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Methodology in
protein science
Yun-Ru (Ruby) Chen 陳韻如 Ph.D.
The Genomics Research Center
(office at 7th floor)
[email protected]
2789-9930 ext 355
Protein synthesis in cell
Incorporate non-natural
amino acid in cell
Protein Expression and Purification
Volume 38, Issue 1, November 2004, Pages 37-44
Preparing protein samples
What is the point?
Endogenous proteins are not enough!
Amplification
Way to go
Molecular cloning
Heterogeneous expression
Harvest and
purification
Hetero expression
Bacteria: E. Coli
Insect cells: baculo virus
infection
Mammalian cells: human
suspension cells

Bioorganism factory!
Expression level, solubility, purification procedures, yield
Detection/Quantification of proteins
Quantification of pure protein
UV absorbance: Tyr, Trp,
Beer’s law A=ebc
e=extinction coefficient
Edhock equation. Abs280=1280*(# of Tyr)+2560*(# of Trp)

•
1.
2.
3.
4.
5.
6.
7.
8.
9.
• Specific Assays (functional based)
Nonspecific assays
1. Catalytic activity
Biuret ( rx peptide backbone)
2. Ligand binding
Lowry( rx peptide backbone)
3. Antibody binding (western blot)
Ninhydrin (rx free amino group)
Fluorescamine (rx free amino group)
Coomassie stain, noncovalent complex, ~10-7g detection
Silver stain, <10-9g detection
Direct blue
Flamingo (fluorescence staining)
SyproRuby (fluorescence staining)
electrophoresis
Agarose gel
DNA

SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (1 SDS molecule
H-bonding with 2 residues)


Native-PAGE
chromatography
Size-exclusion
 Ion-exchange
 Affinity
 hydrophobicity

Size-exclusion chromatography
Ion-Exchange
Anion exchange
 Cation exchange

Affinity Chromatography
Common Fusion Tags and Purification Conditions
Fusion
Tag
Immo
bilize
d
Ligan
d
Glutath
ione Stransfer
ase(GS
T)
Binding Conditions
Elution Conditions
Available Formats
Redu
ced
gluta
thion
e
Neutral (physiologic) pH, and nondenaturing; glutathione must be reduced
and GST must be active
Free reduced
glutathione at
neutral pH
(competitor)
Prepacked column kits, spin
cup column kits, SwellGel
Discs, coated microplates
Histidin
etagged
Chel
ated
Nick
el or
Coba
lt
Neutral (physiologic) pH without reducing
or oxidizing agents; small tag must be
accessible in fusion protein structure; high
ionic strength and denaturants
(chaotropes such as 8 M urea) compatible.
>200 mM
Imidazole, low pH,
or strong
chelators
Prepacked column kits, spin
cup column kits, SwellGel
Discs, Swell- Gel Discs in 96well filter plates, coated
microplates
Maltos
e
Binding
Protein
(MBP)
Dextr
in
Neutral (physiologic) pH and nondenaturing; NaCl added to reduce
nonspecific binding
Maltose at
neutral pH
(competitor)
Gel slurry, coated microplates
Green
Fluores
cent
Protein
(GFP)
AntiGFP
antib
ody
Neutral (physiologic) pH and nondenaturing
Usual
antibody/antigen
elution buffers
(e.g., low pH or
chaotropic salts)
Coated microplates
Peptide synthesis
The longer the more expensive
 Limitation at ~100 residues
 Relatively clean

Methods to detect protein primary
structure
Protein sequencing
By Edman degradation
Phenylisothiocyanate
(PITC)
React with N- free amino
acid
Apply to chromatography
Each amino acid eluted
by Abs254nm
(Error are cumulative)
Instrument setup of Mass
spectrometry
(no detergent, desalt)
•MALDI
•ESI
•Ion bombardment
•Chemical ionization
•Electron impact
ionization
•Magnetic
•Quadrupole
•Ion trap
•TOF(Time of
flight)
•FT-MS
Mass Spectrometry



Combine with Edman degradation
Combine with limited proteolysis (ex:Trypsin digestion)
De novo sequencing is sometimes difficult (tandom mass)
(LC/MS/MS)
Advantage
Protein doesn’t need high purity
Picomoles of sample are required
Also detect post-translational modification
Methods to detect protein
secondary structure
Circular Dichroism
 Infrared spectrometry (FTIR)

Light Waves
farUV CD
Circular dichroism (CD) is a form of
spectroscopy based on the differential
absorption of left- and right-handed
circularly polarized light. It can be used to
determine the structure of
macromolecules (must be asymmetric)
n -> pi* centered around 222 nm
Part of pi -> pi* centered around 208 nm
pi -> pi* centered around 190 nm
n -> pi* involves non-bonding electrons of O
of the carbonyl
pi -> pi* involves the p-electrons of the
carbonyl
Far UV CD spectra
Detect peptide backbone through pi bond formed by
overlapping of 2 p orbital
Alpha helix: min @222 and 208nm
Beta sheet: min @216nm, max @195nm
Random coil: decreasing signal below 200nm, slight
increase @218nm
Signal to noise ratio:
Protein concentration, Path length, salt in buffer, response
time
Units: ellipticity (θ), 32.98 θ = 33.98 ΔAbs
Ellipticity: millidegree
Molar ellipticity ([θ]) is CD corrected for concentration.
molar elliplicity are historical (deg cm2/dmol)
the sample concentration (g/L), cell pathlength (cm), and
the molecular weight (g/mol) must be known
% alpha-helix = (-[θ]222nm +3000)/39000
Biochemistry. 39, 11657-11666, 2000
Secondary Structure Prediction needs spectra down to
at least 200nm (some need 178nm)
Infrared
spectra




The frequencies with which
bonded atoms vibrate relative to
each other determine the
vibrational spectrum of a molecule.
High background of water. Often
use D2O
Amide I is the most sensitive
Spectra need to be deconvoluted
Methods to detect protein
tertiary/quaternary structural changes
Size exclusion chromatography
 Fluorescence spectroscopy
 Near UV Circular dichroism
 Analytical Ultracentrifugation (AUC)
 NMR
 X-ray crystallography

Fluorescence
Fluorescence Wavelength scan
 Fluorescence Anisotropy
 Fluorescence correlation spectrum
 Fluorescence Life Time
 Fluorescence energy transfer

Highly sensitive
 Small amount (ug)
 Give total conformational information

Fluorescence
quantum yield=(# of (3))/(# of (1))
Jablonski diagram
fluorescence intensity (cps/uA)
25E+06
20E+06
15E+06
10E+06
50E+05
0
300
cps/uA / Wavelength (nm)
350
400
450
500
550
600
emission wavelength, nm
File # 2 = Y-M2A
λem＞λex
650
700
750
Stern-Volmer equation, Ksv=t0kq
Fluorescence
instrumentation
Fluorescence Anisotropy

Polarization techniques can provide average
size and shape of rotating fluorophores and
macromolecules
Polarization (P) = (Iv - Ih) / (Iv+ Ih)
Anisotropy (r) = (Iv - Ih) / (Iv+ 2Ih)
where Iv is the intensity parallel to the
excitation plane and Ih is the emission
perpendicular to the excitation plane. They are
interchangeable quantities and only differ in
their normalization. Polarization P ranges
from –0.33 to +0.5 while the range for
anisotropy r is –0.25 to +0.4.
Fluorescence energy transfer
A donor chromophore in its excited state can
transfer energy by a nonradiative, long-range
dipole-dipole coupling mechanism to an
acceptor chromophore in close proximity
(typically <10nm).
Fluorescence correlation spectrum
Using confocal or two photon microscopy, light is focused on a
sample and the measured fluorescence intensity fluctuations
(due to diffusion, chemical reactions, aggregation, etc.). FCS
is the fluorescent counterpart to dynamic light scattering, FCS
obtains quantitative information such as
diffusion coefficients, hydrodynamic radii, average
concentrations, kinetic chemical reaction rates.
Fluorescence Life Time
The fluorescence lifetime refers to the average time the molecule stays in its excited
state before emitting a photon. Fluorescence typically follows first-order kinetics:
where [S]t is the concentration of excited state molecules at time t, [S]0 is the initial
concentration and Γ is the decay rate or the inverse of the fluorescence lifetime.
This is an instance of exponential decay.
[S]t = [S]0 exp (Γ* t)
What Information Can AUC Provide?
Sedimentation
equilibrium
Molecular weight
 Stoichiometry
 Oligomerization
(Kd from 10-3-10-8 M)

The
Sedimentation
velocity



Shape and size
Number of species
Diffusion constant
first model was built in 1924 by Theodore (The) Svedberg.
Diagram of a Sedimentation Experiment
Fc: centrifugal force
Fb: buoyancy
Fd: frictional force
Sedimentation Equilibrium
Change in Concentration Over
Time
Basic Theory of Sedimentation
Equilibrium
C
C: concentration
rm
r: radius
w: angular velocity
JS: Flux of sedimentation
JD: Flux of diffusion (Fick’s Law)
s: sedimentation coefficient
rb

c(r ) = c0e
r 2 r02
( =  )
2 2
Curve fitting with assumed
model or plotting method
Different Cases for
Sedimentation Equilibrium
1.
2.
3.
4.
5.
6.

Single species (monomer)
self-association (homooligomer)
Heterogeneous mixtures (heterooligomer)
Multiple component versus multiple species
Heterogeneous associations
Non ideally
Plotting the residual as a function of radial distance
Sedimentation Velocity
Determining the rate of movement of
a solute under a centrifugal field
The
centrifugal force on the particle
(solute) is equal to the friction of the particle.
dr
M b w r = M (1   )w r = fv = f
dt
2
2
Comparison to Other Useful Techniques
AUC
Mass
Spectrometry
MicroCalorimetry
Fluorescence
spectrometry,
CD, Light
scattering
Solution MW
MW
Thermodynamics
(DG, DH, DC)
Enzyme kinetics,
2nd structures,
solution mass
Stoichiometry,
Assembly Model
Non covalent
interactions
Molar ratio
Stoichiometry
Thermodynamics( D
G)
Stoichiometry
Folding stability
Folding stability
Conformational
changes, shape
Epitope mapping
Conformational
changes
Conformational
changes
Kd 10-3~10-8
Identify unknowns
Kd 10-3~10-11;
Kd 10-6~10-11 M
10-6~10-20 M
Methods to detect atomic
level protein structure
Amount
X-ray
Crystallography
 NMR


10mg/ml
10mg/ml
pro
Atomic level
Large protein
(50-100kD)
con
Magic required
Rigid structure
Mostly native state
Atomic level
Size limit
Flexible proteins
(<50kD)
Dynamics
Over large time scale
EM
Data derived from physical techniques for probing structure, the interpretation is
not unambiguous and entails assumptions and approximations often depending
upon knowledge of the proteins from other sources (biology)
X-ray Crystallography
Bragg’s Law
The interference is constructive when the phase shift is a multiple to 2π; this
condition can be expressed by Bragg's law:
Crystallizing proteins
Both entail a droplet containing purified protein, buffer, and precipitant being allowed
to equilibrate with a larger reservoir containing similar buffers and precipitants in
higher concentrations. Initially, the droplet of protein solution contains an insufficient
concentration of precipitant for crystallization, but as water vaporizes from the drop
and transfers to the reservoir, the precipitant concentration increases to a level
optimal for crystallization.
NMR spectroscopy
(nuclear magnetic resonance)
Determine internuclear distances by measuring
perturbations between assigned resonances from atoms
in the protein in solution
1D NMR
Proton: 1H, Isotope labeled carbon 13C and nitrogen 15N, 19F
Different nuclei in a protein absorb electromagnetic energy
(resonance) at different frequencies because their local
electromagnetic environment differ
Useful parameters
Chemical shift (freq)
Chemical structure
Spin-spin coupling (through
bond)
3J
Signal intensity
Concentration
NHa torsion
angle for structural
constraint
Nuclear overhauser effect (NOE) Intermolecular distance
(through space)
Relaxation time (T1, T2)
Motional dynamics
Signal linewidth
dynamics
2D NMR
Electron Microscope





Electron beam is
stronger than X-ray. No
need for 3D crystal
Achieve atomic level
resolution
electrons interact more
strongly with atoms
than X-rays
Phase problem less
severe
But could destroy
sample
a)
b)
c)
d)
Wire
Ribbon
Ball and Stick
space filling as a sphere of
van der Waals radius
e) surface representation
GRASP image topology of
protein surface (red negative,
blue positive)
Protein structure determination methods
High resolution X-ray crystallography | NMR | Electron crystallography
Medium Cryo-electron microscopy | Fiber diffraction | Mass spectrometry
resolution | SAXS
Spectroscopic NMR | Circular dichroism | Absorbance | Fluorescence |
Fluorescence anisotropy
Translational Analytical ultracentrifugation | Size exclusion chromatography |
Diffusion Light scattering | NMR
Rotational Fluorescence anisotropy | Flow birefringence | Dielectric
Diffusion relaxation | NMR
Chemical Hydrogen-deuterium exchange | Site-directed mutagenesis |
Chemical modification
Thermodynamic Equilibrium unfolding
Computational Protein structure prediction | Molecular docking