1. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Studying protein-protein interactions Ed Evans, T-cell biology group

2. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Studying Protein-Protein Interactions A.INDIRECT (looking for functional association) 1.Correlated mRNA Expression 2.Computational Approaches 3.Phylogenetic Profiling 4.Synthetic Lethality B.QUALITATIVE 1.The Two-Hybrid Method 2.Mass Spectrometry of Affinity-Purified Complexes 3.FRET & BRET C.QUANTITATIVE 1.SPR (BIAcore) 2.AUC 3.Calorimetry

3. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Indirect detection of interactions (looking for implied functional association NOT direct interaction)

4. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching A. 1. Correlated mRNA expression

5. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching A. 2. Computational approaches e.g. “Rosetta Stone”

6. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching A. 2. Computational approaches

7. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching A. 3. Phylogenetic Profiling

8. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching A. 4. Synthetic Lethality

9. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Qualitative detection of protein-protein interactions

10. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching B. 1. The Two-Hybrid Method

11. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching B. 2. Mass Spectrometry of Affinity Purified Complexes

12. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Immunoaffinity TAP tagging 2D gel Formaldehyde crosslinking etc….. Gel MS compatible Silver stain, SYPRO stain Coomassie stain >100 fmol protein Specific Protease e.g. trypsin LC MSMS PROTEIN IDENTIFICATION Q-ToF Micro Mass Spectrometer – LC MSMS Protein Digest Nano HPLC system Nanospray Ion source Quadrupole Time-of-flight mass spectrometer Data acquisition Peptides CID Peptide fragments Peptide sequence Basic Workflow

13. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching “Mass-fingerprint” Indentification

14. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Non covalent protein complex Thiol cleavable cross-linker Covalently cross-linked complex Digest with Protease Thiol reagent MALDI MS Differential peptide mapping Non reduced Reduced Cross-linking the interaction

15. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Summary of current effort in yeast

16. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching...and the bad news

17. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching => BE WARNED! These techniques (along with e.g. Co-immuniprecipitation) give lots of false positives

18. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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

19. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Power of FRET 1.Probe macromolecular interactions Interaction assumed upon fluorescence decay 2.Study kinetics of association / dissociation between macromolecules 3.Estimation of distances (?) 4.In vitro OR on live cells 5.Single molecule studies

20. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching FRET

21. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Live cell FRET imaging Does CD4 specifically associate with the TCR/CD3 complex on triggering? Non-specific peptideSpecific peptide * marks contacts between cells. High FRET signal between CD4 and CD3 when correct antigen is present but not with non-specific antigen.

22. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching DeepBlueC hf 1 hf 2 Luciferase >10nm GFP 2 B. 3. b. BRET: Bioluminescence Resonance Energy Transfer

23. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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

24. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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

25. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching B7-1luc:B7-1YFP CTLA-4luc:CTLA-4YFP B7-1luc B7-1luc:CTLA-4YFP YFP luc B7-1YFPB7-1luc substrate hu2 (530 nm) hu1 (470 nm) e.g. B7-1 BRET

26. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Energy transfer can occur solely by random interactions e.g. BRET on B7 family

27. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Strong dimers Weak dimer Monomers Comparison to T cell surface molecules with known oligomerisation status!

28. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Specific ligand engagement can be observed when receptor is presented in solution or cell-surface bound Ligand binding causes specific increase in dimerisation

29. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Measure Quantitative Properties SPR (BIAcore) AUCITC (microcalorimetry) Surface Plasmon Resonance Analytical Ultracentrifugation Isothermal Calorimetry

30. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Measuring key properties of protein-protein interactions PropertyAUCBIAcoreCalorimetry Affinity++++ Enthalpyno+++ Entropyno+++ Heat capacityno+++ Kineticsno++no Stochiometry++++ Size & Shape+no

31. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching C. 1. SPR / BIAcore (Surface Plasmon Resonance)

32. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Advantages of SPR on the BIAcore 1.No labelling is necessary 2.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 OK 5.All types of binding data obtained – including kinetics as its real-time.

33. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Principle of Surface Plasmon Resonance Angle of ‘dip’ affected by: 1) Wavelength of light 2) Temperature 3) Refractive index n 2 Dip in light intensity

34. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Surface Plasmon Resonance in the BIAcore

35. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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

36. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Sensorgram for ligand binding

37. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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?

38. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Binding curve can be fitted with a Langmuir binding isotherm (assuming a 1:1 binding with a single affinity) 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/K d Equilibrium Binding Analysis

39. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Harder Case: 2B4 binding CD48 Kinetics

40. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Protein Problems:Aggregates (common) Concentration errors Artefacts of construct (eg Fc linked) Importance of controls:Bulk refractive index issues Control analyte Different levels of immobilisation Use both orientations (if pos.) Mass Transport:Rate of binding limited by rate of injection: k on will be underestimated Rebinding:Analyte rebinds before leaving chip k off will be underestimated Last two can be spotted if measured k on and k off vary with immobilisation level (hence importance of controls) Potential pitfalls

41. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 1. Temperature dependence of binding van’t Hoff analysis: Gradient Intercept Less common applications

42. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 1. Temperature dependence of binding Non-linear van’t Hoff analysis: Less common applications

43. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 2. Combination with mutagenesis Q30RQ40KR87A Binding of CD2 by CD48 mutants at 25°C (WT K d = 40  M) Less common applications Reduce / abolish binding Do not affect binding Not tested

44. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 3. Estimation of valency Less common applications

45. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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

46. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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

47. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 2. AUC (Analytical Ultracentrifugation)

48. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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)F C : the centrifugal force b)F B : the buoyant force (Archimedes principle) c)F f : the frictional force between the particle and the liquid 3.Will reach constant velocity where forces balance:

49. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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 M r ) Values for most biomolecules between 1 and 10000 S Theory: The Svedberg equation

50. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching S = D = diffusion coefficient, N = Avogadro’s number or Therefore can directly determine M r 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 M r = Nm 0 ) Theory: The Svedberg equation

51. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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.

52. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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

53. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Equilibrium sedimentation 1.Moderate centrifuge speed 2.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

54. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Data modeling 1.A plot of ln(c) vs r 2 should be a straight line with a slope proportional to molecular weight Single ideal homogeneous species M p (1-  ) = d ln(c) 2RT d r 2  2

55. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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

56. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Protein concentration (mg/ml) 6 5 4 3 2 01.02.0 M w,app (Da/10 4 ) sB7-1 B7-1 : an equilibrium dimer

57. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching sB7-2 sLICOS Concentration (mg/ml) M w (kDa) 0123401234 80 60 40 20 0 80 60 40 20 0 B7-2 and LICOS are monomeric

58. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Velocity sedimentation High centrifuge speed Forms a sharp boundary between solute depleted region (at top) and a region of uniform solute conc n (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

59. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching g(s*) distribution Velocity sedimentation - data analysis

60. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching The example of SLAM (CD150) 1.Claimed to self-associate with nM K d raising serious problems for models of cell surface protein interactions 2.Equilibrium data can’t be fitted – high concentrations! 3.Velocity data confirmed shape of complex and approximate strength of association

61. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 3. ITC (Isothermal Titration Calorimetry)

62. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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.

63. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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

64. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching ITC Data Analysis Get 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 = conc n / K d

65. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Data Analysis – e.g. of B7-1 & CTLA-4 01234 -12 -8 -4 0 kcal/mole of injectant molar ratio  H = -11.6  G = -8.9 T  S = -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 calculateS (entropy).

66. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching 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 (C p )

67. edward.evans@ndm.ox.ac.ukwww.t-cellbiology.org/teaching Studying Protein-Protein Interactions A.INDIRECT 1.Correlated mRNA Expression 2.Computational Approaches 3.Phylogenetic Profiling 4.Synthetic Lethality B.QUALITATIVE 1.The Two-Hybrid Method 2.Mass Spectrometry of Affinity-Purified Complexes 3.FRET & BRET C.QUANTITATIVE 1.SPR (BIAcore) 2.AUC 3.Calorimetry Bulk screening e.g. For database NEED TESTING AFTERWARDS When looking for/at a (or a few) specific interactions