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Forty Years of Crystallographic Studies of Protein Structure, Function and Dynamics or

Forty Years of Crystallographic Studies of Protein Structure, Function and Dynamics or Some Like It Cold - But Should They? Buerger Award Lecture 2015 Gregory A. Petsko Weill Cornell Medical College, New York, NY. Lysozyme (1965). David Phillips Louise Johnson. Tom Alber.

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Forty Years of Crystallographic Studies of Protein Structure, Function and Dynamics or

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  1. Forty Years of Crystallographic Studies of Protein Structure, Function and Dynamics or Some Like It Cold - But Should They? Buerger Award Lecture 2015 Gregory A. Petsko Weill Cornell Medical College, New York, NY

  2. Lysozyme (1965) David Phillips Louise Johnson

  3. Tom Alber 1975

  4. Tom Alber 1975

  5. Tom Alber 1975 2010

  6. Tom Alber: A large number of firsts First direct observation of the structure of an enzyme intermediate* First trapping of an unstable species by cryo-crystallography* First observation of the effect of the glass transition on binding* First direct observation of a Michaelis complex at equilibrium First proposal of “promoting vibrations” in enzyme catalysis First demonstration of the skeleton in the closet of structural biology: the possible perturbing effects of cryo temperatures on protein structure, dynamics and electronic state * Done when he was still an undergraduate

  7. Thermodynamic Trapping: Slowing Reactions By Cooling

  8. Nature 263, 297-300 (1976)

  9. In The Good Old Days…(1,000 reflections/day – one at a time)

  10. Direct Binding Kinetics By Crystallography

  11. Elastase + ZAP at 3.5A Resolution, Tbind=-35oC

  12. Elastase + ZAP at 3.5A Resolution, Tbind=-35oC

  13. Elastase Active Site Before Adding Substrate, data collected at -65oC

  14. Elastase Active Site After Adding Substrate at -65oC and Collecting Data at -65oC

  15. Tony Fink Hans Frauenfelder Martin Karplus Demetrius Tsernoglou

  16. Dagmar Ringe

  17. Dagmar Ringe: A Large Number of Firsts First flash-cooling of a protein crystal (1982) Discovery of first ligand path through a protein (1984) First measurement of thermal expansion of a protein (1987) Invention of fragment-based drug discovery (1992) Mechanism of iron-dependent control of transcription (1995) Structures and mechanisms of all families of PLP enzymes Characterization of role of water in substrate/drug binding

  18. Low Temperature Studies of Protein Dynamics Nature 280, 558-563 (1979)

  19. Low Temperature Studies of Protein Dynamics Biochemistry 26, 254-261 (1987) On heating from 80 to 300 K, the volume occupied by myoglobin increases by approximately 3%.

  20. Low Temperature Studies of Protein Dynamics Effects of Temperature on Protein Structure and Dynamics: X-ray Crystallographic Studies of the Protein Ribonuclease-A at Nine Different Temperatures from 98 to 320 K Robert F. Tilton, Jr.*,John C . Dewan, and Gregory A. Petsko Biochemistry 31, 2469-2481 (1992)

  21. By Early 1990s It Became Apparent That...

  22. Nature 357, 423-424 (1992)

  23. Thermodynamic Trapping: Exploiting the “Glass Transition” at ~220K

  24. Elastase Experiments Were Done on Either Side (Tbind= -35oC and Tbind= -65oC) of the Glass Transition Rasmussen BF, Stock AM, Ringe D & Petsko, GA. Nature 357:423-424 (1992).

  25. Substrates Used

  26. Elastase + ZAP Tbind= -26oC and Tdata = -55oC

  27. Elastase + (Ala)3Tbind= -26oC and Tdata= -50oC

  28. Strategies • Direct observation of E-S complexes at equilibrium • Kinetic trapping: establishment of a pseudo steady-state • Thermodynamic (freeze)-trapping • True time-resolved observation by fast data collection following triggering

  29. Kinetic Trapping: Establishment of a Pseudo Steady-state with a Flow Cell

  30. Rapid Data Collection: The Laue Method

  31. Nature 345, 309-315 (1990)

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