1. 125:583 Biointerfacial Characterization: Protein-Protein Interactions October 30, 2006 Gary Brewer, Ph.D. Dept. of Molecular Genetics, Microbiology & Immunology UMDNJ-RWJMS

2. The flow of “omics” research Genome ↓ Transcriptome ↓ Proteome ↓ Interactome

3. The interactome Proteins rarely (if ever) act alone Components of biomolecular machines Estimate: average of 5 interacting partners per protein For examples of interactomes, see http://bond.unleashedinformatics.com/Action (Must register for free account) http://bond.unleashedinformatics.com/Action

4. Identification and characterization: A formidable problem Proteins have very diverse physiochemical properties Equilibrium dissociation constants can vary over several orders of magnitude Proteins vary in abundance and intracellular localization Differing conditions for purifying individual proteins.

5. Two major considerations relating to protein-protein complexes Have to identify protein-protein interactions Characterize the molecular and biophysical interactions between proteins

6. Molecular and biophysical characterization of complexes: additional considerations Oligomeric state of interacting proteins Stoichiometry of the complex Affinity of interacting partners for each other In vitro analyses require large amounts of pure proteins

7. The two general themes of interest Methods for identification of novel protein- protein interactions (“molecular biology” – we’ll touch on this) Methods for analyses of protein-protein interactions (“cell biology” and “physical biochemistry” – major focus of the lecture)

8. Methods for identification of novel protein-protein interactions

9. Identifying novel protein-protein interactions: tandem affinity purification

10. Large-scale identification of protein- protein complexes Gavin et al. (Nature 415: 141-147, 2002) performed massive TAP strategy using yeast S. cerevisiae Tag one component of 200 different complexes, transformation, perform TAP, identify subunits by mass spectrometry

11. Strategy of Gavin et al. Gavin et al., Nature 415:141-147

12. A partial interactome of S. cerevisiae

13. Methods for analyses of protein-protein interactions: in vitro approaches

14. Surface plasmon resonance (SPR) http://www.astbury.leeds.ac.uk/Facil/SPR/spr_intro2004.htm SPR occurs when light is reflected off thin metal film Fraction of light energy interacts with delocalized electrons in metal and angle  at which this occurs is determined by refractive index on backside of film Molecule binding to surface changes refractive index, leading to change in . Monitored in real time as changes in reflected light intensity to produce a sensorgram

15. Advantages of SPR No labeling of interacting proteins required (can even use cell extracts) Interactions detected in real time Both equilibrium and interaction kinetics can be analyzed But.....one protein must be tethered to the surface

16. Example of SPR: SDF-1 binding to chemokine receptor CXCR4 Stenlund et al. Anal. Biochem. 316: 243-250, 2003

17. Lipid bilayer? yes no

18. Stenlund et al. Anal. Biochem. 316: 243-250, 2003

19. Methods for analyses of protein-protein interactions: in vivo approaches

20. Fluorescence correlation spectroscopy (FCS) G(  ), is a measure of the self-similarity of the signal after a lag time (  ). It resembles the conditional probability of finding a molecule in the focal volume at a later time.

21. Dual color cross-correlation: More effective for protein- protein interaction studies Cross-correlation amplitude is proportional to number of double- labeled molecules Suited to monitoring association and dissociation reactions

22. An example application of FCCS Endocytic pathway: cholera toxin (CTX) CTX has AB 5 subunit structure “B” subunit required for membrane binding and cellular uptake “A” subunit has enzymatic activity (elevates cAMP) → massive efflux of Na + and water Do subunits remain associated throughout vesicular transport?

23. Flow chart of the experiment Label A and B subunits with indocarbo- cyanine dyes Cy3 and Cy5. Double-labeled CTX added to cells Wash away excess toxin Perform FCCS at successive time points and different positions in same cell At what stage in endocytic pathway do “A” and “B” subunits diverge?

24. Bacia et al. Biophys. J. 83: 1184-1193, 2002 FCCS analyses during the first minute

25. Bacia et al. Biophys. J. 83: 1184-1193, 2002 FCCS analyses after 15 minutes

26. Conclusions: endocytosis of CTX, codiffusion in endocytic vesicles, and separation of “A” and “B” subunits within the Golgi apparatus Note: FRET was not suitable here, since the molecules are not necessarily within the “close proximity” required for FRET. Bacia et al. Biophys. J. 83: 1184-1193, 2002 And at yet later times…..

27. A brief review of photophysics: Jablonski Diagram Photoexcitation from the ground state S 0 creates excited states S 1, (S 2, …, S n ) Kasha’s rule: Rapid relaxation from excited electronic and vibrational states precedes nearly all fluorescence emission Internal Conversion: Molecules rapidly (10 -14 to 10 -11 s) relax to the lowest vibrational level of S 1. Intersystem crossing: Molecules in S 1 state can also convert to first triplet state T 1 ; emission from T 1 is termed phosphorescence, shifting to longer wavelengths (lower energy) than fluorescence. Transition from S 1 to T 1 is called intersystem crossing.

28. What is FRET? Fluorescence Resonance Energy Transfer Initial energy absoption (excitation) Loss of some energy (vibration, etc.) Nonradiative movement of energy to second molecule (resonance transfer) Loss of some more energy (vibration, etc.) Bolus release of remaining energy (emission)

29. Initial Energy Absorption donor donor + h Single-photon Fluorescence Excitation h donor +  h Vibrational loss Loss of Some Energy donor + h  represents the fraction of energy that is not rapidly lost, and is equivalent to the quantum yield

30. Transfer of Energy to Second Molecule donor +  h donor Fluorescence Emission  h Transfer Efficiency: how much energy is sent to a second molecule instead of retained and emitted donor acceptor +  h acceptor  h

31. Loss of Some More Energy Fluorescence Emission  h acceptor +  h Release of Remaining Energy Vibrational loss acceptor +  h acceptor +  h acceptor Fluorescence Quenching Complete Dissipation acceptor +  h acceptor  represents the quantum yield of the acceptor

32. Energy Distributions During FRET donor + E T donor Donor Fluorescence Emission (F DA ) E DA donor acceptor + E DA’ acceptor E DA’ Vibrational loss acceptor +  E DA’  E DA’ = E AD acceptor Vibrational loss Acceptor Fluorescence Emission (F AD ) Directly determined: F DA = donor fluorescence in presence of acceptor F AD = acceptor fluorescence in presence of donor  = quantum fluorescent yield of acceptor Calculated: E DA  F DA = energy released by donor E AD  F AD = energy released by acceptor E DA’ = E AD /  = donor energy absorbed by acceptor E T = E DA’ +E DA = total energy released by donor

33. A cartoon explanation of FRET Example: Two membrane- associated proteins. Do they form protein-protein contacts? One fused to CFP, the other to GFP FRET efficiency is a function of scalar distance apart

34. FRET: Single-molecule imaging of Ras activation in living cells Murakoshi et al. PNAS 101: 7317-7322, 2004

36. Total internal reflection fluorescence Mashanov et al. Methods 29: 142-152, 2003 TIRF occurs when light traveling from high- to low-refractive index medium strikes interface at an angle >  c (e.g., ~65° for glass-cytoplasm interface) High-numerical-aperture lens → light at periphery approaches specimen at  i >  c causing total internal reflection Permits single-molecule imaging of cell surface phenomena with unparalleled resolution high low

37. An example application of TIRFM Early events in signal transduction Binding of epidermal growth factor (EGF) to its receptor (EGFR) EGF binds EGFR: EGF-(EGFR) 2 + EGF → (EGF-EGFR) 2 or 2(EGF-EGFR) → (EGF-EGFR) 2 ?? Autophosphorylation of EGFR →→→ cell division Examined Cy3-EGF binding to EGFR by TIRFM

38. The experimental set up of Sako et al. Sako et al. Nature Cell Biol. 2: 168-172, 2000

39. Their data favor the following binding mechanism: EGF-(EGFR) 2 + EGF → (EGF-EGFR) 2