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BIOPHYSICS
Protein kinetics: Structures of intermediates and reaction mechanism from time-resolved x-ray data
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*Physikdepartment E17, Technische Universität München, 85747 Garching, Germany; Department of Biochemistry and Molecular Biology, Consortium for Advanced Radiation Sources, and **Institute of Biophysical Dynamics, University of Chicago, Chicago, IL 60637; ¶Renz Research, Des Plaines, IL 60018; and ||Department of Chemistry and School of Molecular Science (BK21), Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea
Edited by Johann Deisenhofer, University of Texas Southwestern Medical Center, Dallas, TX, and approved January 23, 2004 (received for review September 17, 2003)
We determine the number of authentic reaction intermediates in the later stages of the photocycle of photoactive yellow protein at room temperature, their atomic structures, and a consistent set of chemical kinetic mechanisms, by analysis of a set of time-dependent difference electron density maps spanning the time range from 5 µs to 100 ms. The successful fit of exponentials to right singular vectors derived from a singular value decomposition of the difference maps demonstrates that a chemical kinetic mechanism holds and that structurally distinct intermediates exist. We identify two time-independent difference maps, from which we refine the structures of the corresponding intermediates. We thus demonstrate how structures associated with intermediate states can be extracted from the experimental, time-dependent crystallographic data. Stoichiometric and structural constraints allow the exclusion of one kinetic mechanism proposed for the photocycle but retain other plausible candidate kinetic mechanisms.
chemical, kinetic mechanism | time-resolved crystallography
Abbreviations: PYP, photoactive yellow protein; SV, singular value; SVD, SV decomposition; rSV, right singular vector; lSV, left SV; IS, intermediate state.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1S4S
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To whom correspondence should be addressed at: Physikdepartment E17, Technische Universität München, James Franck Strasse, 85747 Garching, Germany. E-mail: marius{at}hexa.e17.physik.tu-muenchen.de. 
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