
HRP molecules.
m
(A
A) Correlatiion plots betw
ween turnovver event couunts and meaan
waiting time of each single HRP molecules with
w and withhout magnettic pulling foorce appliedd. (B)
Correlatiion plots betw
ween turnov
ver event cou
unts and phooton burst coounts from reeleased prodducts
of each single HRP molecules
m
wiith and witho
out magneticc pulling forrce applied. (C) Correlattion
plots betw
ween turnov
ver event cou
unts and photon burst couunts from reeleased produucts of each
single HR
RP moleculees with and without
w
mag
gnetic pullingg force appliied. (D) Enzzyme-substraate
dissociation constantt of each sing
gle HRP molecules withh and withouut magnetic ppulling forcee
applied.
102
4.3.3. Repetitive Force Pulling-Releasing Manipulation of Enzyme Conformation for
Impacting Enzymatic Activity.
To further demonstrate the reproducibility and effectiveness of the force manipulation via
magnetic tweezers correlated single-molecule TIRFM spectroscopy to the protein function, we
measure the response of HRP enzymatic activity to repetitive force manipulation, toggling
between “pulling” and “releasing” force applications, as shown in Figure 4.6. Under the “pulling”
condition, 1100 Gauss magnetic field is applied to generate 1-3 pN mechanical force to deform
the enzyme conformation; while under the “release” condition, there are no force is applied at all.
In the experiment, the reaction system is first observed by TIRFM without any pulling force
from the magnetic field for 100 seconds, and then, the pulling force is applied for the next 100
seconds; and the process is repeated for a few times for the next 300 seconds. Figure 4.6C shows
that the total photon counts from the product turnover events of a single HRP molecule toggles
between two different levels: reflecting the single molecule enzymatic activity changes due to
the conformational manipulation by the external force pulling and releasing. Such response
demonstrates the reproducible impact of the external force to the single enzyme catalytic
function by affecting substrate binding process via deforming conformation.
103
Figure 4.6.
4 Responsee of HRP en
nzymatic actiivity to repettitive magneetic pulling fforce. (A) (B
B)
Conceptu
ual scheme of
o HRP moleecule at releaased and pullled state, respectively. ((C) Product
counts frrom 20 differrent HRP mo
olecules with
h and withouut being pullled. Errors aare estimatedd as
10% considering the possible inaaccuracies in
nvolved in thhe setting thrreshold methhod in our daata
analysis.
104
4.4. Discussion
In our previous work, we have specifically analyzed an enzyme conformational change
under the same experimental configuration and same magnetic field strength, and we have
reported that the 1-3 pN external force can result that an enzyme from unfolding or deformation
by 30% to even 100%, partially unfolded protein, while by applying such external pulling force,
the enzyme-substrate binding interaction can be weakened, yet not completely diminished.52 We
note that although the applied stretching force on enzyme protein molecules are weaker than
hydrogen bonding force which is typically 6-9 pN, it is a one-direction constant force that is
capable of deviating the conformational fluctuation energy landscape to deform conformation of
single protein molecules.
From the result in Figure 4.5 and Figure 4.6, we can see that when such external pulling
force is applied, the product burst showing a significant decrease indicating the decreasing
catalytic activity of HRP enzyme molecules. This change is as expected, since the conformation
of HRP protein molecules are deformed or partially unfolded by external pulling force. We will
also see from the steered MD simulation in Chapter 5 which also shows supporting result,
illustrating the distortion of active site of a single HRP molecule when being pulled. In
literature, there are studies on enzyme folding and unfolding under the overall denaturing
solutions, where the enzyme either at unfolded condition but not enzymatic conditions or at the
enzymatic reaction conditions but the enzyme is fully folded.60-69 In our experiment, we
achieved actively manipulated single enzyme molecules to partially unfolded conformation
under a physiological enzymatic reaction condition. To the best of our knowledge, our results
present, for the first time, successfully manipulate protein function at single molecule level by
105
conformational manipulation that applying and controlling pulling force on single HRP
molecules under physiological enzymatic reaction conditions with the existence of substrates.
Revealing Significant Tolerance of Enzymatic Activity to Protein Conformational
Deformations.
It is also interesting that the HRP enzyme molecules are not totally lost their catalytic
activity when their conformations are stretched by an external force, although less active than
when in unperturbed condition, as shown in Figure 4.5 and Figure 4.6. In chapter 5, we will also
see that MD simulation results also show a noticeable distortion of the active site in HRP
molecule when the molecule is pulled by magnetic tweezers, yet chemical process in protein
functioning such as electron transfer or proton transfer requires precision at Å in conformation.
However, our result does not contradict the traditional structure-function relationship, because of
conformational fluctuation of protein molecules. The critical factor is conformational
fluctuation. When being deformed by external constant pulling force in our experiment, enzyme
molecules are still capable to temporarily come back by conformational fluctuation to its
substrate-binding accessible conformational subsets, although with a lower possibility compared
with enzymes without pulling force applied on them. For enzyme molecules in enzymatic
reaction conditions, such come-back from conformational fluctuation is most likely further
facilitated by enzyme-substrate interactions. In enzymatic reactions, enzyme-substrate
interaction plays a key role by affecting enzymatic active site conformation and induces the
active site conformational fluctuation towards folded into active-favored subsets from foldingunfolding conformational fluctuation of enzyme molecules. The enzyme–substrate complex
formation can regulate both static and dynamic conformations of the enzyme. In other words,k
substrate-binding process induces the partially unfolded or deformed enzyme active site
106
conformation to be refolded to the active and nature conformation to produce the enzymatic
reaction turnovers. A conceptual picture of this explanation is shown in Figure 4.7.
Such understanding of constant conformational fluctuations of protein molecules are also
in accordance with recent research of intrinsically disordered protein, or intrinsically
unstructured protein. An intrinsically disordered protein typically does not fold in a well-defined
three dimensional structure under physiological conditions.70-73 Such phenomena spurred
extensive studies in recent years.74-84 Significantly different conformations can be detected for
the same type of protein molecules in solution.85-87 Although being observed as lacking stable
tertiary and/or secondary structure, intrinsically disordered proteins are capable to carry out
specific functions. Our results here provides a possible mechanism for the existence of
intrinsically disordered protein by revealing conformational fluctuation of enzyme protein
molecules, and identifying that such conformational fluctuation can be regulated by ligand
binding process. By substrate induced enzyme-substrate interaction, deformed enzyme
molecules can fluctuate from ‘unstructured’ back to ‘structured’ state, still capable to maintain
their function. Similarly, it is possible for intrinsically disordered protein molecules to fluctuate
among different conformations to keep or quit from certain functions.
107
Figure 4.7.
4 Conceptu
ual scheme of
o conformattional fluctuuation of singgle enzyme pprotein whenn
being defformed by ex
xternal forcee. The circleed part conceeptually reprresents the aactive site onn a
single en
nzyme moleccule. When the
t conformaation of an eenzyme moleecule is defoormed by extternal
force, sub
bstrate bindiing induced conformatio
onal changess allow the ennzymatic active site to ccome
back to itts active con
nformation, leading
l
to occcurrence off reaction eveents.
4.5. Conclusion
n this work, we have dem
monstrated th
hat using thee single-mollecule TIRF--magnetic
In
tweezers correlated im
maging specctroscopy, th
he conformattional perturrbations from
m pulling forrce
me function: Structural deformation
d
n of enzyme m
molecules leead to
can maniipulate enzym
correspon
nding chang
ges in their acctivity. Such
h influence rreveals that enzyme has a remarkablle
countenaance and toleerance towarrd conformattional distorttion, i.e., thee enzyme cann still possesss
significan
nt activity ev
ven the enzy
yme conform
mation in its ddeformed orr partially-unnfolded. Ouur
experimeental approacch provides a unique cap
pability: actiively manipuulating an ennzyme to
108
partially unfolded conformation under a physiological enzymatic reaction condition.
Consequentially, we are able to interrogate the enzymatic reactivity in the contest of the protein
structure-function relationship in a unique experiment. This repetitive manipulation of
enzymatic activity reveals the capability of manipulate protein function by applying mechanical
perturbation on its conformation.
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Kissinger C.R.; Bailey R.W.; Griswold M.D.; Chiu W.; Garner E.C.; Obradovic Z. J Intrinsically
Disordered Protein. Mol Graph Model. 2001, 1, 26-59.
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Sugase, K.; Dyson, H.J.; Wright, P.E. Mechanism of Coupled Folding and Binding of an
Intrinsically Disordered Protein. Nature 2007, 447, 1021-1025.
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Tran H.T.; Mao, A.; Pappu, R.V. Role of Backbone-Solvent Interactions in Determining
Conformational Equilibria of Intrinsically Disordered Proteins. J. Am. Chem. Soc. 2008, 130,
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Eliezer, D. Biophysical Characterization of Intrinsically Disordered Proteins. Curr. Opin.
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Elam, W.A.; Schrank, T.P.; Campagnolo, A.J.; Hisler, V.J. Evolutionary Conservation of
the Polyproline II Conformation Surrounding Intrinsically Disordered Phosphorylation Sites.
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Ferreom, A.C.M.; Gambin, Y.; Lemke, E.A.; Deniz, A.A. Interplay of α-Synuclein
Binding and Conformational Switching Probed by Single-Molecule Fluorescence. Proc. Natl.
Acad. Sci. U.S.A.2009, 106, 5645-5650.
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105, 5762–5767.
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88
We note that on a given HRP molecule, biotin or DMS are able to covalently link to
lysine residue in the amino acid sequence, which results in protein immobilization complexity
that a few different tethering conditions are possible for HRP protein molecules when linking to
coverslip or magnetic beads. However, in our single-molecule TIRF measurement, we compare
activity change of each HRP molecules individually under different conditions that with and
119
without magnetic pulling force applied. As a result, although we did not necessarily pinpoint
one specific lysine residue pair on protein molecules for tethering, our observation of different
enzymatic activity associated with various enzyme conformational manipulation conditions are
systematic and well-defined.
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CHAPTER V. STEERED MOLECULAR DYNAMICS SIMULATION STUDIES OF
THE CONFORMATIONALLY DEFORMED ENZYMES MANIPULATED BY
SINGLE MOLECULE MAGNETIC TWEEZERS
5.1. Introduction
Steered Molecular Dynamics (SMD) simulation, sometimes also being named as
force probe simulation, is originally designed for the purpose of applying external force onto
a protein molecule to pull the protein along desired directions.
In our experiments discussed in chapter III and chapter IV, we apply force on protein
by immobilize a single protein at its one given residue position, while pulling at another
residue position which is tethered covalently to magnetic bead. Such force that we apply to
manipulate protein structure allows us to pull a single protein molecule along a given
direction, or a given degree of freedom at atomic level. SMD simulation is ideal for this
type of scenario at molecular level. The SMD simulation is performed using the NAMD
software package, developed by Schulten’s group.
Typically, there are two different types of protocols for simulation by SMD: one in
which the target molecule is pulled at constant velocity, and one in which the target
molecule is pulled with constant force. Although the constant-force simulation will fit the
scenario of our experiments better, considering the computer facility and our goal which
focused on revealing the active domain structural information when the protein is
conformationally deformed, we chose the constant-velocity type method to carry out our
SMD simulation. Details will be discussed in later sections.
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5.2. Estimating conformational stretching extent from HPPK simulation
In chapter III, we have used FRET-Magnetic Tweezers Microscopy to manipulate the
conformation of a single HPPK protein molecule. From the FRET measurement results, we can
clearly see that single-molecule HPPK enzyme is stretched out in conformation under the
external pulling force. And such conformational manipulation has been demonstrated for its
high reproducibility and feasibility of the force manipulation of the conformational changes of
the single HPPK protein molecule via magnetic tweezers. However, since FRET spectroscopy
only reports distance changes between two specific residue positions where dye indicators are
labeled on a protein molecule, what we can really obtain from FRET data is a projection of the
molecular conformational change on the FRET donor-FRET acceptor direction. Such
information is informative for the purpose of dynamical analysis such as conformational
fluctuation rate of protein molecules, yet it does not show atomic details of protein structure with
external mechanical impact is applied on target sample protein molecules.
In this section, we will study what impact such conformational manipulation will give to
HPPK protein structure by Steered Molecular Dynamics (SMD) simulation. As discussed in
chapter 3.2.2, we have labeled dye molecule Cy3 at residue position 48 and Cy5 on residue
position 151 on HPPK protein molecule, as shown in figure 5.1A and 5.1B. A combined
magnetic tweezers and simultaneous FRET spectroscopic microscopy is used to apply and to
monitor conformational manipulation on single HPPK molecule. We observed that when a
single HPPK protein molecule is pulled by our magnetic tweezers, the mean value of FRET
efficiency between Cy3 and Cy5 shifted from 0.5 to 0.3, indicating the single HPPK molecule is
stretched out in conformation leading to an extension between 4Å to 6Å in the distance between
Cy3 and Cy5 residue position, as shown in figure 5.1C. Details have been published in our
122
previous paper.1 To find out how much a protein molecule is stretched while being pulled by
magnetic tweezers in experiment, we performed a steered molecular dynamic (SMD) simulation
of HPPK protein molecule. As we have discussed in chapter 5.1, there are two types of SMD
simulations: constant-force pulling simulation and constant-speed pulling simulation. In our
steered MD simulation, we did constant speed pulling simulation to illustrate the conformational
distortion of HPPK protein molecule responding to external stretching force. There are two
reasons for this choice: (a) the force we apply in experiment is in such a fine scale that
calculation time would be too long for constant force simulation; (b) In chapter III, we have
already demonstrated the capability of the force generated by our magnetic tweezers set to
stretch the conformation of a single protein molecule.
The initial coordinates of HPPK were obtained from Protein Data Bank (PDB code
1HKA), set in aqueous environment during simulation, with periodic boundary condition set for
a rectangular shape water box with 67.9Å in length, 54.7Å in width and 67.3Å in height. MD
simulation is performed using program NAMD, version 2.9. Protein molecule is set in water
solvation condition under CHARMM type force field (par_all27_prot_lipid.inp). Boundary
condition is applied for a time step of 1 fs. Considering the computation time, we set the pulling
speed as 0.5 Å/ps.1-3 Constant temperature at 293 K during the simulation is maintained by
Langevin thermostat, with Langevin damping coefficient set at 1 ps-1. Constant pressure is
maintained at 1atm using Langevin piston. Non-bonded interactions were calculated using
particle mesh Ewald (PME) full electrostatics; cutoff of the van der Waals energy was set at 12.0
Å, with switch distance at 10.0 Å and pair-list distance set at 13.5 Å. PME grid spacing is set at
1.0 Å. We have also run 20 times the HPPK molecule under same conditions but without any
123
pulling events to test the HPPK conformational thermal fluctuation at its equilibrium condition
as a controlling group test.
The results are shown in figure 5.1D and 5.1E. Figure 5.1D shows that for a single
HPPK molecule in aqueous environment without any perturbation applied on it, the distance
between residue 48 and 151 displays a fluctuation within 2Å induced by thermal motion. Figure
5.1E shows that upon being stretched, this distance will be distorted, which is 4Å to 6Å from
FRET measurement in our previous paper.
One point needs mentioning was there are 5 lysine residues in an HPPK protein molecule
as possible tethering positions: residues 23, 85, 119, 154 and 157. Two out of these five lysine
residues participate in the conformational manipulation: for each single HPPK protein molecule,
it is immobilized on coverslip through one lysine residue, and being tethered to magnetic beads
via another lysine residue. Since the conformational stretching by magnetic tweezers has to be
achieved through applied force on magnetic beads, in magnetic tweezers pulling experiment, the
HPPK molecule will be stretched in the direction formed by two out of these five lysine residues.
For residue pair 85-154, it will need to be stretched for 17Å to 18Å to make the distance between
residues 48-151 extend for 4Å to 6Å, while stretching through any other possible lysine residue
pair on HPPK molecule requires significantly larger distortion to achieve similar effect. In other
words, the HPPK protein will be stretched no less than for 17Å to 18Å when FRET shift from
0.5 to 0.3 is observed. Therefore, in HRP simulation, we also assumed that our HRP molecule is
stretched 17Å to 18Å when being pulled by magnetic tweezers.
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Fig. 5.1. SMD simulation of HPPK molecule pulling by magnetic tweezers. The magnetic
tweezers pulling experiment is described in our previous publication.1 (A) Natural HPPK protein
molecule. (B) HPPK protein molecule being pulled by magnetic tweezers. (C) FRET efficiency
distributions of single HPPK molecules under pulling (Red) and releasing (Blue) manipulation.
(D) Distortion of distance between residue 48 and 151 induced by thermal fluctuation of HPPK
protein molecule. (E) Distortion of distance between residue 48 and 151 induced by pulling
force applied on HPPK protein molecule.
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5.3. SMD simulation study of HRP protein molecule
5.3.1. SMD simulation of HRP protein molecule in one tethering condition
In chapter III, we have achieved conformational manipulation of a single HPPK
protein molecule using magnetic tweezers. Based on this demonstration of capability that
our magnetic tweezers is able to apply pico-newton scale force on single protein molecule,
we can start to study the impact of protein function from such fine-scale conformational
manipulation. The first choice will be testing the impact of such applied force on HPPK
protein function. Unfortunately, the reaction that HPPK serve as enzyme does not release
fluorescence product available for detection in our lab. Hence we chose another protein,
HRP, instead, as we have discussed in chapter IV. On the other hand, for FRET
measurement, labelling dye molecules on protein requires mutation to introduce cysteine
residues. Yet for HRP molecules, mutation on cysteine residue will significantly affect its
conformation leading to change in its function; not to mention that there are 6 cysteine
residues on an HRP molecule, making it hard to do selective mutation. As a result, we take
a compromise to estimate the stretching extent of protein molecule under magnetic tweezers
manipulation from our previous HPPK-FRET study.
To quantitatively understand the impact of structural distortion on protein function,
we performed a steered molecular dynamic (SMD) simulation to help our understanding on
how the conformational manipulation by magnetic tweezers affects the active domain on a
protein molecule.4, 5 From the single-molecule FRET measurement result in our recent
publication, we can identify that a single protein molecule is typically stretched no less than
18Å using our magnetic tweezers under the same experimental conditions reported (see
supporting information for details).6 For HRP protein molecule, since HRP can only be
126
linked to modified glass or tethered to magnetic beads through lysine residue, we set lysine
residue 65 and lysine residue 174 on HRP protein molecule stretched 17Å as an example to
discover the corresponding active site conformational change. As shown in Figure 6, we
use the distance between residue 68 and residue 178 to characterize conformational
distortion on active site of HRP protein molecule.
In the steered MD simulation, we did constant speed pulling simulation to illustrate the
conformational distortion of HRP protein molecule responding to external stretching force.
Similarly to SMD simulation of HPPK in section 5.2, here the HRP protein molecules are
stretched in the same pulling condition by the same single molecule fluorescence magnetic
tweezers microscopic approach. The goal for this simulation is to characterize the distortion
scale of the active site of a HRP protein once the HRP protein molecules are in the same
stretching condition via our magnetic tweezers, compared with the results from our previous
published work.36 The initial coordinate of HRP protein molecule is taken from Protein Data
Bank (PDB code 1W4Y). MD simulation is performed using program NAMD, version 2.9.
Protein molecule is set in water solvation condition under CHARMM type force field
(par_all27_prot_lipid.inp). Boundary condition is applied for a time step of 1 fs. Considered the
computation time, we set the pulling speed as 0.5 Å/ps.1-3 Constant temperature at 293 K during
the simulation is maintained by Langevin thermostat, with Langevin damping coefficient set at 1
ps-1. Constant pressure is maintained at 1atm using Langevin piston. Non-bond interaction is
calculated using particle mesh Ewald (PME) full electrostatics; cutoff of the van der Waals
energy is set at 12.0 Å, with switch distance at 10.0 Å and pair-list distance set at 13.5 Å. PME
grid spacing is set at 1.0 Å. We have also run 20 times MD simulation of HRP molecule at same
127
condition without any pulling events to test the HRP conformational thermal fluctuation at its
equilibrium condition as a controlling group test.
128
Figure 5.2. SMD simulation results show the scheme of the distortion of active site when
the protein is pulling by magnetic tweezers. (A) Natural HRP protein molecule. (B) HRP
protein molecule being pulled by magnetic tweezers. (C) Distortion of distance between
residue 68 and 178 induced by thermal fluctuation of HRP protein molecule. (D) Distortion
of distance between residue 68 and 178 induced by pulling force applied on HRP protein
molecule. (E)(G)(I) Projection on Cartesian coordinate of the distance distortion between
residue 68 and residue 178 for HRP protein induced by thermal fluctuation in unperturbed
condition. (F)(H)(J) Projection on Cartesian coordinate of distance distortion between
residue 68 and residue 178 for HRP protein in stretched condition.
As shown in Figure 5.2A, the initial condition of the HRP protein molecule is under
equilibrium conformation in aqueous environment without the force pulling perturbation
applied. In about 20 independent simulation events, the distance between residue 68 and
residue 178 is observed to have a fluctuation within 2Å due to conformational thermal
fluctuation of HRP protein, as shown in Figure 5.2C, while the corresponding projections on
spatial Cartesian coordinate are shown in Figure 5.2E, 5.2G and 5.2I. Figure 5.2B illustrates
how the distance of the residue pair 68-178 gets distorted when the HRP molecule is
stretched in experiment by magnetic tweezers, indicating distortion of active site in HRP
protein. Figure 5.2D shows the statistical results from 20 simulation events that the distance
extension of residue 68 and residue 178 gets extended for about 8-10Å when the
experimentally tethered lysine residue 65 and residue 174 on protein molecule are stretched
for 17Å. The corresponding projections on spatial Cartesian coordinate are shown in Figure
5.2F, 5.2H and 5.2J. Such responses from SMD simulation conceptually reveal that in our
129
experiment condition, when being stretched by magnetic tweezers, the active site on HRP
protein molecule is distorted in an extent that significantly beyond its thermal
conformational fluctuation.
We also note here that because HRP protein in experiment is linking to either
coverslip or magnetic bead via connection with lysine. Therefore, similar to HPPK case,
there are multiple possible tethering conditions of the protein molecule to the coverslip or
magnetic beads, leading to multiple possible stretching types for different HRP protein
molecules too. The active site distortion response shown in Figure 5.2 is from one possible
pattern to stretch HRP molecules. Hence, we tested all the possible stretching condition for
HRP protein molecule to reveal that conformational distortion beyond thermal fluctuation
range of active site occurs for most stretching types for HRP molecule, which will be
discussed in section 5.3.2.
5.3.2. SMD study on all possible stretching type of HRP protein molecule
There are 6 lysine residues in the HRP amino acid sequence. As being discussed earlier,
HRP molecule can only be linked to modified glass or tethered to magnetic beads through lysine
residue, and in experiment the HRP protein can only be stretched through direction formed by
two out of these lysine residues. From mathematical combination, there are 15 possible lysine
pair combination, leading to 15 different possible stretching types for different HRP protein
molecules. We studied all these 15 possible stretching situations by SMD simulation, as shown
in figure 5.3. We set three residue pairs to illustrate distortion on the active domain of HRP
molecule: residue pair 68-178, residue pair 140-228 and residue pair 30-252, as shown in figure
5.3A. The SMD environment parameters are all the same as described in materials and methods
130
section. From the sim
mulation results shown in
i figure 5.3B
B, 5.3C andd 5.3D, we find that whenn an
HRP mollecule is streetched by maagnetic tweeezers, its actiive domain w
will have disstortion
significan
ntly greater than
t
thermall conformatiional fluctuaation for mosst of the possible stretchhing
types.
ue pairs to illlustrate disto
ortion on thee active dom
main when ann HRP molecule
Fig. 5.3. Three residu
hed by magneetic tweezers. All the bllack dashed line are therrmal fluctuattion range off
is stretch
selected residue
r
pairss, obtained from
f
statisticcal result fro m 20 simulaation events of HRP prottein
in aqueou
us equilibriu
um condition
n. Blue dash
hed lines are doubled rannge of black ones, set as
stricter th
hreshold. (A
A) Scheme of the three seelected residdue pairs. (B
B) Distortion of distance
between residue 68 and
a residue 178
1 in all 15 possible strretching condditions for H
HRP moleculle.
ortion of disttance betweeen residue 14
40 and residdue 228 in alll 15 possiblee stretching
(C) Disto
condition
ns for HRP molecule.
m
(D
D) Distortion
n of distance between ressidue 30 andd residue 2522 in
all 15 possible stretch
hing conditio
ons for HRP
P molecule.
131
5.3.3. Disstortion in unfolding
u
sim
mulation
To
T quantitativ
vely comparre difference in impact o n active dom
main betweenn our
experimeental stretchiing condition
n and unfold
ding conditioon for HRP pprotein moleecule, we tesst the
active sitte conformattional distorttion in largerr unfolding ssituation by simulation. We still sellect
the three residue pairr 68-178, 140
0-228 and 30
0-252 to chaaracterize coonformationaal change of
hown in figu
ure S3A and S3B, when the distancee between lyssine
active sitte of HRP prrotein. As sh
residue 65
6 and residu
ue 174 is streetched rough
hly 50Å, the distance bettween residuue 68 and 1778 is
extended
d for 38Å, the distance beetween resid
due 140 and 228 is extennded for 14Å
Å, while the
distance between residue 30 and 252 decreassed for 1Å. S
Such active site distortioons show thaat the
conformaational distortion achieved in our maagnetic tweeezers experim
ment is signiificantly smaaller
from protein unfoldin
ng, indicatin
ng that the co
onformationaal distortion is more likeely to be on
tertiary structure rath
her than protein unfoldin
ng, which is iin accordancce with our pprevious studdy. 1
onal distortio
on in larger uunfolding sittuation. Thee initial
Fig. 5.4. Active site conformatio
A)
coordinatte of HRP prrotein moleccule is taken from Proteiin Data Bankk (PDB codee 1W4Y). (A
132
Equilibrium conformation of HRP protein in aqueous solution. (B) Conformation of HRP protein
when its lysine residue 65 and 174 is stretched for 48.8Å.
5.4. References
1
Gao, M.; Wilmanns, M.; Schulten, K. Steered Molecular Dynamics Studies of Titin I1
Domain Unfolding. Biophys. J. 2002, 83: 3435-3445.
2
Lu H, Isralewitz, B.; Krammer, A.; Vogel, V.; Schulten, K. Unfolding of Titin
Immunoglobulin Domains by Steered Molecular Dynamics Simulation. Biophys.1998, J. 75:
662-671.
3
Gräter, F., Shen, J.; Jiang, H.; Gautel, M.; Grubmüller, H. Mechanically Induced Titin
Kinase Activation Studied by Force-Probe Molecular Dynamics Simulations. Biophys. J.2005,
88: 790-804.
4
NAMD was developed by the Theoretical and Computational Biophysics Group in the
Beckman Institute for Ad-vanced Science and Technology at the University of Illinois at
Urbana-Champaign.
5
Phillips J.C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.;
Skeel, R.D.; Kalé, L.; Schulten, K. Scalable Molecular Dynamics with NAMD. J.Comput.
Chem.,2005, 26:1781-1802, 2005.
6
Guo, Q.; He, Y.; Lu, H.P. Manipulating and Probing Enzymatic Conformational
Fluctuations and Enzyme-Substrate Interactions by Single-Molecule FRET-Magnetic Tweezers
Microscopy. Phys. Chem. Chem. Phys. 2014, 16: 13052-13058.
133
CHAPTER VI. DESIGN AND IMPLEMENTATION OF A QUADRUPOLE MAGNETIC
TWEEZERS
In this chapter, we discussed a newly developed technical improvement for higher
controlling ability for next generation of magnetic tweezers.
6.1 History of Instrumental Design for Magnetic Tweezers
Magnetic tweezers has been widely acknowledged for its capability to apply pN scale
force on multiple biological specimens and single molecules simultaneously, as we have already
discussed in chapter II. It is from 1996 that the word ‘magnetic tweezers’ started to be widely
used for one certain type of experiment using magnetic field to apply external force onto single
molecule, especially DNA or protein molecule, through paramagnetic nanoparticles tethered on
those molecules. For example, Bensimon and Croquette have used magnetic tweezers to study
DNA topological properties since 1996;1 Ingber’s group has made a lot of achievements using
magnetic tweezers for transportation in living cell condition;2‐3 Fernandez and Sheetz has
developed magnetic tweezers as a force spectroscopy method to study protein unfolding
problems, etc.4
Scientific aim of magnetic tweezers when being used as a force approach to apply
mechanical manipulation.in biophysics studies focuses on two aspects. The first one is using
magnetic tweezers to manipulate target molecules such as DNA or protein to study their roles in
biological processes.5‐6 The second aspect is using magnetic tweezers to manipulate
paramagnetic beads for the purpose of drug delivery or cell mechanics applications.7‐8 Therefore,
versatile force to be applied on target specimen and precise manipulation of magnetic beads has
been a central topic in the field of magnetic tweezers for a long time.
134
In practical, there are three technical requirements for magnetic tweezers to be
successfully applied in biophysical experiments, especially single molecule biophysical research.
Firstly it needs to be able to achieve force ranged from sub-pN to nN, depending on specific
sample system experimental measurements: for molecule manipulation purpose, such as single
molecule studies of DNA or protein molecules, force range from sub-pN to 100 pN are always
preferred, while for the purpose of transportation of magnetic particles in biological specimen,
larger force from a few hundreds of pN to even nN is necessary. Secondly, since magnetic
tweezers is a force approach without the capability to provide spatial information of target
sample molecules, a correlated optical observation method is always needed to provide spatial
information of the sample system simultaneously, which requires that the setup of magnetic
tweezers needs to be not too large to be launched with correlated optical observation instruments.
Thirdly, since the core part of magnetic tweezers is always the magnet which generating
magnetic fired, for the purpose of the capability to move magnetic beads in arbitrary directions, a
classical design of the magnet part is multi-channel magnetic tweezers, which applies
multidimensional force with several magnetic poles.
Since 1996, many research works has been made to develop various types of magnetic
tweezers aiming at these requirements. A two-pole electromagnet combination has been
developed back in 1992 by Gore’s group to generate large force onto paramagnetic metal
particles with 10 µm diameter for biological tissue studies.9 In 1996, a computer controlled fourpole magnetic manipulator that capable to generate force at pN scale onto magnetic beads in 2.8
µm size has been developed by Leiber’s group.10 The force scale it can achieve at that time is
already ideal for single molecule experiments. However, the design of multidimensional magnet
setup in 1990s was always very space consuming, limiting technical developments from being
135
directly benefit scientific research. For example, scientists had to rely on simple one piece of
permanent magnet to apply force onto magnetic beads for cell-substrate studies in 1998.11
After 2000s, some scientists started to focus their effort on designing magnetic tweezers
setup that small enough to be correlated into single molecule measurements in experiment, while
still being able to generate force at pN scale, which is always required for molecule level
manipulation. In 2000, a smaller, yet still complicated design of two-pole electromagnet coil
was developed by Habor and Wirtz.12 In 2002, Croquette’s group designed a six-coiled
electromagnet with 2cm in diameter, which is small enough to be launched above a normal
inverted microscope, yet just generating twisting force specifically for DNA topological
research.13 In 2003, a computer controlled two-pole electromagnet setup in a compactible size
that capable to be fitted onto the stage of a normal inverted microscope was developed by
Forgacs’s group.14 Meanwhile, from 2000 to 2004, needle shaped electromagnet has been
studied throughout by Ingber’s group.2‐3 In 2005, Fisher et al for the first time developed a four
pole needle shaped electromagnet which can apply force in a wide range from a few pN to
thousands of pN, yet still having a few mechanical trade-offs in its design.15 In 2006, a
symmetric face-centered-cubic pole combination has been designed by Fisher et al to apply
magnetic force manipulation which can be easily correlated with optical observation in
experiments. Yet the force they achieved via the six-pole setup was still large from a few
hundreds of pN to nN scale onto paramagnetic beads small as 1 µm size.16 In 2007, Fabry’s
group developed a single needle shaped electromagnet with strong near-tip magnetic field
gradient and hysteresis compensation design to achieve large force up to 10 nN.17 By 2008,
Driel’s group developed a four-pole needle shaped electromagnet with even smaller size making
the magnet part to be better fitted with normal inverted microscope.18 Meanwhile, there are also
136
some theoretical studies to modeling magnetic field for various types of magnets.19 However,
there are still some technical limitations in each design of those multi-pole electromagnet setups
in different aspects: hysteresis effect, durability of the needle shaped electromagnet, lacking of
spatial freedom of the electromagnets involved in the multi-pole design, etc. As a result, for
biophysical experiments, scientists still rely on the simple way to generate reliable magnetic
force, although in a limited range. For example, until 2009, a few pieces of simple permanent
magnets were still used for manipulation research for DNA molecules.20
6.2
6.2.1
The Multi-Channel Magnetic Tweezers
An Introduction of the Multi-Dimensional Magnetic Tweezers Setup in Our Lab
In our studies, we also developed a multi-channel electromagnet to apply magnetic force
onto paramagnetic beads in in arbitrary directions. A conceptual figure of the design of our
quadrupole magnetic tweezers is shown in figure 6.1. Four magnetic poles are launched in a
quadrupole configuration on an alumni plate. Each magnetic pole is an electromagnet which is
custom-made in the Instrument Development Laboratory (IDL) located in the Environmental
Molecular Sciences Laboratory (EMSL) facility of Pacific Northwestern National Lab (PNNL).
The electromagnet is built by twining coils on a sharpened metal rod as probe. Magnet field will
be generated and sent out through the tip of the metal rod once current is applied. The coils have
been tested to be able to bear voltages up to 5V before showing obvious temperature change
from heating effect of applied current. In this way, the four poles can generate magnetic field
and hence to apply force on paramagnetic beads in arbitrary directions in 2D plane.
137
Figure
F
6.1. A conceptuall scheme of the
t quadrupoole magneticc tweezers seetup. Four
electromagnets poless are set on an
a alumni plaate which caan be launchhed on a simpple inverted
microsco
ope. The tip end of each poles are cu
ustom-made to be sharpeen metal to ggenerate stroong
magnet flux
f
gradientt. The four poles
p
are insttalled on thee plate via foour independdent 2D
micrometers, allowin
ng us to adju
ust physical position
p
of eeach magnet pole independently. Heence
nce among four
f
metal tip
ps of poles are
a tunable inn a range froom touching each other tto a
the distan
few centiimeters. Thee controller was
w used to be a joystickk with a few
w limitations in its function,
while currrently it hass been upgraade to a digittalized contrroller, whichh will be disccussed in secction
6.2.2.
The
T function of this apparratus to applly external fo
force onto paaramagnetic beads has
already been
b
tested by
b Jason J. Han
H and Alex
x Li from W
Washington Sttate Universsity in 2004. 3‐4
In brief, a few λ‐DNA
A molecules are immobillized at one end on glasss coverslip inn aqueous
138
environm
ment, while being
b
tethereed on the oth
her end by m
magnetic beadds with 1.5 µ
µm diameterr. An
additionaal permanentt magnet is needed
n
on th
he top to helpp in obtaininng repetitive response froom
the tetherred beads. In plane 2D-m
manipulation
n can be appplied by elecctromagnet ppoles on the setup
illustrated in figure 6.1.
6 DNA molecules
m
sen
nse the magnnetic force viia tethered bbeads. Net fforce
our differentt directions has
h been testted. Flash laamp has beenn used as ligght source, w
while
toward fo
the respo
onse of magn
netic beads can
c be observ
ved from eyeepiece on thhe microscoppe.
6 Concepttual scheme of an experiiment testingg the functioon electromagnet poles.
Figure 6.2.
The
T result of this testing experiment
e
is
i shown in ffigure 6.3. W
When applyinng mechaniccal
pulling fo
orce via mag
gnetic field, the regulated
d movementt of the magnnetic beads ccan be obserrved
by a norm
mal optical microscope.
m
The moving
g pattern of m
magnetic beeads can be m
manipulated by
changing
g the directio
on of externaal net total magnetic
m
fieldd. Firstly, thhere are no m
magnetic fielld
139
applied onto
o
the sample system, and with thee help of addditional perm
manent magnnet, Browniaan
fluctuatio
on of the teth
hered bead is limited. Then, magnettic field towaard differentt net directioons
are applieed consecutiively: firstly
y, the net field is set tow
ward west, theen toward eaast, afterwarrd,
toward west
w again; laater, net field
d is set towaard southeastt direction, thhen southweest direction,,
finally to
oward northeeast direction
n before it iss totally remooved. The teesting result of magneticc
beads is shown
s
in fig
gure 6.3B.
Figure 6.3.
6 A testing
g experimentt examinatio
on the electroomagnet funnctions. (A) Sample systtem
for the teest. (B) Expeerimental ressults show th
he response oof the param
magnetic beadds toward
external net
n magneticc field, preseenting the caapability of eelectromagneet poles to aapply force
140
manipulating movements of magnetic beads. (1) No field; (2) Net field toward West; (3) Net
field toward East; (4) Net field toward West; (5) Net field toward Southeast; (6) Net field toward
southwest; (7) Net field toward northeast; (8) Field removed.
A limitation of this multi-pole magnetic tweezers is that its voltage driven inside is not
well characterized. A joystick was used to provide qualitative control of the applied voltage, just
capable to show which direction of the net total voltage is applied toward. As a result, an
additional permanent magnet is needed on the top to help in obtaining repetitive response from
the tethered beads. As shown in figure 6.2, there is a permanent magnet constantly set on top of
the sample plane for reproducible manual manipulation around the translational stage. This is
not unacceptable for DNA manipulation experiments, since it makes the optical observation of
magnetic beads easier. However, to study protein conformational dynamics or enzymatic
activities, a quantitative controlling of the applied magnetic field is needed, and our work is
focused on making a quantitative controlling system of the four magnet poles.
6.2.2
An Improvement: Developing the New Generation Magnetic Tweezers
To develop a quantitative controller of the four magnet poles, our goal is to build an
electro-controlling system that can generate current to the wires on electromagnets to provide
magnetic field. A simultaneous quantitative real-time digital readout is also needed. The design
of quantitative voltage controller is illustrated in figure 6.4. The system includes two parts: the
electric controlling module and the output module, as labeled out in the figure. Firstly, an
external potential at 15V is applied through power supply. One coarse adjustment knob and one
fine adjustment knob are set for different precise level of tuning potential in experiments. After
going through an operational amplifier (OPA), the electric potential output in unit of Volt will be
141
translated
d into curren
nt output in unit
u of Ampeere by the cuurrent modulle. The currrent will be tthe
output to
o the wires on
n the electro
omagnet, while 1% of thee current willl be sent to a parallel circuit
to generaate a digital signal
s
on thee display mo
odule.
Figure
F
6.4. A circuit diag
gram of the new
n controllling system ffor the quaddrupole
electromagnet. This controlling
c
circuit
c
is for the purpose of quantitattively controolling one ouut of
142
the four electromagnet poles. And there are four sets of this circuit built in the controlling box
independently in charge of each magnet pole. Acronyms stand for: PCB for printed circuit board;
IC stands for integrated circuit; inverted triangles for ‘connected to ground’; CT for course
tuning; FT for fine tuning; Polar C for polarized capacitance; Non-Polar C for non-polarized
capacitance; LM324 for the operational amplifier.
The newly built controller can achieve up to 1 Amperes output, which allows the
electromagnet to generate more than 300 Gauss magnetic field. The controlling precision can
achieve 0.01 Ampere, which can be directly shown in the digital display module on the
controller. Since the capability of the electromagnet poles to manipulate paramagnetic beads has
already been tested previously, this newly designed digital controller can be used to provide
precise quantitative magnetic field which is beneficial for future single molecule biophysical or
cell-mechanical studies using magnetic tweezers. This controller is built thanks to great helpful
effort by Mr. Douglas Martin, the Design Engineer from Department of Chemistry at Bowling
Green State University. And I sincerely acknowledge Mr. Jason J. Han and Mr. Alex Li from
Washington State University for providing testing result of the electromagnet poles.
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143
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