Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds |
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Authors: | Ihee Hyotcherl Rajagopal Sudarshan Srajer Vukica Pahl Reinhard Anderson Spencer Schmidt Marius Schotte Friedrich Anfinrud Philip A Wulff Michael Moffat Keith |
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Affiliation: | Department of Chemistry and School of Molecular Science (BK21), Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, South Korea. hyotcherl.ihee@kaist.ac.kr |
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Abstract: | Determining 3D intermediate structures during the biological action of proteins in real time under ambient conditions is essential for understanding how proteins function. Here we use time-resolved Laue crystallography to extract short-lived intermediate structures and thereby unveil signal transduction in the blue light photoreceptor photoactive yellow protein (PYP) from Halorhodospira halophila. By analyzing a comprehensive set of Laue data during the PYP photocycle (forty-seven time points from one nanosecond to one second), we track all atoms in PYP during its photocycle and directly observe how absorption of a blue light photon by its p-coumaric acid chromophore triggers a reversible photocycle. We identify a complex chemical mechanism characterized by five distinct structural intermediates. Structural changes at the chromophore in the early, red-shifted intermediates are transduced to the exterior of the protein in the late, blue-shifted intermediates through an initial "volume-conserving" isomerization of the chromophore and the progressive disruption of hydrogen bonds between the chromophore and its surrounding binding pocket. These results yield a comprehensive view of the PYP photocycle when seen in the light of previous biophysical studies on the system. |
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Keywords: | intermediates mechanism signal transduction time-resolved crystallography singular value decomposition |
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