| Research Abstract |
This research project aims at revealing the correlation between structure and function in rhodopsins by means of polarized infrared spectroscopy. The target molecules are bacteriorhodopsin (bR) as a light-driven proton pump, halorhodopsin (hR) as a light-driven chloride pump, rhodopsin (Rh) as a light-sensor in twilight vision, and color visual pigments (Chicken-Red and Chicken-Green) as light-sensors in color vision. Infrared spectroscopy of these proteins as well as their mutants and isotope-labeled molecules provided the following results.
(1) Bridged water stretching vibrations inside bR under strong hydrogen bond were directly observed by highly refined polarized infrared spectroscopy. Rotation of a water molecule was observed accompanying retinal isomerization.
(2) Polarized infrared spectroscopy of bR provides information on the structural changes of protein side upon photoisomerization (K intermediate). Investigation of isotope-labeled sample on threonine side chain and mutants r
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evealed that 3 of the threonines of a total of 18 change their hydrogen bonding. The structural change of Thr17, which is located > 11 Å from the retinal chromophore, implicates a specific perturbation channel in the protein that accompanies the retinal motion.
(3) Polarized infrared spectroscopy of bR provides information on the structural changes of protein side before and after the primary proton transfer (L and M intermediates). It was found that hydrogen bond of Thr89 is strengthened upon photoisomerization, and the strong hydrogen bond is persistent even after the "switch" event for proton pump. The results implicate that the Thr89-Asp85 region is not involved in the switch, suggesting that the switch is indeed local machinery.
(4) Protein structural changes of hR, Chicken-Red, Chicken-Green, and photoactive yellow protein were extensively studied by low-temperature infrared spectroscopy.
(5) Excited-state dynamics of bovine rhodopsin was studied by femtosecond fluorescence spectroscopy. Less
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