MOLECULAR MECHANISMS IN VISUAL TRANSDUCTION

我们的长期目标是了解分子 视觉传导和适应的机制。这项建议 主要集中在视紫红质作用的分子机制上。之后 视紫红质吸收一个光子，它通过一系列 光谱定义的构象状态。一项或多项 这些构象状态(R*)催化GTP的交换 对于GDP盘中相关的G蛋白导致的激活 后者。类似的，如果不是完全相同的，信息传递 步骤的出现是许多因素相互作用的结果 荷尔蒙和药物及其受体。这项提议在很大程度上是 与定义视紫红质的分子变化有关 使激活的视紫红质R*达到。一项重大利益 这里是在R*和 光谱定义的中间体。这项工作将是 既与我们对视觉的理解有关，也与 药物与受体的相互作用。 视紫红质席夫碱可以质子化的事实是 在这种蛋白质的功能中具有极其重要的作用。这个 发色团的质子化希夫碱，与 活性部位的带负电荷的氨基酸，被认为是 在波长调节和通电方面具有首要重要性 视紫红质。希夫碱的最终去质子化是 也被认为在后视紫红质的形成中起重要作用 II，推定R*的光谱特征。沿着这些思路， 视蛋白的活性部位赖氨酸也必须去质子化才能形成 具有11顺式视网膜的席夫碱基。这里描述的实验 旨在证明电荷和电荷运动在 视紫红质的功能，在很小的程度上， 细菌视紫红质。这里使用的方法是生物有机的。 化学和生化性质的，并将涉及修饰 结合视网膜类比研究。通过引入 化学探针活性部位赖氨酸上的化学探针，如甲基 我们将确定这种赖氨酸上的全部正电荷是否为 光化学能量存储、波长调节所需 以及R*的形成。此外，活动站点的PK 将测定视紫红质的赖氨酸。氨基酸 与生色团发生关键作用的反离子将是 通过设计使用活性部位的结构研究确定 视蛋白的赖氨酸将伪交联剂定向到这些 反离子。最后，形成色素的新型视网膜类似物 将利用光学蛋白来探索能量储存的机制。

Our long-term objectives are to understand the molecular mechanisms of visual transduction and adaptation. This proposal is focused on the molecular mechanism of rhodopsin action. After rhodopsin absorbs a photon it proceeds through a series of spectroscopically defined conformation states. One or more of these conformational states (R*) catalyzes the exchange of GTP for GDP in a disk associated G-protein resulting in the activation of the latter. Similar, if not identical , informational transducing steps occur as a consequences of the interaction of many hormones and drugs with their receptors. The proposal is largely concerned with defining the molecular changes in rhodopsin which enable activated rhodopsin R* to be reached. A major interest here is to establish a structural link between R* and the spectroscopically defined intermediates. This work will be of relevance both to our understanding of vision and the nature of drug-receptor interactions. The fact that Schiff base of rhodopsin can be protonated is of immense importance in the functioning of this protein. The protonated Schiff base of the chromophore, interacting with negatively charge amino acids at the active-site, is thought to be of primary importance in wavelength regulation and energization of rhodopsin. The ultimate deprotonation of the Schiff base is assumed also to be important in the formation of metarhodopsin II, the presumed spectroscopic signature of R*. Along these lines, the active-site lysine of opsin must also be deprotonated to form a Schiff base with 11-cis-retinal. The experiments described here are designed to prove the role of charge and charge movement in the functioning of rhodopsin and, to a much lesser extent, bacteriorhodopsin. The approaches used here are bio-organic chemical and biochemical in nature and will involve modification of the protein along with retinal analog studies. By introducing chemical probes, such as a methyl group, at the active-site lysine we will determine if a full positive charge on this lysine is required for photochemical energy storage, wavelength regulation and the formation of R*. Furthermore the pK of the active-site lysine of rhodopsin will be determined. The amino acid counterions which critically interact with the chromophore will be identified by structural studies designed to use the active-site lysine of opsin to direct a pseudo cross-linking reagent to these counterions. Finally, novel retinal analogs which form pigments with opsin will be used to probe the mechanism of energy storage.