Download Single Molecule Slides

Single Molecule Biophysics
(Mainly fluorescence spectroscopy)
Reading: van Holde Chapter 16
Homework: due Wednesday, April 23 (in class)
Van Holde: 16.1, 16.2, 16.3, 16.4 (consult original papers)
Overview:
1. Why single-molecule biophysics?
2. Single-molecule fluorescence, TIRF illumination & applications
3. Atomic Force Microscopy (AFM) & applications (AFM imaging, AFM singlemolecule force measurements)
4. Optical tweezers (laser tweezers, laser traps) & applications
Single molecule biophysics – Why?
• Observation and physical manipulation of single, dynamic biomolecules
• A recently emerged, new field offering much deeper analysis of molecular behavior.
Most previous studies have been on the average, ensemble behavior of molecules.
Why study single molecules?
• How any one molecule behaves is not revealed in bulk studies.

An individual enzyme may exist in two or more states of activity that are not revealed by
bulk studies. (e.g speed of RNA polymerase transcription).

Analogy: How insurance actuary and novelist look at human behavior.

Static heterogeneity: Different enzyme molecules function at different rates (e.g. a lame
population and a fast population)

Dynamic heterogeneity: A given single enzyme can switch between different rates.
• Can take force measurements (force spectroscopy) on single molecules (previously
impossible). E.g. motor proteins, unfolding-refolding proteins
Quick example:
The graph shows the speed of a population of polymerase molecules.
What is the approximate, ensemble-average speed (as measured by bulk
experiments)?
What additional information can you obtain from single molecules?
Single molecule fluorescence
•
Like Chapter 11 (bulk fluorescence), except on a single fluorophores
•
Challenges:
•
–
Photobleaching: Most fluorophores photobleach, i.e. after absorbing many photons ( usually
a few million), they chemically rearrange and stop fluorescing (end of experiment).
–
Signal to noise ratio (need to reduce noise and have sensitive detection)
–
Must have very, very clean sample.
–
Detecting weak signals requires very sensitive instrumentation
Needed:
–
A strong fluorophore (high absorbance and Q-yield).
–
Intense light source (often laser).
–
Very sensitive camera/detector
–
Eliminate all background light.
–
Illuminate a very small region only
•
Confocal microscopy (images only slices (stacks) of a sample)
•
Use TIR (total internal reflection).(On white board): From Snell’s law : When light encounters a lower
index of refraction medium the light gets totally reflected when the incident angle is larger than the
critical angle.  We get an evanescent wave (evanescent = tending to vanish like vapor).
Prism-based TIR illumination
Distance above
cover slip
Relative light intensity
132 nm
0.125
88 nm
0.25
No signal
(background) from
molecules far from
surface.
Laser
44 nm
0.5
Only molecules
close to surface
will fluoresce
Microscope objective
Prism and cover slip
I( z )  I 0 e

TIRF
z
d
penetration depth:
d

4 n12 sin 2 1  n2 2
n1 …index of refraction of glass slide
n2 …index of refraction of water
 … incident angle
 … wavelength of light
No TIRF
Objective-based TIRF illumination
Ray paths (schematic):
Angle of incidence smaller than the
critical angle.
www.zeiss.com
Total reflection
1: Objective,
2: Immersion oil n = 1.518,
3: Cover slip n = 1.518,
4: Evanescent field,
5: Mountant n = 1.33…1.38
See applet and info at: http://www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfhome.html
Application: Single molecule FRET
G. Bokinsky et al. “Single-molecule transition-state analysis of RNA folding” (2003) PNAS 100: 9302
The hairpin ribozyme:
The docking and undocking conformations
and a single molecule FRET trace:
SA
FRET ratio =
IA
I A  ID
Application: Single molecule FRET
G. Bokinsky et al. “Single-molecule transition-state analysis of RNA folding” (2003) PNAS 100: 9302
From figure on previous page:
The ribozyme exhibits fluctuations between two states, docked and
undocked  FRET signal changes abruptly when a transition occurs.
2. Rate constants can be extracted from dwell times!!
N  N0  e

t

 N0  e k1 t
For single rate.
3. They found a single rate constant for docking kd = 0.018 sec-1.
Occurence
•
4. They found four different rate constants for undocking
(ku = 0.01, 0.1, 0.8, 6 sec-1), which would have been hard to
find in bulk studies).
On-time (msec)
Schematic of an AFM
Photodetector
Laser
Cantilever
Piezoelectric
transducer
Sample
Substrate
Force controlled by feedback
Images from NT-MDT web page
Atomic Force Microscopy
Advantages:
• Can achieve atomic resolution on hard, crystalline surfaces.
• Can often achieve nanometer resolution on biological samples.
• Imaging can be done in buffer  can image (biological) processes.
• Can also be used to mechanically manipulate molecules (more in a bit).
Gold surface
(atomic resolution)
scan
Tapping
Contact mode AFM
4 nm
On
Off
On
scan
Off
0 nm
On
Tapping mode AFM
Off
On
2 mm
Contact mode (constant force mode): Use cantilever deflection as feedback
signal, (use softer cantilevers, can still have lateral forces pushing molecules
around)
Tapping mode: oscillated cantilever at its resonance frequency (10 kHz to 300
kHz), use cantilever amplitude or phase as feedback signal. (Lateral forces
mostly eliminated).
Transcribing RNA Polymerase Imaged by AFM1,2
T=0s
T = 80 s
A
T = 210 s
T = 130 s
B
T = 250 s
E
T = 170 s
C
T = 290 s
F
G
100 nm
D
5.0 nm
0 nm
H
Kasas movie
1. Kasas et al. (1997) Biochemistry 36(3), 461-468
2. Guthold et al. (1999) Biophys. J. 77, 2284-2294
Normal force measurements.
Example 1. Protein unfolding
(a) The principal AFM components. (b) Mechanical unfolding of repeating immunoglobulin-like domains (1). As the
distance between the surface and tip increases (from state 1 to state 2), the molecule extends and generates a
restoring force that bends the cantilever. When a domain unfolds (state 3), the free length of the protein increases,
relaxing the force on the cantilever. Further extension again results in a restoring force (state 4). The last peak
represents the final extension of the unfolded molecule before detachment from the SFM tip (state 5).
(1) Carrion-Vazquez et al. “Mechanical and chemical unfolding of a single protein: a comparison” (1999) PNAS 96 3694-99
Figure from Bustamante, Macosko, Wuite “Grabbing the cat by the tail: Manipulating molecules one by one. Nature reviews
Molecular Cell Biology 1 131-6
Normal force measurements.
Example 2. Ligand binding forces
and how they related to the koff rate (force spectroscopy).
• Protein-ligand is spanned between
the tip and the substrate.
• The tip is then retracted, and,
thus, applying a force to the bonds
under investigation.
• If the force is measured as a
function of the pulling rate, it is
termed force spectroscopy.
Connection between rupture force and off-rate k-1
Bell model: an applied force
lowers the activation energy.
Assume a two-state model for the reaction.
 B
A 
k 1
k1

 unbound
bound 
Dissociation rate without an applied force:
Dissociation rate with applied force:
k1( 0 )   1e
 G1
k BT
k1( F )  k1( 0 )e
F  x1
k BT
G. Bell (1978) Science 200, 616-627; E. Evans & K Ritchie (1997) Biophys. J. 72, 1541-55
Connection between rupture force and off-rate, k-1
Experiment: Measure rupture
force as a function of pulling rate.
(here done of two different
proteins).
For this treatment, we assume the
reaction proceeds far from
equlibrium.
The faster you pull the higher the
rupture force.
The rupture
force is related
to the off-rate


k BT
r
F
 ln 
x1
 k ( 0 )  k BT
 1
x1







F … rupture force
T … temperature
k-1 … off-rate
x-1 … width of potential
kB …Boltzmann constant
Data from F. Schwesinger et al. (2000) PNAS 97, 9972-77, First done by Rief at al. Science (1997) 276, 1109-12
How does a laser trap work?
• Light “consists of photons, which carry
Laser beam
lens
Bead is
below center
of focus
 force on
bead toward
focus
momentum. Momentum is conserved.
 When light is absorbed, reflected or
refracted, tiny forces on the order of
piconewtons are generated.
• For a laser trap we need a light
gradient (light is focused).
• Opposing scattering and gradient
forces, trap a bead in the focus.
• If moved from the focus, bead is pulled
back toward the focus.
 Particle radius has to be larger than
wavelength of light (Mie scattering
regime).
Fscatt.
Fgrad
 Need transparent dielectric bead with
index of refraction larger than
surrounding medium.
Ray-diagram for a bead to the left
and higher than laser focus
Restoring force of a laser trap
A trap exerts a linear restoring force
proportional to trap stiffness (force is
linear to displacement).
Using optical tweezers, one can apply
pico-newton sized loads and measure
nanometer level displacements.
From Hubmayr lab, Mayo Clinic):
http://mayoresearch.mayo.edu/mayo/research/hubmayr/
Laser tweezers
• Force clamp: Force on molecule is kept
constant by always having bead at the
same position in laser trap ( feedback
loop moving bead or stage).
• Position clamp: Position of molecule is
kept constant  bead is pulled out of trap
and, thus, force increases.
Applications of laser tweezers
1. Transcription by single RNA polymerase.
Set-up for measuring force-velocity relation
of a transcribing RNA polymerase (Wang et
al. (1998) Science 282, 902-7.
Stall force is about 20 nN.
Applications of laser tweezers
2. Transcription by single RNA polymerase.
Individual RNA polymerase
molecules switch between a fast
and a slow mode
(Davenport et al. (2000) Science
287, 2497-500
Dumbbell set-up used for some experiments
From Block lab: http://www.stanford.edu/group/blocklab/RNAP.html
Mechanical properties of DNA
Stretching of double-stranded phage DNA; Length for B DNA
~ 16 mm).
1. Up to a length of about 15 mm:
worm-like chain is straightened
(entropy), little force needed.
2. Steep part of curve
corresponds to elastic
stretching of extended chain.
3. At ~ 17 mm a major
conformational change occurs
 conversion to S-DNA
(stretched-DNA).
4. Then DNA denatures and
becomes single-stranded.
From Smith et al. (1996) Science 271, 795
A few more applications
• Force-producing properties of various molecular motors,
including kinesin moving along microtubules 13,
actomyosin complexes 28, RNA polymerase 36, 48 and
DNA polymerase 11 (Fig. 4b). Mechanical properties of
DNA 47
• Mechanical unfolding of proteins 52, 53
• Unfolding of RNA molecules (54)
• For a somewhat recent review see: Bustamante et al.
“Grabbing the cat by the tail: Manipulating molecules one
by one.” (2000) Nature reviews Molecular Cell Biology 1
131-6
11. Wuite, G. J., Smith, S. B., Young, M., Keller, D. & Bustamante, C. Single-molecule studies of the
effect of template tension on T7 DNA polymerase activity. Nature 404, 103-106
(2000). | Article | PubMed | ISI | ChemPort |
13. Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S. M. Direct observation of kinesin stepping
by optical trapping interferometry. Nature 365, 721-727
(1993). | Article | PubMed | ISI | ChemPort |
28. Essevaz-Roulet, B., Bockelmann, U. & Heslot, F. Mechanical separation of the complementary
strands of DNA. Proc. Natl Acad. Sci USA 94, 11935-11940
(1997). | Article | PubMed | ChemPort |
47. Smith, S. B., Cui, Y. & Bustamante, C. Overstretching B-DNA: the elastic response of individual
double-stranded and single-stranded DNA molecules. Science 271, 795-799
(1996). | PubMed | ISI | ChemPort |
48. Yin, H. et al. Transcription against an applied force. Science 270, 1653-1657
(1995). | PubMed | ISI | ChemPort |
52. Kellermayer, M. S., Smith, S. B., Granzier, H. L. & Bustamante, C. Folding-unfolding transitions in
single titin molecules characterized with laser tweezers. Science 276, 1112-1116 (1997); erratum
277, 1117 (1997). | PubMed | ISI | ChemPort |
53. Tskhovrebova, L., Trinick, J., Sleep, J. A. & Simmons, R. M. Elasticity and unfolding of single
molecules of the giant muscle protein titin. Nature 387, 308-312
(1997). | Article | PubMed | ISI | ChemPort |
54. (J. Liphardt et al. “Reversible Unfolding of Single RNA Molecules by Mechanical Force” (2001)
Science 292, 733-737