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Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved X-ray Diffraction and Solution Scattering

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Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved X-ray Diffraction and Solution Scattering


P. Anfinrud1*, F. Schotte1, H.S. Cho1

1National Institutes of Health, Laboratory of Chemical Physics, Bethesda, MD, USA

To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in proteins with 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore. Briefly, a picosecond laser pulse photoexcites pCA and triggers a structural change in the protein, which is probed with a suitably delayed picosecond X-ray pulse. When the protein is studied in a crystalline state, this “pump-probe” approach recovers time-resolved diffraction “snapshots” whose corresponding electron density maps can be stitched together into a real-time movie of the structural changes that ensue [1]. However, the actual signaling state is not accessible in the crystalline state due to crystal packing constraints. This state is accessible in time-resolved small- and wide-angle X-ray scattering studies, which probe changes in the size, shape, and structure of the protein [2]. The mechanistically detailed, near-atomic resolution description of the complete PYP photocycle developed from these studies provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics [3]. This research was supported in part by the Intramural Research Program of the NIH, NIDDK.

References:

[1] F. Schotte, H.S. Cho, V.R. Kaila, H. Kamikubo, N. Dashdorj, E.R. Henry, T. Graber, R. Henning, M. Wulff, G. Hummer, P.A. Anfinrud Proc. Natl. Acad. Sci. U.S.A. 109, 19256 (2012).

[2] H.S. Cho, F. Schotte, N. Dashdorj, J. Kyndt, and P.A. Anfinrud, J. Phys. Chem. B, 117, 9161 (2013).

[3] V.R.I. Kaila, F. Schotte, H.S. Cho, G. Hummer, and P.A. Anfinrud, Nature Chemistry, 6, 258 (2014).