Protein kinetics: structures of intermediates and reaction mechanism from time-resolved x-ray data |
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Authors: | Schmidt Marius Pahl Reinhard Srajer Vukica Anderson Spencer Ren Zhong Ihee Hyotcherl Rajagopal Sudarshan Moffat Keith |
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Affiliation: | Physikdepartment E17, Technische Universit?t München, 85747 Garching, Germany. marius@hexa.e17.physik.tu-muenchen.de |
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Abstract: | 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 micros 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. |
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