Photobiology of Rhodopsin and the Cone Pigments

描述（由申请人提供）：视锥细胞负责感光视觉，即正常光照条件下的视觉过程。锥体感受器必须在宽范围的光强度下工作，并覆盖可见光谱的全部范围。在这些不同条件下发挥作用的能力主要是由于高度优化的GPCR光转导蛋白，非正式地称为视锥色素。这些蛋白质的最大吸收范围为350至660纳米，在吸收光后，经历有效的光漂白序列以产生活性蛋白质。转导蛋白随后与活化蛋白结合，产生神经冲动和视觉。在之前的NIH资助的研究中，一个关键的观察是，视锥色素在光激活过程中经历了一个反离子开关。本研究的一个关键目的是探索反离子开关机制是否在红色和蓝色视锥色素中也有活性，如果有，则表征分子细节。为了实现这一目标，我们将在从10K到环境温度下使用振动和电子光谱来捕获和表征光漂白中间体。位点定向诱变将用于确定负责波长选择的关键残基和反离子开关的性质。从同源性研究中可以清楚地看出，许多红色视锥细胞与绿色、蓝色和紫外线视锥细胞在性质和反开关的实施上有所不同。事实上，红色视锥细胞可能完全缺乏这种机械特征。本研究的另一个目的是系统地确定紫外线，蓝色，绿色和红色锥细胞的波长选择机制。虽然我们的研究确定了蓝色和紫外线锥细胞波长选择的关键特征，但仍有许多有待了解。我们在这项研究中纳入红色视锥细胞是新的，我们对这个话题的热情部分取决于我们相信红色视锥细胞是根本不同的。在我们的初步研究讨论中，我们有初步的证据表明，深红色视锥细胞至少使用了一种新的波长选择机制，涉及对发色团环构象的操纵。独特的波长选择和明显不同（或不存在）的反离子开关机制的结合使红色锥体成为重要的目标。我们的研究将包括使用分子轨道理论来探测锥体色素的结构-功能关系，并计算结合的发色团的光谱特性。我们将重构我们的MNDO-PSDCI代码并改进接口，使这些程序对科学界更有用。和以前一样，我们将免费向感兴趣的研究人员提供这些程序。公共卫生相关性：人们越来越需要了解锥状光感受器中与光暴露相关的光漂白和恢复机制。由于这些细胞对人类光视觉至关重要，因此了解相关光转导色素的结构和功能关系非常重要。本研究的项目目标可能有助于了解黄斑疾病，包括锥体细胞的丧失、锥体营养不良和其他眼部疾病，包括视网膜光感受器的损伤或功能减弱。

DESCRIPTION (provided by applicant): Cone cells are responsible for photopic vision, the visual process under normal light conditions. The cone receptors must operate over a wide range of light intensities and cover the full range of the visible spectrum. The ability to function under these diverse conditions is due primarily to the highly optimized GPCR light-transducing proteins informally called cone pigments. These proteins have absorption maxima that range from 350 to 660 nm, and upon the absorption of light, undergo an efficient photobleaching sequence to produce an activated protein. Subsequent binding of transducin to the activated protein results in a nerve impulse and vision. A key observation made during the previous NIH funded study was that cone pigments undergo a counterion switch during photoactivation. A key aim of this study is to explore whether a counterion switch mechanism is also active in the red and blue cone pigments, and if so, to characterize the molecular details. To achieve this goal, we will use vibrational and electronic spectroscopy at temperatures from 10K to ambient to trap and characterize the photobleaching intermediates. Site directed mutagenesis will be used to identify the key residues responsible for wavelength selection and the nature of the counterion switch. It is clear from homology studies that many of the red cones differ from the green, blue and UV cones in nature and implementation of the counterion switch. Indeed, it is possible that the red cones lack this mechanistic feature entirely. An additional aim of this study is to systematically identify the mechanisms of wavelength selection in the UV, blue, green and red cones. Although our research identified key features of wavelength selection in the blue and UV cones, much remains to be understood. Our inclusion of the red cones in this study is new, and our enthusiasm for this topic rests in part on our belief that the red cones are fundamentally different. We have preliminary evidence, presented in our preliminary studies discussion, that the deep red cones use at least one new mechanism for wavelength selection involving manipulation of the chromophore ring conformation. The combination of unique wavelength selection and a significantly different (or absent) counterion switching mechanism make the red cones an important target. Our studies will include the use of molecular orbital theory to probe structure-function relationships in the cone pigments, and to calculate the spectroscopic properties of the bound chromophores. We will refactor our MNDO-PSDCI code and improve the interface to make these procedures more useful to the scientific community. As before, we will provide these procedures to interested researchers without charge. PUBLIC HEALTH RELEVANCE: There is a growing need to understand the photobleaching and recovery mechanisms associated with light exposure in cone photoreceptors. Because these cells are essential for human photopic vision, it is important to understand the structure and function relationships in the associated light transducing pigments. The project goals of this research may help understand macular disease, which involves loss of cone cells, cone dystrophy, and other eye diseases, which involve damage or diminished function of retinal photoreceptors.