Development of high time resolution CCD spectrophotometer with weak light sources

生物系统中的信号转导是由受体蛋白的构象变化引发的，然后在大多数情况下与其他蛋白相互作用。本研究旨在开发一种新的设备，用于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冷却CCD相机作为多通道检测器。我们成功地开发了一种分光光度计，它可以连续记录光谱，时间分辨率为10 ms，这比具有单通道检测器的传统分光光度计高出约10，000倍。通过引入监测光束和激发激光，优化光学系统和软件，该装置可以在光激发前后100小时的记录时间内测量视色素视紫红质的200个吸收光谱（量子产率≤ 0.67），具有低漂白（≤ 3%）和低基线漂移（≤ 0.002 OD）。记录条件下获得的数据的准确性是可比的，从传统的光电倍增管，除了时间分辨率。该系统使我们能够监测视紫红质与G-蛋白转导蛋白在感光细胞膜的生理相关条件下的相互作用过程。此外，我们能够监测的结合和释放过程中的配体视网膜进入/从蛋白质的内源性色氨酸残基的荧光变化。这些结果表明，该分光光度计将是一个有用的工具，进一步了解在生理条件下的生物信号转导的分子机制。

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.