Biological systems utilize light as the source of signals and energy. In photoreceptive proteins, which are responsible for these functions, a chromophore molecule undergoes unique photophysical and photochemical processes. Dr. Kandori was interested in the primary reactions of photoreceptive proteins when he was a graduate student and a postdoctoral fellow. By applying ultrafast spectroscopy, he and his colleagues revealed that ultrafast and efficient reactions take place in photoreceptive proteins. Altered chromophore-protein interaction by light finally leads to each function. It is not easy to understand these processes because details of protein structural changes have to be analyzed at the atomic level. Dr. Kandori started to use FTIR spectroscopy in 1993 to understand what happens in photoreceptive proteins after light absorption until functional expression.

1. Construction of highly accurate difference FTIR measuring system for biomolecules
IR spectroscopy is a sensitive and informative method. However, strong absorption of water is always problematic and there were many limitations for biomolecules. Dr. Kandori applied light-induced difference FTIR spectroscopy at low temperatures. This method itself was not his original, but he 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. This enabled information on hydrogen-bonding donors such as O-H, N-H and S-H groups to be obtained.
Using the new spectroscopic window, Dr. Kandori studied hydrogen-bonding alteration in many photoreceptive proteins. One of his research highlights was the detection of protein-bound water molecules. By using hydration between ¹⁶O and ¹⁸O waters, water vibrations were successfully identified, and hydrogen-bonding alteration of protein-bound waters were linked to each function. The analysis of protein-bound water was extended to room temperature by step-scan time-resolved FTIR spectroscopy. Dr. Kandori also applied polarized FTIR spectroscopy, which determines angles of dipole moments to the membrane normal in membrane proteins, and ATR-FTIR spectroscopy, which provides ligand-binding induced structural changes even for non-photoreceptive proteins.

2. FTIR spectroscopy of animal rhodopsins, microbial rhodopsins and flavoproteins
A highly accurate measuring system of light-induced difference FTIR spectroscopy was applied to various photoreceptive proteins. Our vision is composed of twilight vision mediated by a single pigment and color vision achieved by three color pigments (red, green, and blue). A common chromophore molecule, 11-cis retinal, is used to distinguish different colors in vision. In contrast to twilight vision receptor, no structural studies were performed for color pigments, because of difficulties in sample preparation and the lack of suitable methods in structural analysis. By applying low-temperature difference FTIR spectroscopy, Dr. Kandori obtained structural information of primate red, green, and blue sensitive pigments for the first time.
While animal rhodopsins are all G-protein coupled receptors, the functions of microbial rhodopsins become highly diverse: light-driven ion-pump, light-gated ion-channel, light sensor, and light-activated enzyme. Interestingly, their structures and photoreaction cycles are similar to each other, suggesting that small difference in structure and structural changes determine their function. Light-induced FTIR spectroscopy provided structural insight related to each function. In particular, comprehensive FTIR analysis revealed that proton-pumping rhodopsins possess strongly hydrogen-bonded water molecules, from which 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 photoreactions other than cis-trans isomerization initiate protein structural changes to activate their functionality. Dr. Kandori 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.

3. Discovery, creation and conversion of functions in photoreceptive proteins
In studies on rhodopsin, Dr. Kandori contributed to the discovery of new functions. In some cases, he focused on unique amino acid sequences of rhodopsins, and discovered a light-driven sodium-ion pump, an inward proton pump, and a light-activated enzyme. In another case, he was requested to characterize the molecular properties of a new rhodopsin, heliorhodopsin, which was a new family of rhodopsin. FTIR spectroscopy is used for molecular characterization of new rhodopsins. These new rhodopsins offer promising tools in optogenetics that revolutionized brain sciences. A light-driven sodium-ion pump can transport lithium ion while it pumps protons in potassium and larger cations. Based on structural information, Dr. Kandori successfully designed mutants that pump potassium, rubidium, and cesium ions.
Dr. Kandori also challenged the functional conversion of photoreceptive proteins by a limited number of mutations. He converted a light-driven outward proton-pump BR into an inward chloride-ion pump by replacing a single amino acid. Regarding light-driven eubacterial proton, sodium, and chloride pumps, he found that successful functional conversions of these ion pumps was achieved exclusively when mutagenesis reversed the evolutionary changes in amino acid sequence. Dependence of the observed functional conversions on the direction of evolution strongly suggests that the essential structural mechanism of an ancestral function was retained even after gaining a new function during natural evolution which can be evoked by a few mutations. Asymmetric functional conversion was also the case for photolyases, where a mutant (6-4) photolyase repairs a CPD photoproduct, but the reverse mutant of CPD photolyase never repairs the (6-4) photoproduct.

As highlighted above, Dr. Kandori has provided new insights into the structure/function relationship of biomolecules by unique difference FTIR spectroscopy. In particular, by studying photoreceptive proteins such as rhodopsins, he clarified the mechanism of how light is taken into proteins, and how it leads to their function. Furthermore, by utilizing spectroscopic data, Dr. Kandori achieved discovery, creation and conversion of functions. These world-leading achievements now attract attention not only in basic research fields but also in applied research fields such as optogenetics. Therefore, he deserves The Chemical Society of Japan Award.