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FTIR Spectroscopy of Flavin-Binding Photoreceptors

  • Daichi Yamada
  • Hideki KandoriEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

FTIR Difference spectroscopy Double difference spectroscopy BLUF domain LOV domain Cryptochrome Photolyase Hydrated film Redissolved sample Concentrated solution 

Notes

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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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Frontier MaterialsNagoya Institute of TechnologyNagoyaJapan

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