Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-ray Scattering.
The capacity to respond to environmental changes is crucial to an organism's survival. Halorhodospira halophila is a photosynthetic bacterium that swims away from blue light, presumably in an effort to evade photons energetic enough to be genetically harmful. The protein responsible for this response is believed to be photoactive yellow protein (PYP), whose chromophore photoisomerizes from trans to cis in the presence of blue light. We investigated the complete PYP photocycle by acquiring time-resolved small and wide-angle X-ray scattering patterns (SAXS/WAXS) over 10 decades of time spanning from 100 ps to 1 s. Using a sequential model, global analysis of the time-dependent scattering differences recovered four intermediates (pR0/pR1, pR2, pB0, pB1), the first three of which can be assigned to prior time-resolved crystal structures. The 1.8 ms pB0 to pB1 transition produces the PYP signaling state, whose radius of gyration (Rg = 16.6 Å) is significantly larger than that for the ground state (Rg = 14.7 Å) and is therefore inaccessible to time-resolved protein crystallography. The shape of the signaling state, reconstructed using GASBOR, is highly anisotropic and entails significant elongation of the long axis of the protein. This structural change is consistent with unfolding of the 25 residue N-terminal domain, which exposes the β-scaffold of this sensory protein to a potential binding partner. This mechanistically detailed description of the complete PYP photocycle, made possible by time-resolved crystal and solution studies, provides a framework for understanding signal transduction in proteins and for assessing and validating theoretical/computational approaches in protein biophysics.
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892, United States.
Hyun Sun
HS
Cho
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892, United States.
Friedrich
F
Schotte
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892, United States.
Naranbaatar
N
Dashdorj
College of Science and Technology, Bellevue University , Bellevue, Nebraska 68005, United States.
John
J
Kyndt
Center for Advanced Radiation Sources, University of Chicago , Chicago, Illinois 60637, United States.
Robert
R
Henning
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892, United States.
Philip A
PA
Anfinrud
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D020607:Halorhodospira halophila; D008958:Models, Molecular; D018462:Photobiology; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D053838:Scattering, Small Angle; D015398:Signal Transduction; D013237:Stereoisomerism; D013997:Time Factors; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria