1. Kinetics of Protein-Protein Interactions November 2002

2. 2Content n Preview – Basic kinetics n Protein-protein Kinetics – Basic view n Electrostatic steering – study review

3. 3 1.Preview – Basic kinetics Reaction Rate (V) – Change of concentration over time n Basic Reaction A C A C Rate slows as concentration concentration of A decreases Rate slows as concentration concentration of A decreases

4. 4 Reaction Rate constant n A + B C V = K [A] [B] V = K [A] [B] Rate (at first stage of reaction) Rate (at first stage of reaction) Kinetic Constant ConstantKinetic Rate is dependant on preliminary concentration of reactants preliminary concentration of reactants Rate is dependant on preliminary concentration of reactants preliminary concentration of reactants n Example 1: H 2 + F 2 2HI H 2 + F 2 2HI V = K [F 2 ] [H 2 ] V = K [F 2 ] [H 2 ] (K1 – Slow) F 2 + NO 2 NO 2 F + F Fast Equilibrium NO 2 + F NO 2 F Would expect [NO 2 ][NO 2 ] [NO 2 ][NO 2 ] Would expect [NO 2 ][NO 2 ] [NO 2 ][NO 2 ] It appears things are not that simple: MechanismMechanism Different K ’ sDifferent K ’ s It appears things are not that simple: MechanismMechanism Different K ’ sDifferent K ’ s K1 n Example 2: F 2 + 2NO 2 2NO 2 F F 2 + 2NO 2 2NO 2 F V = K [F 2 ] [NO 2 ] V = K [F 2 ] [NO 2 ]

5. 5 Constant: SizeSize OrientationOrientation SolventSolvent electrostaticselectrostaticsConstant: SizeSize OrientationOrientation SolventSolvent electrostaticselectrostatics Activation Energy – Limiting Barrier Activation Energy – Limiting Barrier n Arrhenius Factors Influencing K

6. 6 2.Protein-Protein Kinetics – Basic View n Kd= Kdissociation / Kassociation (dissociation=off, association=on) n ΔG = -RTln(Kd) A B A B n Physiological conditions z Possible concentration of a unique Protein in a cell 10^-6 – 10^-8 M z Protein diameter 50 – 100 A (Protein surface ~8,000 A) z Free Walk collision with interacting designated protein ~ 1000A – 2000A A 1000 – 2000A A A A B B B B

7. 7 A more elaborate representation Diffusion + Possible Steering Desolvation, VDW, Electrostatics Intermediate Transition-State Random Diffusion Electrostatic Steering Encounter Complex Final Complex Transition A B A + - B +- A B A B A B A B Intermediate ???√√√

8. 8 Reaching the Encounter Complex n Random diffusion according to the Smolochowski-Einstein equation - ~ 10^9 - 10^10 1/MS n With geometrical constraints - ~ 10^5 – 10^6 1/MS n Adding electrostatic steering could enhance rate to 10^9 1/MS A B A + - B +- A B Random Diffusion Electrostatic Steering Encounter Complex ??√ n Energetic factors: ∆S Electrostatic AttractionAttractionSteeringSteering

9. 9 An example of electrostatic steering

10. 10 Barnase-Barstar Electrostatic potential Landscape

11. 11 3.Evaluation of steering effect (Camacho, Vajda) A. Chymotrypsin with turkey ovomucoid third domain (1CHO); B. human leukocyte elastase with turkey ovomucoid third domain (1PPF), ionic strength 0.15 M and protein dielectric 4; C. kallikrein A and pancreatic trypsin inhibitor (2KAI), ionic strength 0.15 M and protein dielectric 4; D. barnase and barstar (1BGS); E. subtilisin and chymotrypsin inhibitor (2SNI); F. subtilisin and eglin-c (1CSE), ionic strength 0.15 M and protein dielectric 4; G. trypsin and bovine pancreatic trypsin inhibitor (2PTC). A. Chymotrypsin with turkey ovomucoid third domain (1CHO); B. human leukocyte elastase with turkey ovomucoid third domain (1PPF), ionic strength 0.15 M and protein dielectric 4; C. kallikrein A and pancreatic trypsin inhibitor (2KAI), ionic strength 0.15 M and protein dielectric 4; D. barnase and barstar (1BGS); E. subtilisin and chymotrypsin inhibitor (2SNI); F. subtilisin and eglin-c (1CSE), ionic strength 0.15 M and protein dielectric 4; G. trypsin and bovine pancreatic trypsin inhibitor (2PTC). Complex separation (5A) + XY rotation

12. 12 Evaluation of steering effect (Wade) ccp:cc - cytochrome c peroxidase:cytochrome c ache:fas - acetylcholinesterase: fasciculin-2 Bn:bs - Barnase-Barstar hyhel5:hel - HyHEL-5 antibody: hen egg white lysozyme; hyhel10:hel - HyHEL-10 antibody:hen egg white lysozyme ccp:cc - cytochrome c peroxidase:cytochrome c ache:fas - acetylcholinesterase: fasciculin-2 Bn:bs - Barnase-Barstar hyhel5:hel - HyHEL-5 antibody: hen egg white lysozyme; hyhel10:hel - HyHEL-10 antibody:hen egg white lysozyme

13. 13 Evaluation of steering effect (Wade)

14. 14 Evaluation of steering effect (Wade)

15. 15 Structure – Tem1 β Lactamase

16. 16 Structure – Tem1 β Lactamase

17. 17 Structure – BLIP-ΙΙ

18. 18 Bound – Blip-ΙΙ & TEM1

19. 19 Bound – Blip-ΙΙ & TEM1

20. 20 Mutations on BLIP outside the active site

21. 21Results

22. 22Results

23. 23 Possible Transition state orientation n Still water molecules awaiting extraction n Possibly a core of atoms in proximity with final orientation

24. 24 Encounter complex modeling n Bound Model n Camacho/wade – Electrostatic minima Barnase - Barstar

25. 25 Encounter complex modeling n Bound Model n Janin – 50% surface area + rotational limit Barnase - Barstar

26. 26 Encounter complex modeling n Bound Model n Vijayakumar – solvent separation + (2 angles – 3dg limit) Barnase - Barstar