Trans-to-cis isomerization, the key reaction in photoactive proteins, usually cannot occur through the standard one-bond-flip mechanism. Owing to spatial constraints imposed by a protein environment, isomerization probably proceeds through a volume-conserving mechanism in which highly choreographed atomic motions are expected, the details of which have not yet been observed directly. Here we employ time-resolved X-ray crystallography to visualize structurally the isomerization of the p-coumaric acid chromophore in photoactive yellow protein with a time resolution of 100 ps and a spatial resolution of 1.6 Å. 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 through E46Q mutation, which switches off the bicycle-pedal pathway.
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We acknowledge extensive support from M. Wulff of beamline ID09 at ESRF during data collection there. We thank M. Wulff, P. Anfinrud, F. Schotte and H. S. Cho for their earlier contributions to this research. This work was supported by the Research Center Program (CA1201) of Institute for Basic Science (IBS) in Korea and by Creative Research Initiatives (Center for Time-Resolved Diffraction) of MEST/NRF of Korea. M.S. is supported by National Science Foundation grants 0952643 (Career) and 0843459. K.M. is supported by National Institutes of Health (NIH) grant GM036452. Use of the BioCARS Sector 14 at the APS was supported by NIH National Institute of General Medical Sciences grant P41GM103543. The time-resolved set up at Sector 14 was funded in part through collaboration with P. Anfinrud (NIH/NIDDK) through the Intramural Research Program of the NIDDK. Use of the APS was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under Contract No. DE-AC02-06CH11357.
The authors declare no competing financial interests.
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Jung, Y., Lee, J., Kim, J. et al. Volume-conserving trans–cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography. Nature Chem 5, 212–220 (2013). https://doi.org/10.1038/nchem.1565
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