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Structure of the I1 early intermediate of photoactive yellow protein by FTIR spectroscopy

Nature Structural Biology volume 8, pages 265–270 (2001)Cite this article

Abstract

To understand how proteins translate the energy of sunlight into defined conformational changes, we have measured the photocycle reactions of photoactive yellow protein (PYP) using time-resolved step scan Fourier transform infrared (FTIR) spectroscopy. Global fit analysis yielded the same apparent time constants for the reactions of the chromophore, the protonation changes of protein side chains and the protein backbone motions, indicating that the light cycle reactions are synchronized. Changes in absorbance indicate that there are at least four intermediates (I1, I1′, I2, I2′). In the intermediate I1, the dark-state hydrogen bond from Glu 46 to the aromatic ring of the p-hydroxycinnamoyl chromophore is preserved, implying that the chromophore undergoes trans to cis isomerization by flipping, not the aromatic ring, but the thioester linkage with the protein. This excludes an I1 structural model proposed on the basis of time resolved Laue crystallography, but does agree with the cryotrapped structure of an I1 precursor.

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Figure 1: Overall view of the PYP ground state structure.
Figure 2: Three-dimensional representation of the fitted infrared absorbance changes of PYP.
Figure 3: I1 − P FTIR difference spectrum and FT Raman spectrum of P.
Figure 4: I2 − P and I2 / I1′ − P FTIR difference spectra.
Figure 5: Infrared kinetics at specific wavenumbers.
Figure 6: Stereo view of PYP's active site.

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Acknowledgements

This work was supported by grants from the NIH to E.D.G. and the DFG to K.G. R.B. gratefully acknowledges a fellowship from the DFG.

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Authors and Affiliations

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

    Ronald Brudler, Tammy T. Woo & Elizabeth D. Getzoff

  2. Lehrstuhl für Biophysik, Ruhr-Universität Bochum, D-44780, Bochum, Germany

    Robin Rammelsberg & Klaus Gerwert

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Correspondence to Ronald Brudler or Klaus Gerwert.

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Brudler, R., Rammelsberg, R., Woo, T. et al. Structure of the I1 early intermediate of photoactive yellow protein by FTIR spectroscopy. Nat Struct Mol Biol 8, 265–270 (2001). https://doi.org/10.1038/85021

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