TIME-RESOLVED VIBRATIONAL SPECTROSCOPY OF HALORHODOPSIN

氯离子跨膜转运的生理学意义 最近的一项研究表明， 在参与氯离子转运的单跨膜蛋白中 引起囊性纤维化。 然而，尽管遗传学方面取得了这些进展， 氯离子膜转运的详细机制 对蛋白质的了解很少，很大程度上是因为缺乏 关于阴离子之间相互作用动力学的基本信息 以及它们在转运蛋白上的结合位点。 我们将应用 时间分辨傅里叶变换红外（TR-FTIR）光谱， 生物膜对氯离子主动转运的研究。 的 待研究的模型系统是盐视紫红质（hR），一种光驱动的阴离子 在几个物种的膜中发现的转运蛋白 古细菌 比较了hR和 它的瞬态光中间体将有助于阐明结构 在这种蛋白质的功能发生变化。 广大 时间分辨FTIR差谱的灵敏度应允许 检测发色团中的瞬时结构变化，以及 肽骨架的局部构象改变; 可电离氨基酸侧链的质子化或去质子化;和 改变阳离子残基与 氯离子或其它阴离子。 这些测量的时间分辨率将 同位素标记的和突变的hR的光谱在1-10 μ s的范围内 还将获得蛋白质，以便为个体分配角色。 氨基酸在各种阴离子结合和释放步骤中的作用。 结果 应该允许开发一个详细的机制模型， 这个特殊系统的氯化物运输，以及 适用于哺乳动物阴离子转运蛋白的一般原则。

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.