Structure/Function Study of Photoreceptive Proteins by FTIR Spectroscopy

Abstract

Abstract Light-induced difference Fourier-transform infrared (FTIR) spectroscopy is a powerful, sensitive and informative method for studying protein structural changes in photoreceptive proteins. Strong absorption of water in the IR region is always an issue in this method. However, if water content in the sample is controlled during measurements, this method can provide detailed structural information on a single protein-bound water molecule. We optimized the measuring conditions of light-induced difference FTIR spectroscopy to hydrated film samples. In doing so, highly accurate difference FTIR spectra were successfully obtained for a light-driven proton-pump bacteriorhodopsin (BR), not only in the conventional 1800–800 cm−1 region, but also in the 4000–1800 cm−1 region. A highly accurate measuring system of light-induced difference FTIR spectroscopy was applied to various photoreceptive proteins such as animal and microbial rhodopsins, and comprehensive FTIR analyses revealed that proton-pumping rhodopsins possess strongly hydrogen-bonded water molecules. It was concluded that a strongly hydrogen-bonded water molecule is the functional determinant of a proton pump. FTIR spectroscopy was also applied to flavin-binding photoreceptors, where we elucidated the molecular mechanisms of adduct formation in the LOV domain, hydrogen-bonding alteration in the BLUF domain, and activation and DNA-repair mechanisms in photolyases. In studies on rhodopsin, we contributed to the discovery and creation of new functions, where FTIR spectroscopy was used for the molecular characterization of new rhodopsins. These new rhodopsins offer promising tools in optogenetics that revolutionized brain sciences. As highlighted in this review article, we provided new insights into the structure/function relationship of biomolecules by unique difference FTIR spectroscopy. In particular, by studying photoreceptive proteins such as rhodopsins, we clarified the mechanism of how light is taken into proteins, and how it leads to their function.