COFACTOR PROTEIN INTERACTIONS IN RHODOPSIN

视紫红质中辅助因子蛋白的相互作用

• 批准号: 2420612
• 负责人: ELISABETH A MORLINO
• 金额: $ 2.43万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-23 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2420612
• 关键词: aminoacid; chemical transfer reaction; cofactor; conformation; electronic spectra; infrared spectrometry; laser spectrometry; photoactivation; photochemistry; protein structure function; rhodopsin; site directed mutagenesis

The proposal describes four specific aims which will test the ultrafast responses of native rhodopsin and rhodopsin analogues using visible, near, and mid infrared spectroscopy. The first specific aim concerns isotope effects on the ultrafast processes involved in isomerization. The candidate will utilize an ultrafast laser system with 35 fs resolution to monitor the dynamics of the initially-produced intermediates involved in the primary processes of isomerization. Theses experiments will test the possibility that local charge density surrounding retinal controls the dynamics of isomerization. If this is so, then a proton transfer, which may precede retinal isomerization, would change the charge density in the vicinity of retinal and affect isomerization dynamics. The candidate should be able to determine whether a proton shift or a change in hydrogen bonding strength has occurred by correlating the dynamics of isomerization, using visible light pulses, and proton shifting, using infrared light pulses. These experiments are will-designed and should provide valuable information. In the second aim, the candidate, using short excitation/probe pulses, will concentrate on following the isomerization process in real time in the spectral region to the red of 650 nm and to the blue of 480 nm. Recent results on bacteriorhodopsin, which has substantial homology with rhodopsin, indicate that important information concerning the kinetic signatures of intermediates crucial to the initial isomerization process may be contained in these spectral regions. These well- designed experiments should provide this information, as well as determine from which electronically excited state isomerization occurs. In the third specific aim, the candidate will use techniques developed to monitor conformational changes of retinal during isomerization. Specifically, she will use measurements of the vibrational characteristics and dipole moment changes of retinal to track isomerization in real time. The experiments are well-designed and should provide a clearer picture of the dynamics of isomerization as seen from changes in the anisotropy signal and vibrational spectra. In addition, these results may provide insight into the relative importance of the individual motions of specific substituents of retinal of the initial isomerization process. In the fourth specific aim, the candidate will use site directed mutagenesis to modify specific amino acid residues that reside within the cofactor binding pocket in rhodopsin. These experiments could reveal whether particular sites on the opsin interact with the cofactor. In fact, the candidate provides reasonable hypotheses with respect to the possible important of two specific opsin amino acid residues that she proposes to test. However, she does not explain how mutations of these amino acid residues will be obtained, except to state that various named individuals will develop these rhodopsin mutants or supply the resources to do so. These individuals have not provided letters of collaboration for this proposal, however. It is therefore not clear whether this last specific aim will be successfully performed.

该提案描述了四个特定目标，这些目标将测试超快 使用可见的天然视紫红质和视紫红质类似物的反应， 附近和中红外光谱法。 第一个特定目的涉及对超快的同位素影响 涉及异构化的过程。候选人将利用 超快激光系统具有35 FS分辨率，以监测 最初生产的中间体参与了主要过程 异构化。这些实验将测试本地的可能性 电荷密度围绕视网膜控制 异构化。如果是这样，则可以在 视网膜异构化，将改变附近的电荷密度 视网膜和影响异构化动力学。候选人应该是 能够确定质子移位还是氢的变化 通过关联的动力学来实现粘结强度 异构化，使用可见光脉冲和质子转移，使用 红外光脉冲。这些实验将是设计的，应该 提供有价值的信息。 在第二个目标中，候选人使用简短的激发/探针脉冲， 将专注于实时遵循异构化过程 光谱区域为650 nm的红色和480 nm的蓝色。 细菌紫红质的最新结果 使用Rhodopsin，指出有关 中间体的动力学特征对初始异构化至关重要 过程可能包含在这些光谱区域中。这些 设计的实验应提供此信息以及 确定从哪些电子激发态异构化发生。 在第三个特定目标中，候选人将使用开发的技术 监测异构化期间视网膜的构象变化。 具体来说，她将使用振动的测量值 视网膜的特性和偶极矩的变化 实时异构化。实验是精心设计的，并且 应该更清楚地描绘出异构化动力学 从各向异性信号和振动光谱的变化中可以看出。在 此外，这些结果可能会洞悉亲戚 视网膜特定取代基的单个运动的重要性 初始异构化过程。 在第四个特定目标中，候选人将使用定向网站 诱变，以修改驻留在内的特定氨基酸残基 Rhodopsin中的辅因子结合口袋。这些实验可以 揭示Opsin上的特定站点是否与 辅因子。实际上，候选人提供了合理的假设 尊重两个特定的Opsin氨基酸的重要重要性 她建议测试的残留物。但是，她没有解释如何 除非 指出各种命名的人会发展这些视紫红质 突变者或提供资源。这些人没有 但是，为此提案提供了协作信。这是 因此，尚不清楚最后一个特定目标是否会成功 执行。