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Structure at 0.85 Å resolution of an early protein photocycle intermediate

Nature volume 392, pages 206–209 (1998)Cite this article

Abstract

Protein photosensors from all kingdoms of life1,2 use bound organic molecules, known as chromophores, to detect light. A specific double bond within each chromophore is isomerized by light, triggering slower changes in the protein as a whole. The initial movements of the chromophore, which can occur in femtoseconds, are tightly constrained by the surrounding protein, making it difficult to see how isomerization can occur, be recognized, and be appropriately converted into a protein-wide structural change and biological signal. Here we report how this dilemma is resolved in the photoactive yellow protein (PYP). We trapped a key early intermediate in the light cycle of PYP at temperatures below −100 °C, and determined its structure at better than 1 Å resolution. The 4-hydroxycinnamoyl chromophore3,4 isomerizes by flipping its thioester linkage with the protein, thus avoiding collisions resulting from large-scale movement of its aromatic ring during the initial light reaction. A protein-to-chromophore hydrogen bond that is present in both the preceding dark state5 and the subsequent signalling state6 of the photosensor breaks, forcing one of the hydrogen-bonding partners into a hydrophobic pocket. The isomerized bond is distorted into a conformation resembling that in the transition state. The resultant stored energy is used to drive the PYP light cycle. These results suggest a model for phototransduction, with implications for bacteriorhodopsin7,8, photoactive proteins1,2, PAS domains9, and signalling proteins.

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Figure 1: Structural consequences of light activation.
Figure 2: Active-site structures of the (a) ground state, (b) early intermediate, and (c) bleached state of PYP.
Figure 3: The environment of the thioester oxygen atom (O2) of the chromophore in the early intermediate.

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Acknowledgements

We thank C. D. Mol and C. D. Putnam for testing the PYP crystals for sub-Ångstrom diffraction; B. E. Honig for discussing low-temperature approaches to the study of photosensors; J.Berendzen for discussing appropriate illumination conditions; C. L. Brooks III, B. N. Dominy, J.-L. Pellequer and D. A. Case for exploring possibilities of energy calculation; and T. Woo for growing the crystal used for microspectrophotometry. This research was supported by a grant from the N.I.H. (to E.D.G.) and a scholarship from Boehringer Ingelheim Fonds (for U.K.G.). This work is based on research conducted at the Stanford Synchrotron Radiation Laboratory (SSRL), which is funded by the Department of Energy, Office of Basic Energy Sciences. The Biotechnology Program is supported by the NIH, National Center for Research Resources, Biomedical Technology Program and the Department of Energy, Office of Biological and Environmental Research.

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Authors and Affiliations

  1. Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    Ulrich K. Genick, Ilona L. Canestrelli & Elizabeth D. Getzoff

  2. Stanford Synchrotron Radiation Laboratory, PO Box 4349, Bin 69, Stanford, 94309, California, USA

    S. Michael Soltis & Peter Kuhn

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  1. Ulrich K. Genick
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  5. Elizabeth D. Getzoff
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Correspondence to Elizabeth D. Getzoff.

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Genick, U., Soltis, S., Kuhn, P. et al. Structure at 0.85 Å resolution of an early protein photocycle intermediate. Nature 392, 206–209 (1998). https://doi.org/10.1038/32462

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