Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds.
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.
Ihee
Hyotcherl
H
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
Rajagopal
Sudarshan
S
Srajer
Vukica
V
Pahl
Reinhard
R
Anderson
Spencer
S
Schmidt
Marius
M
Schotte
Friedrich
F
Anfinrud
Philip A
PA
Wulff
Michael
M
Moffat
Keith
K
D001426:Bacterial Proteins / Q000378:metabolism* / Q000502:physiology; D003373:Coumaric Acids / Q000378:metabolism; D003461:Crystallography; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D017785:Photons; D020283:Photoreceptors, Microbial / Q000378:metabolism* / Q000502:physiology; D011422:Propionates; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors
D001426:Bacterial Proteins; D003373:Coumaric Acids; D020283:Photoreceptors, Microbial; D011422:Propionates; C050162:photoactive yellow protein, Bacteria; C495469:p-coumaric acid