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[email protected] www.t-cellbiology.org/teaching
Studying protein-protein interactionsEd Evans, T-cell biology group
[email protected] www.t-cellbiology.org/teaching
Studying Protein-Protein Interactions
A. INDIRECT (looking for functional association)1. Correlated mRNA Expression2. Computational Approaches3. Phylogenetic Profiling4. Synthetic Lethality
B. QUALITATIVE1. The Two-Hybrid Method2. Mass Spectrometry of Affinity-Purified Complexes3. FRET & BRET
C. QUANTITATIVE1. SPR (BIAcore)2. AUC3. Calorimetry
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Indirect detection of interactions(looking for implied functional
association NOT direct interaction)
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A. 1. Correlated mRNA expression
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A. 2. Computational approaches
e.g. “Rosetta Stone”
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A. 2. Computational approaches
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A. 3. Phylogenetic Profiling
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A. 4. Synthetic Lethality
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Qualitative detection of protein-protein interactions
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B. 1. The Two-Hybrid Method
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B. 2. Mass Spectrometry of Affinity Purified Complexes
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•Immunoaffinity•TAP tagging•2D gel•Formaldehyde crosslinking•etc…..
Gel
MS compatibleSilver stain,SYPRO stainCoomassie stain
>100 fmol protein
Specific Proteasee.g. trypsin
LC MSMSPROTEIN IDENTIFICATION
Q-ToF Micro Mass Spectrometer – LC MSMS
ProteinDigest
Nano HPLC system
NanosprayIon source
Quadrupole Time-of-flight mass spectrometer
Data acquisition
Peptides
CID
Peptidefragments
Peptide sequence
Basic Workflow
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“Mass-fingerprint” Indentification
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Non covalent protein complex
Thiol cleavable cross-linker
Covalently cross-linked complex
Digest with Protease
Thiol reagent
MALDI MS
MALDI MS
Differential peptide mapping
Non reduced
Reduced
Cross-linking the interaction
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Summary of current effort in yeast
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...and the bad news
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=> BE WARNED!
These techniques (along with e.g. Co-immuniprecipitation) give lots of false positives
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Förster (Fluorescence) Resonance Energy Transfer (FRET)
In this strategy, excitation of GFP will result in emission from a nearby protein such as blue fluorescent protein (BFP) if it is physically close enough. The best FRET pairs are actually the cyan and yellow mutants of GFP, referred to as CFP and YFP.
B. 3. a. FRET
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Power of FRET
1. Probe macromolecular interactionsInteraction assumed upon fluorescence decay
2. Study kinetics of association / dissociation between macromolecules
3. Estimation of distances (?)4. In vitro OR on live cells5. Single molecule studies
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FRET
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Live cell FRET imagingDoes CD4 specifically associate with the TCR/CD3 complex on triggering?
Non-specific peptide Specific peptide
* marks contacts between cells. High FRET signal between CD4 and CD3 when correct antigen is present but not with non-specific antigen.
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DeepBlueC hf1 hf2
Luciferase >10nm
GFP2
B. 3. b. BRET: Bioluminescence Resonance Energy Transfer
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• BRET analysis can be achieved at physiological levels of protein expression
• No problems with photobleaching or photoconversion as seen in FRET techinques (no laser stimulation)
• Both methods involve the same physical processes and so can be analysed in a similar manner
• BRET cannot be used in microscopy-based techniques such as FRAP or FLIP, or FACS-based analysis
BRET vs FRET
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• The gene of interest is fused to both luciferase (donor) and GFP (acceptor) in two separate vectors
• A positive control is used to determine maximal BRET
Construction of Fusion Proteins
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B7-1luc:B7-1YFP
CTLA-4luc:CTLA-4YFP
B7-1luc
B7-1luc:CTLA-4YFP
YFP
luc
B7-1YFPB7-1luc
substratehu2 (530 nm)
hu1 (470 nm)
e.g. B7-1 BRET
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Energy transfer can occur solely by random interactions
e.g. BRET on B7 family
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Strong dimers
Weak dimer
Monomers
Comparison to T cell surface molecules with known oligomerisation status!
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0 1 2 3 4 50.0
0.1
0.2
0.3
0.4
0.5
BR
ET
Rat
io
GFP / Rluc
hCD80 - CTLA-4 hCD80 + CTLA-4 hCD86 - CTLA-4 hCD86 + CTLA-4
Specific ligand engagement can be observed when receptor is presented in solution or cell-surface bound
Ligand binding causes specific increase in dimerisation
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Measure Quantitative Properties
SPR(BIAcore)
AUC ITC(microcalorimetry)
Surface Plasmon
Resonance
AnalyticalUltracentrifugatio
n
IsothermalCalorimetry
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Measuring key properties of protein-protein interactions
Property AUC BIAcore Calorimetry
Affinity + ++ +
Enthalpy no + ++
Entropy no + ++
Heat capacity no + ++
Kinetics no ++ no
Stochiometry + + ++
Size & Shape + no no
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C. 1. SPR / BIAcore(Surface Plasmon Resonance)
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Advantages of SPR on the BIAcore
1. No labelling is necessary2. Real-time analysis allows equilibrium
binding levels to be measured even with extremely rapid off-rate.
3. Small volumes allow efficient use of protein. Important when very high concentrations are required.
4. No wash steps => weak interactions OK5. All types of binding data obtained –
including kinetics as its real-time.
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Principle of Surface Plasmon Resonance
Angle of ‘dip’ affected by:1) Wavelength of light2) Temperature3) Refractive index n2
Dip in light intensity
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Surface Plasmon Resonance in the BIAcore
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2 Main options:• Direct:
Covalently bind your molecule to the chip• Indirect:
First immobilise something that binds your molecule with high affinity e.g. streptavidin / antibodies
Direct: Indirect:
Immobilisation
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Sensorgram for ligand binding
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• Each chip has four ‘flow-cells’• Immobilise different molecules in each flow-cell• Must have a ‘control’ flowcell• ‘Specific binding’ is the response in flow-cell of
interest minus response in the control flowcell
“Specific” Binding
Response in control / empty flowcell due to viscosity of protein solution injected – therefore ‘control’ response DOES increase with concentration (this is NOT binding!!)
Specific response in red flowcell
Measured response
Is it specific?
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Binding curve can be fitted with a Langmuir binding isotherm (assuming a 1:1 binding with a single affinity)
d
Max
KA
ARBound
][
][
Scatchard plot: rearrangement of binding isotherm to give a linear plot. Not so good for calculating Kd, as gives undue weight to least reliable points (low concentration)
Plot Bound/Free against BoundGradient = 1/Kd
dd
Max
K
Bound
K
R
A
Bound
][
Equilibrium Binding Analysis
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• Protein Problems: Aggregates (common)Concentration errorsArtefacts of construct (eg Fc
linked)• Importance of controls: Bulk refractive index issues
Control analyteDifferent levels of immobilisationUse both orientations (if pos.)
• Mass Transport: Rate of binding limited by rate of injection: kon will be
underestimated• Rebinding: Analyte rebinds before leaving
chipkoff will be underestimated
Last two can be spotted if measured kon and koff vary with immobilisation level (hence importance of controls)
Potential pitfalls
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1. Temperature dependence of binding
van’t Hoff analysis: STHKRTG a )ln(
R
S
TR
HKa
1
)ln(
Gradient
Intercept
Less common applications
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1. Temperature dependence of binding
Non-linearvan’t Hoff analysis:
0,0,,, ln)(
00 T
TCTTTCSTHG vHpvHpTvHTvH
Less common applications
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2. Combination with mutagenesis
Q30R Q40K R87A
Binding of CD2 by CD48 mutants at 25°C (WT Kd = 40M)
Less common applications
Reduce / abolish bindingDo not affect bindingNot tested
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4. Screening
Newer BIAcore machines are capable of high throughput injection. With target immobilised, many potential partners / drugs can be tested for binding.
5. Identification of unknown ligands
Mixtures e.g. cell lysates, tcs, food samples etc. can be injected over a target and bound molecules can then be eluted into tandem mass spectroscopy for identification.
Less common applications
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CD48 binding to immobilised CD2(van der Merwe et al.)
What a lot of people would have used(straight out of the freezer)
Correct result
One last warning: take care
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2. AUC(Analytical Ultracentrifugation)
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Theory: The Svedberg equation
1. Consider a particle m in a centrifuge tube filled with a liquid.
2. The particle (m) is acted on by three forces:
a) FC: the centrifugal force b) FB: the buoyant force
(Archimedes principle) c) Ff: the frictional force
between the particle and the liquid
3. Will reach constant velocity where forces balance:
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• Define s, the sedimentation coefficient:
s =
• s is a constant for a given particle/solvent, has units of seconds, but use Svedberg (S) units (10–13 s).
• Cytochrome c has s=1S, ribosome s=70S, composed of 50S and 30S subunits (s does not vary linearly with Mr)
• Values for most biomolecules between 1 and 10000 S
Theory: The Svedberg equation
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S =
f RT
ND
D = diffusion coefficient, N = Avogadro’s number
sm0(1 )
RT NDor
RTs NDm0(1 )
Mr RTs
D(1 )
• Therefore can directly determine Mr in solution by measuring physical properties of the particle (s and v) under known experimental conditions (D, T and ),
• c.f. PAGE, chromatography – comparative & non-native
(Because Mr = Nm0)
Theory: The Svedberg equation
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AUC – analytical ultracentrifugation
•Spin down protein at various concentrations and follow its distribution in the cell by OD.
•Equilibrium Analysis:Spin slowly - centrifugal force and back-diffusion reach equilibrium. Distribution depends on average mass. If this increases with concentration then association is occurring and affinity can be estimated.
•Velocity Analysis: Spin fast & follow speed of boundary descent. Depends on mass and shape– can fit multiple distributions to estimate number of species and their properties. Dependence on concentration again gives affinity.
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AUC – analytical ultracentrifugation
• Generally less precise than others.
• Key advantages are:
1. Works well for homomeric association, which is hard to follow with other techniques
2. Estimates size & shape – useful. In its own right and also for quality assessment
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Equilibrium sedimentation
1. Moderate centrifuge speed2. After sufficient time, an
equilibrium is reached between sedimentation & diffusion, resulting in a montonic solute distribution across the cell
Cell bottomMeniscus
• Non-linear curve fitting can rigorously determine:– the solution molecular
weight– association state– equilibrium constant for
complex formation
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Data modeling
1. A plot of ln(c) vs r2 should be a straight line with a slope proportional to molecular weight
Single ideal homogeneous species Mp(1- ) = d ln(c) 2RT d r2 2
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Testing for monomorphic protein
little or no curvature
10 ºC, 200 mM NaCl 40 ºC, 100 mM NaCl
26K
19K
31K
40K
obvious curvature = variation in mass i.e. unstable protein leading to aggregation
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Protein concentration (mg/ml)
6
5
4
3
20 1.0 2.0
Mw
,ap
p(D
a/1
04)
sB7-1
B7-1 : an equilibrium dimer
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sB7-2 sLICOS
Concentration (mg/ml) Concentration (mg/ml)
Mw(k
Da)
Mw(k
Da)
0 1 2 3 4 0 1 2 3 4
80
60
40
20
0
80
60
40
20
0
B7-2 and LICOS are monomeric
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Velocity sedimentation
• High centrifuge speed• Forms a sharp boundary between
solute depleted region (at top) and a region of uniform solute concn (at bottom)
• The concentration gradient (dc/dr) defines the boundary position
Non-linear curve fitting can rigorously determine:• number of mass species • molecular weight • shape information for a molecule of known mass
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g(s*) distribution
Velocity sedimentation - data analysis
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The example of SLAM (CD150)1. Claimed to self-associate with nM Kd raising serious
problems for models of cell surface protein interactions2. Equilibrium data can’t be fitted – high concentrations!3. Velocity data confirmed shape of complex and
approximate strength of association
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3. ITC(Isothermal Titration Calorimetry)
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Isothermal Titration Microcalorimetry:Using the heat of complex formation to
report on a binding interaction.The Basic Experiment:1. Fill the upper syringe with ligand at
high concentrations.2. Fill the larger lower reservoir with
protein at a lower concentration.3. Titrate small aliquots of ligand into
protein.4. After each addition, the instrument
returns the reservoir temperature to the temperature of the control cell and measures the heat required to cause this change.
5. Typically, subtract appropriate blank titrations (ligand into buffer & buffer into protein) to control for heats of dilution.
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Microcalorimetry
1. Two proteins are mixed and the heat release upon binding is measured
2. Provides a direct measure of the H (whereas van’t Hoff analysis is indirect)
3. Allows more accurate measurement of C
4. Can also determine G and => T S
5. Its disadvantage compared with the BIAcore is that very large amounts of protein are required and no kinetic data are provided
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ITC Data AnalysisGet a plot of heat (J or Cal) / s following each injection, integrate peaks for total heat released and plot against concentration of protein injected – binding isotherm.
c = concn / Kd
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Data Analysis – e.g. of B7-1 & CTLA-4
0 1 2 3 4
-12
-8
-4
0
kcal/m
ole
of
inje
ctant
molarratio
H = -11.6 G = -8.9 TS = -2.7 kcal/mol-1
1. Curve fitting gives values for H (enthalpy) and G (Gibbs free energy, related to affinity) – from these one can also calculateS (entropy).
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Calculating heat capacity
1. H and S are not constant with temperature, hence direct measurement by ITC is better than deriving them from binding data across several temperatures (e.g. by SPR)
2. Relationship of H to temperature can be used to calculate heat capacity change on binding (Cp)
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Studying Protein-Protein Interactions
A. INDIRECT1. Correlated mRNA Expression2. Computational Approaches3. Phylogenetic Profiling4. Synthetic Lethality
B. QUALITATIVE1. The Two-Hybrid Method2. Mass Spectrometry of Affinity-Purified Complexes3. FRET & BRET
C. QUANTITATIVE1. SPR (BIAcore)2. AUC3. Calorimetry
Bulk screeninge.g. For databaseNEED TESTINGAFTERWARDS
When looking for/at a (or a few) specificinteractions