Protein Kinetics: Structures of Intermediates and Reaction Mechanism from Time-Resolved X-Ray Data
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 s...
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Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2004-04, Vol.101 (14), p.4799-4804 |
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Sprache: | eng |
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Zusammenfassung: | 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. |
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ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.0305983101 |