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FTIR Spectroscopy of Flavin-Binding Photoreceptors

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Book cover Flavins and Flavoproteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1146))

Abstract

Light-induced difference Fourier transform infrared (FTIR) spectroscopy is a powerful, sensitive, and informative method to study structure–function relationships in photoreceptive proteins. Strong absorption of water in the IR region is always problematic in this method, but if water content in the sample is controlled during measurements, this method can provide useful information on a single protein-bound water molecule. We established three kinds of sample preparations: hydrated film, redissolved sample, and concentrated solution. Hydrated films were used for the measurements of LOV and BLUF domains, where accurate difference FTIR spectra were obtained in the whole mid-IR region (4,000–800 cm−1). Vibrations of S–H stretch of cysteine, O–H stretch of water, and O–H stretch of tyrosine provide important information on hydrogen bonds in these proteins. Redissolved samples were used for the measurements of (6-4) photolyase, in which enzymatic turnover takes place. From the illumination time-dependence of excess amount of substrate, it is possible to isolate the signal originating from the binding of enzyme to substrate. If proteins are less tolerant to drying, as for example cryptochromes of the DASH type, concentrated solution is used. Detailed methodological aspects in light-induced difference FTIR spectroscopy are reviewed by mainly focusing on our results.

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Acknowledgments

We thank Drs. Tatsuya Iwata and Yu Zhang for their efforts to establish the FTIR study of flavin-binding photoreceptors. We also thank our many collaborators that contributed to our publications (see "References").

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Authors and Affiliations

  1. Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya, 466-8555, Japan

    Daichi Yamada & Hideki Kandori

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  1. Daichi Yamada
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  2. Hideki Kandori
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Correspondence to Hideki Kandori .

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Editors and Affiliations

  1. Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany

    Stefan Weber

  2. Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Germany

    Erik Schleicher

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Yamada, D., Kandori, H. (2014). FTIR Spectroscopy of Flavin-Binding Photoreceptors. In: Weber, S., Schleicher, E. (eds) Flavins and Flavoproteins. Methods in Molecular Biology, vol 1146. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0452-5_14

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  • DOI: https://doi.org/10.1007/978-1-4939-0452-5_14

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