Protein Interaction Analysis

Stefan Schauer

FGCZ Tutorial Day

September 8th, 2010

introduction

what is it good for ?

• Biomolecular interactions are intrinsic and vital to many cellular processes

• Most of the work of living organisms is performed by proteins

• Proteins do their work by acting on other macromolecules: nucleic acids, carbohydrates, lipids, and, especially,other proteins

Biomolecular Interaction Analysis provides qualitativeand quantitative information. Basis for

understanding many biological processes

• Interaction Trap/Two-Hybrid System to Identify Interacting Proteins• High-Throughput Screening for Protein-Protein Interactions Using Yeast Two-Hybrid

Arrays• Phage-Based Expression Cloning to Identify Interacting Proteins• Detection of Protein-Protein Interactions by Coprecipitation• Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)

Microscopy• Identification of Protein Interactions by Far Western Analysis• Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions• Identifying Protein Interactions by Hydroxyl-Radical Protein Footprinting• Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence

Complementation (BiFC) Analysis• Production and Use of Trimeric Isoleucine Zipper Fusion Proteins to Study Surface

Receptor Ligand Interactions• Fluorescence Quenching Methods to Study Lipid-Protein Interactions• Determination of Protein Contacts by Chemical Cross-Linking With EDC and Mass

Spectrometry• Membrane-Based Yeast Two-Hybrid System to Detect Protein Interactions• Detection and Analysis of Protein-Protein Interactions of Organellar and Prokaryotic

Proteomes by Blue Native and Colorless Native Gel Electrophoresis

introduction

qualitative methods

• Measuring Protein Interactions by Optical Biosensors

• Analytical Ultracentrifugation: Equilibrium Approach

• Analytical Ultracentrifugation: Sedimentation Velocity Analysis

• Titration Microcalorimetry

• Reduced-Scale Large-Zone Analytical Gel-Filtration Chromatography for Measurement of Protein Association Equilibria

• Size-Exclusion Chromatography with On-Line Light Scattering

• Spectroscopic Methods for the Determination of Protein Interactions

• Application of Amide Proton Exchange Mass Spectrometry for the Study of Protein-Protein Interactions

• Circular Dichroism to Study Protein Interactions

• Quantitative Determination of Protein Stability and Ligand Binding by Pulse Proteolysis

introduction

quantitative methods

introduction

http://onlinelibrary.wiley.com/book/10.1002/0471140864

Wiley Online Library Current Protocols in Protein Science

http://www.els.net/

Encyclopedia of Life Sciences

• The strength of an noncovalent interaction between two molecules is described by equilibrium parameters

A + B [AB]

equilibrium dissociation constant: Kd = [A][B]/[AB] = koff /kon

where [AB] is the concentration of the complexed species and [A] and [B] are the concentrations of the noncomplexed species. Concentrations are given in molar terms, as is the Kd

basic concepts

how does it work ?

kon

koff

a low Kd corresponds toa high „affinity“

or better, „binding strength“

• Equilibrium Dissociation Constants for Some Biologically Significant Interactions

basic concepts

• Quick yes or no answer

• SPR range for affinity (Kd) measurement: 10pM to 1mM

• Affinity maturation

• Comparison of different mutants/constructs

• Full kinetic and thermodynamic characterisation (conf. state

trapping, comparison MD/NMR/kinetics)

• Confirmation of affinities obtained by other methods

• Epitope mapping

• Concentration measurements

• Quality control

introduction

SPR / what is it good for ?

basic concepts

measuring principle

• SPR detects refractive index changes close to the surface (0 – 100nm)

• accumulation of 1 pg substance/mm2 gives a change of 1 µRIU or 1 RU

basic concepts

correlation of SPR response with absolute surface protein concentration

• signal proportional to mass

• same specific response for different proteins

Protein

Chymotrypsinogen ATransferrinanti-Transferrinanti-β2microglobulin

MW

2570084000150000150000

Symbol

▲

basic concepts

information content of a sensogram

dR/dt = kon ∙ C ∙ [Rmax - R] - koff ∙ R

RU/s M-1s-1 M RU s-1 RU

d[AB]/dt = kon ∙ [A] ∙ [B] - koff ∙ [AB]

basic concepts

information content of a sensogram

kon

koff

A + B [AB]

• All four compounds have the same affinity KD = 10 nM = 10-8M

• The same affinity can be the result from different kinetics !

time

RU

10-5103

10-4104

10-3105

10-2106

kd

[s-1]

ka

[M-1s-1]

KD 10 nM

basic concepts

kinetic resolution of affinities with SPR

basic concepts

kinetic resolution of affinities with SPR

koff (s-1)

k on

(M-1

s-1 )

104

0.0001 0.001 0.01 0.1 1

107

106

105

102

103

1 nM100 pM 10 nM10 pM

100 nM

1 µM

1 mM

100 µM

10 µM

KD

• HIV-1 protease inhibitors: on-off rate map

koff (s-1)

k on

(M-1

s-1 )

104

0.0001 0.001 0.01 0.1 1

107

106

105

102

103

1 nM100 pM 10 nM10 pM

100 nM

1 µM

1 mM

100 µM

10 µM

KD

B435

B408

B347

B365

B425

B439

A016

B249

B277

A018

A015

A017

B322

B440

B429A03

7

B409

B388

B268

A038

B412

B295B355

B369

Nelf

RitSaq

Amp

U75875

Ata

B268

basic concepts

kinetic resolution of affinities with SPR

• HIV-1 protease inhibitors: on-off rate mapDevelopment of Saquinavir, Amprenavir and Atazanavir

investigating biomolecular interactions with SPR sensors

applications

• Protein – Protein Interactions

− recombinant mouse prion protein – IgG POM1

− L. lactis multidrug exporter LmrCD – DARPins

− arginyl-tRNA synthetase – ribosomal protein L3

− human Nogo-A – Nogo-A receptor

− human IL18 and IL33 – IL18BP

− PhyR – NepR

− p150 – hMSH6

− SicP/SptP complex – ATPase InvC

− AcrB (multidrug efflux pump membrane protein) – DARPins

− CitS (transport membrane protein) – DARPins

− Caspase-1, -2, -3, -5 and -8 (Proteases in Apoptosis) – DARPins

− Caspase-1, -2, -3, -5 and -8 – PRYSPRY (C-terminal domain of Pyrin)

investigating biomolecular interactions with SPR sensors

applications

• Protein – Protein Interactions

− HLA B27 – Fab (reagent to investigate dimerization, impact on Bechterew's disease)

− rhCAIX – Fab (tumor targeting)

− rhFAP (fibroblast activating protein) – aFAPhGITR-L (ligand for GITR on T cells)

− Nimak – HcpC

− HIV Capsid Protein – PRYSPRY (C-terminal domain of antiviral factor TRIM5α)

− Cellular receptor CD46 – Ad3 (7,11,35) - Fiber Knobs

− VEGF – VEGF receptor

− MSG1 – Actin

− chaperone SicP – effector protein SptP

− S.aureus Orf1055 – unknown protein interaction partner (ligand fishing)

investigating biomolecular interactions with SPR sensors

applications

• Protein – Peptide Interactions

− prion protein PrP – amyloid-β− single chain Fv – biotinylated-nitrotyrosine containing 10-14mers− anti-PrP monoclonal antibodies – PrP peptide set (12mers)

• Protein – Small Molecule Interactions

− M. tuberculosis secreted chorismate mutase – transition state analogon− polyclonal IgGs – polythiophene acetic acid− cell wall binding domain protein CBDP35 – N-acetyl-D-glucosamine− M. tuberculosis IspF – thiazolopyrimidines− hCarbonic Anhydrase Ι – sulfonamide inhibitors

• Protein – RNA Interactions

− SerRS – seryl-tRNA

investigating biomolecular interactions with SPR sensors

applications

• Protein – Carbohydrate Interactions

− Influenza A Virus H5N1 hemagglutinin – synthetic trimeric sialylglycan− Nkp30 – synthetic heparin oligosaccharides− Moesin – Glycosylphosphatidylinositol

• Protein – Lipid Interactions

− ApoM – lauryl-oleyl-phosphatidylethanolamine

• Protein – Cell Interactions

− cell adhesion protein fasciclin – whole M. extorquens cells

• Large RNA – Small Molecule Interactions

− E.coli btuB riboswitch (205nt) – adenosyl-cobalamine

• DNA – DNA interactions

− Biotin-TTTCCTCAGCATCTTATCCGAGTTT – ATGCTGAGG (and variants)

applications

example: E.coli btuB riboswitch – cobalamine (vitamin B12)

5`- untranslated region of btuB transcript

R =

vitamin B12

C72H100CoN18O17P 1579,60 g/mole

P. Choudhary / Sigel Group / UZH

applications

example: riboswitch – cobalamine

applications

example: E.coli btuB riboswitch – cobalamine (vitamin B12)

applications

example: riboswitch – cobalamine

Rmax: 10 RUChi2: 0.0569

applications

example: riboswitch – cobalamine

applications

example: riboswitch – cobalamine

23°C 25°C 27°C 29°C

31°C 33°C 35°C 37°C

39°C 41°C

applications

example: riboswitch – cobalamine

• Surface plasmon resonance detects binding events as changes in refractive index in the volume

between 0 and 100 nm from the chip surface

• Real-time kinetic measurements

• No labeling of interactants necessary

• Low sample amounts (micrograms) required

• High Throughput SPR needs are covered by BioRad`s ProteOnXPR36

summary

• importance of sample preparation:

1. purity

2. homogeneity

3. stability

• further reading on surface plasmon resonance (SPR):

Surface Plasmon Resonance Based Sensors, Homola, Springer 2006

take home message

acknowledgments

• Pallavi ChoudharyGroup Roland SigelUniversity Zurich