Volume-conserving trans–cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography

Abstract

Trans-to-cis isomerization, the key reaction in photoactive proteins, cannot usually occur through the standard one-bond-flip mechanism. Due to spatial constraints imposed by a protein environment, isomerization is likely to proceed via a “volume-conserving” mechanism in which highly-choreographed atomic motions are expected, the details of which have not yet been directly observed. Here we employ time-resolved X-ray crystallography to structurally visualize isomerization of the p-coumaric acid chromophore in photoactive yellow protein with 100 picosecond time resolution and 1.6 Å spatial resolution. The structure of the earliest intermediate (IT) resembles a highly-strained transition state in which the torsion angle is located halfway between the trans and cis isomers. The reaction trajectory of IT bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue via E46Q mutation, which switches off the bicycle-pedal pathway.

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

“…A second fundamental difference between both papers concerns the bifurcation which is proposed by Jung et al from the first IT state to form the 'pR1 and 'ITC' states with 3 ns and 1.7 ns, respectively (Jung et al [1], figure 1a). The ITC state further decays in 20 ns to form a pR2 state, such that delays from 50 ns typically show a product state that is a mixture of pR1 and pR2 where the phenol rings have different orientations (the 'pR1' species corresponding to the phenol position that is eventually developed in the long-lived millisecond intermediate 'pB').…”

“…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].…”