Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

Abstract

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 10 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. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography 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.time-resolved X-ray diffraction | photoreceptor | light sensor

Populations and coherence in femtosecond time resolved X-ray crystallography of the photoactive yellow protein

“…In contrast, Schotte et al [2] and Kaila et al [10] argue on the basis of DFT calculations that the structure should be closer to having a 33 degree dihedral angle.…”

“…Based on very similar data, different interpretations have also been argued, in particular with regard to the chromophore conformation of the early, 100 ps, PYP structure, but also to propose different reaction schemes for nanosecond and microsecond intermediates [1,2]. Regarding the early, sub-nanosecond, chromophore structure, discussion has focussed on the chromophore dihedral angle C1-C2-C3-C1'.…”

“…Using synchrotron sources, a time resolution for TR-Laue of up to 100 ps is possible, and several groups have reported on sub-nanosecond measurements and slower time scales [1,2].…”

“…Regarding the early, sub-nanosecond, chromophore structure, discussion has focussed on the chromophore dihedral angle C1-C2-C3-C1'. In one study, this dihedral angle was found by X-ray structural refinement with application of Density Functional Theory (DFT) derived structural restraints to have a value of 33 degrees in the 'pR0' structure at 100 ps (4B9O pdb [2]) Jung et al used structure refinement avoiding additional restraints to arrive at a structure with a 90 degree dihedral angle for the 'IT' state (4I38 pdb [1]) (corresponding to the 'pR0' in 4B9O pdb [9]). …”

“…The Photoactive Yellow Protein (PYP) was one of the first light-sensitive proteins to be used for time-resolved pump-probe X-ray crystallography, initially by the TR-Laue method [1][2][3][4][5][6][7].…”