Watching a Signaling Protein Function in Real Time via Picosecond Time-Resolved Laue Crystallography

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 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans to cisphotoisomerization of its p-coumaric acid (pCA) chromophore over ten decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90° out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. This novel structure, which corresponds to a strained cis intermediate, is short lived (~600 ps), has not been observed in prior cryo-crystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins and for assessing and validating theoretical/computational approaches in protein biophysics.

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Schotte, F., Cho, H. S., Kamikubo, H., Kataoka, M., and Anfinrud, P. A.
Watching a Signaling Protein Function in Real Time via Picosecond Time-Resolved Laue Crystallography
In Molecular Science of Fluctuations Toward Biological Functions (Terazima, M., Kataoka, M., Ueoka, R., and Okamoto, Y., Eds.),
pp 65–85. Springer Japan (2015)

Pump-probe geometry employed to acquire time-resolved diffraction snapshots. The PYP crystal is sealed in a thin-walled glass capillary. Because the laser penetration depth in PYP is shallow, an orthogonal pump-probe geometry is employed in which the top edge of the protein crystal is positioned at the top edge of the focused x-ray pulse. This geometry ensures optimal overlap between the laser and x-ray-illuminated volumes of the crystal. The protein crystal acts as a monochromator with various line spacings (d) and diffracts different x-ray colors (λ) in different directions (θ) according to Bragg’s law (λ¼2d sinθ). Approximately 3000 spots are found in each time-resolved diffraction image. The spots in this figure are annotated according to integrated photons (spot dimension) and x-ray wavelength (spot color)