Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 33, Pages 11742-11747Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0804828105
Keywords
acetylcholinesterase; kinetic crystallography; structure-dynamics-function relationships; energy landscape; synchrotron radiation
Categories
Funding
- Commissariat l'Energie Atomique
- Centre National de la Recherche Scientifique
- Universite Joseph Fourier, Agence Nationale de la Recherche [JC05_45685]
- National Institutes of Health CounterACT Program
- U.S. Army Defense Threat Reduction Agency
- Nalvyco Foundation
- Kimmelman Center for Biomolecular Structure and Assembly (Rehovot, Israel)
- Minerva Foundation
- Benoziyo Center for Neuroscience
- Israel Ministry of Science, Culture, and Sport (the Israel Structural Proteomics Center)
- Divadol Foundation
- European Commission VIth Framework SPINE2-COMPLEXES [LSHG-CT-2006-031220]
- Universite Joseph Fourier
- European Molecular Biology Organization [ASTF230-2006]
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Although x-ray crystallography is the most widely used method for macromolecular structure determination, it does not provide dynamical information, and either experimental tricks or complementary experiments must be used to overcome the inherently static nature of crystallographic structures. Here we used specific x-ray damage during temperature-controlled crystallographic experiments at a third-generation synchrotron source to trigger and monitor (Shoot-and-Trap) structural changes putatively involved in an enzymatic reaction. In particular, a nonhydrolyzable substrate analogue of acetylcholinesterase, the off-switch at cholinergic synapses, was radiocleaved within the buried enzymatic active site. Subsequent product clearance, observed at 150 K but not at 100 K, indicated exit from the active site possibly via a backdoor. The simple strategy described here is, in principle, applicable to any enzyme whose structure in complex with a substrate analogue is available and, therefore, could serve as a standard procedure in kinetic crystallography studies.
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