Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 102, Issue 20, Pages 7145-7150Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0409035102
Keywords
intermediates; mechanism; signal transduction; time-resolved crystallography; singular value decomposition
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Funding
- NCRR NIH HHS [RR07707, P41 RR007707] Funding Source: Medline
- NIGMS NIH HHS [R37 GM036452, R01 GM036452, GM36452] Funding Source: Medline
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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 timer-esolved 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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