Photobiology of Rhodopsin and the Cone Pigments

描述(申请人提供)：视锥细胞负责明视视觉，即正常光线条件下的视觉过程。视锥感受器必须在广泛的光强度范围内工作，并覆盖可见光光谱的全部范围。在这些不同的条件下发挥作用的能力主要归功于高度优化的GPCR光传导蛋白，非正式地称为锥体色素。这些蛋白质的最大吸收波长从350 nm到660 nm，在吸收光后，经过一系列有效的光漂白过程来产生活性蛋白质。随后，转导蛋白与激活的蛋白质结合，产生神经冲动和视觉。在NIH资助的前一项研究中，一个关键的观察结果是，锥体色素在光活化过程中经历了反离子开关。这项研究的一个关键目的是探索反离子开关机制是否也在红色和蓝色锥体颜料中活跃，如果是，则表征分子细节。为了实现这一目标，我们将使用从10K到环境温度下的振动和电子光谱来捕获和表征光漂白中间体。定点突变将被用来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，许多红色锥体在性质和反离子开关的实现方面不同于绿色、蓝色和UV锥体。事实上，有可能红色圆锥体完全缺乏这种机械特征。这项研究的另一个目的是系统地识别紫外锥、蓝锥、绿锥和红锥的波长选择机制。尽管我们的研究确定了蓝光和紫外光视锥中波长选择的关键特征，但仍有许多需要了解。我们将红锥体纳入这项研究是新的，我们对这个话题的热情在一定程度上取决于我们的信念，即红锥体是根本不同的。在我们的初步研究讨论中，我们有初步证据表明，深红色锥体至少使用了一种新的波长选择机制，涉及到对发色团环构象的操纵。独特的波长选择和显著不同(或不存在)的反离子开关机制相结合，使红色圆锥体成为重要目标。我们的研究将包括使用分子轨道理论来探索锥体颜料中的结构-功能关系，并计算结合生色团的光谱性质。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。像以前一样，我们将免费向感兴趣的研究人员提供这些程序。与公共健康相关：人们越来越需要了解与锥状光感受器中的光暴露相关的光漂白和恢复机制。由于这些细胞对人类的明视视觉是必不可少的，因此了解相关光传导色素的结构和功能关系是很重要的。这项研究的项目目标可能有助于了解黄斑部疾病，包括视锥细胞丢失、视锥细胞营养不良和其他眼部疾病，这些疾病涉及视网膜光感受器功能受损或降低。

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.