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Forty Years of Crystallographic Studies
of Protein Structure, Function and
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
Tom Alber
Tom Alber
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
Thermodynamic Trapping: Slowing
Reactions By Cooling
Nature 263, 297-300 (1976)
In The Good Old Days…
(1,000 reflections/day – one at a time)
Direct Binding Kinetics By Crystallography
Elastase + ZAP at 3.5A Resolution, Tbind=-35oC
Elastase + ZAP at 3.5A Resolution, Tbind=-35oC
Elastase Active Site Before Adding
Substrate, data collected at -65oC
Elastase Active Site After Adding Substrate at -65oC and
Collecting Data at -65oC
Tony Fink
Hans Frauenfelder
Demetrius Tsernoglou
Martin Karplus
Dagmar Ringe
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
Low Temperature Studies of Protein
Nature 280, 558-563 (1979)
Low Temperature Studies of Protein
Biochemistry 26, 254-261 (1987)
On heating from 80 to 300 K, the volume occupied by myoglobin increases
by approximately 3%.
Low Temperature Studies of Protein
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)
By Early 1990s It Became Apparent That...
Nature 357, 423-424 (1992)
Thermodynamic Trapping: Exploiting the
“Glass Transition” at ~220K
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
Substrates Used
Elastase + ZAP Tbind = -26oC and Tdata = -55oC
Elastase + (Ala)3 Tbind = -26oC and Tdata = -50oC
• Direct observation of E-S complexes at
• Kinetic trapping: establishment of a
pseudo steady-state
• Thermodynamic (freeze)-trapping
• True time-resolved observation by fast
data collection following triggering
Kinetic Trapping: Establishment of a
Pseudo Steady-state with a Flow Cell
Rapid Data Collection: The Laue Method
Nature 345, 309-315 (1990)