Development of high time resolution CCD spectrophotometer with weak light sources

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Signal transductions in biological systems are initiated by conformational changes of receptor proteins followed by the interaction with, in most case, other proteins. In this research project, we aimed to develop new equipment for monitoring the conformational changes f photoreceptor proteins and their interactions with other proteins under the physiological conditions. We tried to develop a high performance absorption/fluorescence spectrophotometer using a cooled CCD camera as a multi-channel detector. We have succeeded in developing a spectrophotometer that can continuously record the spectra with a time resolution of 10 ms, which is about 10,000 times higher than that of a conventional spectrophotometer that has a single-channel detector. By introducing the monitor beam and excitation laser, optimization of the optics and software, this equipment allowed to measuring the 200 absorption spectra of the visual pigment rhodopsin (quantum yield 〜0.67) with low bleaching (〜3 %) and low baseline drift (〜0.002 OD) during 〜1hr recording time before and after light excitation. The accuracy of the data obtained by the recording condition is comparable to that obtained from conventional spectrophotometers except for the time resolution.This system allowed us to monitor the interaction process of rhodopsin with G-protein transducin in photoreceptor membranes under the physiologically relevant conditions. In addition, we were able to monitor the binding and the releasing processes of ligand retinal into/from protein as fluorescence changes of the intrinsic tryptophan residues. These results indicate that this spectrophotometer would be a useful tool for furthering our understanding of the molecular mechanisms of biological signal transduction under the physiological conditions.