Time-Resolved Fourier Transform Infrared Study on Photoadduct Formation and Secondary Structural Changes within the Phototropin LOV Domain |
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Authors: | Anna Pfeifer Kazunori Zikihara Satoru Tokutomi Tilman Kottke |
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Institution: | † Biophysical Chemistry, Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany ‡ Institute of Neuroscience and Biophysics 2, Research Center Jülich, 52425 Jülich, Germany § Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan ¶ Research Center for Environmental Genomics, Kobe University, Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan |
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Abstract: | Phototropins are plant blue-light photoreceptors containing two light-, oxygen-, or voltage-sensitive (LOV) domains and a C-terminal kinase domain. The two LOV domains bind noncovalently flavin mononucleotide as a chromophore. We investigated the photocycle of fast-recovery mutant LOV2-I403V from Arabidopsis phototropin 2 by step-scan Fourier transform infrared spectroscopy. The reaction of the triplet excited state of flavin with cysteine takes place with a time constant of 3 μs to yield the covalent adduct. Our data provide evidence that the flavin is unprotonated in the productive triplet state, disfavoring an ionic mechanism of bond formation. An intermediate adduct species was evident that displayed changes in secondary structure in the helix or loop region, and relaxed with a time constant of 120 μs. In milliseconds, the final adduct state is formed by further alterations of secondary structure, including β-sheets. A comparison with wild-type adduct spectra shows that the mutation does not interfere with the functionality of the domain. All signals originate from within the LOV domain, because the construct does not comprise the adjacent Jα helix required for signal transduction. The contribution of early and late adduct intermediates to signal transfer to the Jα helix outside of the domain is discussed. |
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