Watching a Signaling Protein Function in Real Time Via 100-Ps 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 cis photoisomerization 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.