Studying protein protein interactions ed evans t cell biology group
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Studying protein-protein interactions Ed Evans, T-cell biology group. Studying Protein-Protein Interactions. INDIRECT (looking for functional association) Correlated mRNA Expression Computational Approaches Phylogenetic Profiling Synthetic Lethality QUALITATIVE The Two-Hybrid Method

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Studying protein protein interactions ed evans t cell biology group

Studying protein-protein interactionsEd Evans, T-cell biology group


Studying protein protein interactions

Studying Protein-Protein Interactions

  • INDIRECT (looking for functional association)

    • Correlated mRNA Expression

    • Computational Approaches

    • Phylogenetic Profiling

    • Synthetic Lethality

  • QUALITATIVE

    • The Two-Hybrid Method

    • Mass Spectrometry of Affinity-Purified Complexes

    • FRET & BRET

  • QUANTITATIVE

    • SPR (BIAcore)

    • AUC

    • Calorimetry


Studying protein protein interactions ed evans t cell biology group

Indirect detection of interactions(looking for implied functional association NOT direct interaction)


A 1 correlated mrna expression

A. 1. Correlated mRNA expression


A 2 computational approaches

A. 2. Computational approaches

e.g. “Rosetta Stone”


A 2 computational approaches1

A. 2. Computational approaches


A 3 phylogenetic profiling

A. 3. Phylogenetic Profiling


A 4 synthetic lethality

A. 4. Synthetic Lethality


Qualitative detection of protein protein interactions

Qualitative detection of protein-protein interactions


B 1 the two hybrid method

B. 1. The Two-Hybrid Method


B 2 mass spectrometry of affinity purified complexes

B. 2. Mass Spectrometry of Affinity Purified Complexes


Basic workflow

Basic Workflow

  • Immunoaffinity

  • TAP tagging

  • 2D gel

  • Formaldehyde crosslinking

  • etc…..

MS compatible

Silver stain,

SYPRO stain

Coomassie stain

>100 fmol protein

LC MSMS

PROTEIN

IDENTIFICATION

Specific

Protease

e.g. trypsin

Gel

Q-ToF Micro Mass Spectrometer – LC MSMS

Quadrupole

Time-of-flight mass

spectrometer

Protein

Digest

Peptides

Peptide

fragments

Data

acquisition

CID

Nanospray

Ion source

Nano HPLC

system

Peptide sequence


Mass fingerprint indentification

“Mass-fingerprint” Indentification


Cross linking the interaction

Cross-linking the interaction

Non covalent protein complex

Thiol cleavable cross-linker

Covalently cross-linked complex

Digest with Protease

MALDI MS

Non reduced

Thiol reagent

MALDI MS

Reduced

Differential peptide mapping


Summary of current effort in yeast

Summary of current effort in yeast


And the bad news

...and the bad news


Be warned

=> BE WARNED!

These techniques (along with e.g. Co-immuniprecipitation) give lots of false positives


B 3 a fret

B. 3. a. FRET

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.


Power of fret

Power of FRET

  • Probe macromolecular interactions

    Interaction assumed upon fluorescence decay

  • Study kinetics of association / dissociation between macromolecules

  • Estimation of distances (?)

  • In vitro OR on live cells

  • Single molecule studies


Studying protein protein interactions ed evans t cell biology group

FRET


Live cell fret imaging

Live cell FRET imaging

Does 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.


B 3 b bret bioluminescence resonance energy transfer

B. 3. b. BRET: Bioluminescence Resonance Energy Transfer

hf1

hf2

DeepBlueC

GFP2

Luciferase

>10nm


Bret vs fret

BRET vs FRET

  • 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


Construction of fusion proteins

Construction of Fusion Proteins

  • 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


E g b7 1 bret

e.g. B7-1 BRET

B7-1luc

B7-1YFP

substrate

YFP

luc

hu2 (530 nm)

hu1 (470 nm)

B7-1luc:B7-1YFP

B7-1luc

CTLA-4luc:CTLA-4YFP

B7-1luc:CTLA-4YFP


E g bret on b7 family

e.g. BRET on B7 family

  • Energy transfer can occur solely by random interactions


Comparison to t cell surface molecules with known oligomerisation status

Comparison to T cell surface molecules with known oligomerisation status!

Strong dimers

Weak dimer

Monomers


Ligand binding causes specific increase in dimerisation

Ligand binding causes specific increase in dimerisation

  • Specific ligand engagement can be observed when receptor is presented in solution or cell-surface bound


Measure quantitative properties

Measure Quantitative Properties

SPR

(BIAcore)

AUC

ITC

(microcalorimetry)

Surface Plasmon Resonance

Analytical

Ultracentrifugation

Isothermal

Calorimetry


Measuring key properties of protein protein interactions

Measuring key properties of protein-protein interactions


C 1 spr biacore surface plasmon resonance

C. 1. SPR / BIAcore(Surface Plasmon Resonance)


Advantages of spr on the biacore

Advantages of SPR on the BIAcore

  • No labelling is necessary

  • Real-time analysis allows equilibrium binding levels to be measured even with extremely rapid off-rate.

  • Small volumes allow efficient use of protein. Important when very high concentrations are required.

  • No wash steps => weak interactions OK

  • All types of binding data obtained – including kinetics as its real-time.


Principle of surface plasmon resonance

Principle of Surface Plasmon Resonance

Dip in light intensity

Angle of ‘dip’ affected by:

1) Wavelength of light

2) Temperature

3) Refractive index n2


Surface plasmon resonance in the biacore

Surface Plasmon Resonance in the BIAcore


Immobilisation

Immobilisation

Direct:

Indirect:

  • 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


Sensorgram for ligand binding

Sensorgram for ligand binding


Specific binding

“Specific” Binding

Specific response in red flowcell

Response in control / empty flowcell due to viscosity of protein solution injected – therefore ‘control’ response DOES increase with concentration (this is NOT binding!!)

Measured response

Is it specific?

  • 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


Studying protein protein interactions ed evans t cell biology group

Equilibrium Binding Analysis

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 Bound

Gradient = 1/Kd

Binding curve can be fitted with a Langmuir binding isotherm (assuming a 1:1 binding with a single affinity)


Kinetics

Kinetics

Harder

Case:

2B4 binding CD48


Potential pitfalls

Potential pitfalls

  • Protein Problems:Aggregates (common)Concentration errorsArtefacts of construct (eg Fc linked)

  • Importance of controls:Bulk refractive index issuesControl 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)


Less common applications

Less common applications

van’t Hoff analysis:

Gradient

Intercept

1. Temperature dependence of binding


Studying protein protein interactions ed evans t cell biology group

Less common applications

1. Temperature dependence of binding

Non-linear

van’t Hoff analysis:


Studying protein protein interactions ed evans t cell biology group

Less common applications

Q30R

Q40K

R87A

2. Combination with mutagenesis

Binding of CD2 by CD48 mutants at 25°C (WT Kd = 40mM)

Reduce / abolish binding

Do not affect binding

Not tested


Less common applications1

Less common applications

3. Estimation of valency


Studying protein protein interactions ed evans t cell biology group

Less common applications

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.


One last warning take care

One last warning: take care

What a lot of people would have used

(straight out of the freezer)

Correct result

CD48 binding to immobilised CD2

(van der Merwe et al.)


2 auc analytical ultracentrifugation

2. AUC(Analytical Ultracentrifugation)


Theory the svedberg equation

Theory: The Svedberg equation

  • Consider a particle m in a centrifuge tube filled with a liquid.

  • The particle (m) is acted on by three forces:

    • FC: the centrifugal force

    • FB: the buoyant force (Archimedes principle)

    • Ff: the frictional force between the particle and the liquid

  • Will reach constant velocity where forces balance:


Theory the svedberg equation1

Theory: The Svedberg equation

  • 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 (sdoes not vary linearly with Mr)

  • Values for most biomolecules between 1 and 10000 S


Theory the svedberg equation2

Theory: The Svedberg equation

S =

D = diffusion coefficient, N = Avogadro’s number

or

(Because Mr = Nm0)

  • Therefore can directly determine Mr in solution by measuring physical properties of the particle (s and v) under known experimental conditions (D, T and r),

  • c.f. PAGE, chromatography – comparative & non-native


Auc analytical ultracentrifugation

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.


Auc analytical ultracentrifugation1

AUC – analytical ultracentrifugation

  • Generally less precise than others.

  • Key advantages are:

  • Works well for homomeric association, which is hard to follow with other techniques

  • Estimates size & shape – useful. In its own right and also for quality assessment


Equilibrium sedimentation

Equilibrium sedimentation

  • Moderate centrifuge speed

  • After sufficient time, an equilibrium is reached between sedimentation & diffusion, resulting in a montonic solute distribution across the cell

Meniscus

Cell bottom

  • Non-linear curve fitting can rigorously determine:

    • the solution molecular weight

    • association state

    • equilibrium constant for complex formation


Data modeling

Data modeling

d ln(c) 2RT

d r2w2

Mp(1- ) =

  • A plot of ln(c) vs r2 should be a straight line with a slope proportional to molecular weight

Single ideal homogeneous species


Testing for monomorphic protein

Testing for monomorphic protein

19K

26K

31K

40K

40 ºC, 100 mM NaCl

10 ºC, 200 mM NaCl

obvious curvature = variation

in mass i.e. unstable protein leading to aggregation

little or no curvature


B7 1 an equilibrium dimer

B7-1 : an equilibrium dimer

6

5

Mw,app(Da/104)

4

3

sB7-1

2

0

1.0

2.0

Protein concentration (mg/ml)


B7 2 and licos are monomeric

B7-2 and LICOS are monomeric

80

80

60

60

40

40

20

20

0

0

0

0

1

1

2

2

3

3

4

4

sLICOS

sB7-2

Mw(kDa)

Mw(kDa)

Concentration (mg/ml)

Concentration (mg/ml)


Velocity sedimentation

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


Velocity sedimentation data analysis

Velocity sedimentation - data analysis

g(s*) distribution


The example of slam cd150

The example of SLAM (CD150)

  • Claimed to self-associate with nM Kd raising serious problems for models of cell surface protein interactions

  • Equilibrium data can’t be fitted – high concentrations!

  • Velocity data confirmed shape of complex and approximate strength of association


3 itc isothermal titration calorimetry

3. ITC(Isothermal Titration Calorimetry)


Studying protein protein interactions ed evans t cell biology group

Isothermal Titration Microcalorimetry:Using the heat of complex formation to report on a binding interaction.

The Basic Experiment:

  • Fill the upper syringe with ligand at high concentrations.

  • Fill the larger lower reservoir with protein at a lower concentration.

  • Titrate small aliquots of ligand into protein.

  • 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.

  • Typically, subtract appropriate blank titrations (ligand into buffer & buffer into protein) to control for heats of dilution.


Microcalorimetry

Microcalorimetry

  • Two proteins are mixed and the heat release upon binding is measured

  • Provides a direct measure of the H (whereas van’t Hoff analysis is indirect)

  • Allows more accurate measurement of C

  • Can also determine G and => T S

  • Its disadvantage compared with the BIAcore is that very large amounts of protein are required and no kinetic data are provided


Itc data analysis

ITC Data Analysis

Get a plot of heat (mJ or mCal) / s following each injection, integrate peaks for total heat released and plot against concentration of protein injected – binding isotherm.

c = concn / Kd


Data analysis e g of b7 1 ctla 4

Data Analysis – e.g. of B7-1 & CTLA-4

  • Curve fitting gives values for DH (enthalpy) and DG (Gibbs free energy, related to affinity) – from these one can also calculate DS (entropy).

0

-4

H = -11.6

G = -8.9

TS = -2.7

kcal/mol-1

kcal/mole of injectant

-8

molar

ratio

-12

0

1

2

3

4


Calculating heat capacity

Calculating heat capacity

  • DH and DS 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)

  • Relationship of DH to temperature can be used to calculate heat capacity change on binding (DCp)


Studying protein protein interactions1

Studying Protein-Protein Interactions

  • INDIRECT

    • Correlated mRNA Expression

    • Computational Approaches

    • Phylogenetic Profiling

    • Synthetic Lethality

  • QUALITATIVE

    • The Two-Hybrid Method

    • Mass Spectrometry of Affinity-Purified Complexes

    • FRET & BRET

  • QUANTITATIVE

    • SPR (BIAcore)

    • AUC

    • Calorimetry

Bulk screening

e.g. For database

NEED TESTING

AFTERWARDS

When looking for/at a (or a few) specific

interactions


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