Gloeobacter rhodopsin (GR) is a light-driven proton-pump protein similar to bacteriorhodopsin (BR), found in Gloeobacter violaceus PCC 7421, a primitive cyanobacterium. In this paper, structural changes of GR following retinal photoisomerization are studied by means of low-temperature Fourier-transform infrared (FTIR) spectroscopy. The initial motivation was to test our hypothesis that proton-pumping rhodopsins possess strongly hydrogen-bonded water molecules in the active center. Water O-D stretching vibrations at <2400 cm(-1) in D(2)O have been regarded as coming from such strongly hydrogen-bonded water, and there is a strong correlation between the proton-pumping activity and the presence of such water molecule. Since GR pumps protons, we expected that GR also possesses strongly hydrogen-bonded water molecule(s), and the FTIR results clearly show that this is indeed the case. In addition, another unexpected finding was gained from the frequency region of protonated carboxylic acids in the GR(K) minus GR spectra at 77 K, where we observed the unique bands of a protonated carboxylic acid at 1735 (+)/1730 (-) cm(-1). Comprehensive mutation study revealed that the vibrational bands originate from the carboxylic C=O stretch of Glu132 at the position corresponding to Asp96 in BR. Glu132 presumably functions as an internal proton donor for the retinal Schiff base, but they may be located far apart (ca. 12 A in BR). The present study demonstrates the long-range structural changes of GR along the proton pathway, even though the protein matrix is frozen at 77 K.

We propose and demonstrate a newly designed optical delay line (W-type delay line) that improves the resolution of optical path length, namely, time resolution, by an order of more than 3 in comparison to the spatial resolution of a conventionally used translational stage. Using a conventional mechanical translational stage having a spatial resolution of 1 mum, the performance of the W-type delay line was evaluated by interferometric measurements with a diode laser, and the time resolution of 16 as was confirmed.

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