TIME-RESOLVED VIBRATIONAL SPECTROSCOPY OF HALORHODOPSIN

盐视紫红质的时间分辨振动光谱

• 批准号: 3306337
• 负责人: MARK S BRAIMAN
• 金额: $ 11.2万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306337
• 关键词: Archaea; Raman spectrometry; active transport; adenosinetriphosphatase; anions; arginine; bacterial proteins; bacteriorhodopsins; chemical binding; chlorine; cis trans isomerization; conformation; infrared spectrometry; interferometry; ion transport; ionic strengths; mathematical model; membrane activity; membrane transport proteins; mutant; protein structure function; retinaldehyde; rhodopsin; site directed mutagenesis; statistics /biometry; stereochemistry; time resolved data

The physiological importance of chloride transport across cell membranes has been underscored recently by the demonstration that genetic defects in a single transmembrane protein involved in chloride ion transport cause cystic fibrosis. However, despite such advances in genetic information the detailed mechanisms of chloride transport by membrane proteins are understood only poorly, in large part because of a paucity of basic information about the dynamics of interactions between anions and their binding sites on transport proteins. We will apply time-resolved Fourier transform infrared (TR-FTIR) spectroscopy to the study of the active chloride transport by biological membranes. The model system to be studied is halorhodopsin (hR), a light-driven anion transport protein found in the membranes of several species of archaebacteria. Comparisons of TR-FTIR vibrational spectra from hR and its transient photointermediates will help to elucidate structural changes occurring during the functioning of this protein. The broad sensitivity of time-resolved FTIR difference spectroscopy should allow detection of transient structural changes in the chromophore, as well as localized conformational alterations of the peptide backbone; protonations or deprotonations of ionizable amino acid side chains; and alterations in non-covalent interactions of cationic residues with chloride or other anions. The time resolution of these measurements will be in the range of 1-10 mus. Spectra of isotope-labeled and mutant hR proteins will also be obtained, in order to assign roles for individual amino acids in various anion binding and release steps. The results should allow the development of a detailed mechanistic model for active chloride transport for this particular system, and the derivation of general principles applicable to mammalian anion transport proteins.

氯离子跨细胞膜转运的生理重要性 最近有证据表明，遗传缺陷 参与氯离子转运的单个跨膜蛋白 引起囊性纤维化。 然而，尽管遗传技术取得了如此大的进步 信息通过膜传输氯离子的详细机制 人们对蛋白质了解甚少，很大程度上是因为缺乏 有关阴离子之间相互作用动力学的基本信息 及其在转运蛋白上的结合位点。 我们将申请 时间分辨傅里叶变换红外 (TR-FTIR) 光谱 生物膜主动氯离子转运的研究。 这 要研究的模型系统是盐视紫红质 (hR)，一种光驱动的阴离子 在多种物种的细胞膜中发现了转运蛋白 古细菌。 hR 和 TR-FTIR 振动光谱的比较 其瞬态光中间体将有助于阐明结构 该蛋白质发挥作用期间发生的变化。 广义的 时间分辨 FTIR 差值光谱的灵敏度应允许 检测发色团的瞬时结构变化，以及 肽主链的局部构象改变； 可电离氨基酸侧链的质子化或去质子化；和 阳离子残基非共价相互作用的改变 氯离子或其他阴离子。 这些测量的时间分辨率将 范围为1-10亩。 同位素标记和突变 hR 的光谱 还将获得蛋白质，以便为个体分配角色 各种阴离子结合和释放步骤中的氨基酸。 结果 应允许开发主动的详细机制模型 该特定系统的氯离子传输，以及推导 适用于哺乳动物阴离子转运蛋白的一般原则。