Development of high time resolution CCD spectrophotometer with weak light sources

在生物系统中，信号转导是由受体蛋白的构象变化启动的，随后在大多数情况下，与其他蛋白相互作用。在本研究项目中，我们旨在开发一种新的设备来监测生理条件下光感受器蛋白的构象变化及其与其他蛋白的相互作用。我们试图开发一种高性能的吸收/荧光分光光度计，使用制冷的CCD摄像机作为多通道探测器。我们已经成功地研制出一种可以连续记录光谱的分光光度计，其时间分辨率为10ms，比传统的具有单通道探测器的分光光度计高出约1万倍。通过引入监视光束和激发激光，优化光学系统和软件，该装置可以测量光激发前后1小时记录时间内低漂白(~3%)和低基线漂移(~0.002 OD)的视觉色素视紫红质的200个吸收光谱(量子产率~0.67)。该系统能够监测生理条件下视紫红质与光感受器膜上G蛋白转导蛋白的相互作用过程。此外，我们还能够监测配体视黄醇与蛋白质的结合和释放过程，以及内源性色氨酸残基的荧光变化。这些结果表明，该分光光度计将为我们进一步了解生理条件下生物信号转导的分子机制提供有用的工具。

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.