Light-Driven Ion Transport in Bacterial Rhodopsins

细菌视紫红质中的光驱动离子传输

• 批准号: 8325065
• 负责人: JANOS K LANYI
• 金额: $ 52.95万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8325065
• 关键词: Active Ion Transport; Active Sites; Affinity; Archaeal Proteins; Aspartate; Bacterial Rhodopsins; Bacteriorhodopsins; Binding; Biological; Biological Models; Carrier Proteins; Cell membrane; Cells; Cleaved cell; Complex; Coupling; Crystallization; Cytoplasmic Tail; Development; Devices; Digestive System Disorders; Elements; Escherichia coli; Family; Fluorescence; Health; Heart Diseases; Histidine; Homologous Gene; Hydrogen Bonding; Intervention; Ion Transport; Ions; Life; Light; Location; Measures; Membrane; Membrane Proteins; Methods; Modeling; Optics; Organelles; Pathway interactions; Pharmacologic Substance; Process; Proteins; Proton Pump; Protons; Publishing; Pump; Reporting; Retinal; Rhodopsin; Role; Schiff Bases; Site; Solutions; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Spin Labels; Structure; Surface; Testing; Water; Work; absorption; aqueous; base; extracellular; insight; ionic balance; mutant; nervous system disorder; novel; public health relevance; uptake

DESCRIPTION (provided by applicant): Ionic and electrical gradients across cell and organelle membranes are created by pumps, whose correct functioning is essential to all biological and biomedical processes. Dysfunctional membrane proteins of this kind underlie many cardiac, neurological and digestive diseases, and they have been prominent targets of pharmaceutical intervention. We will study and describe proton transfer mechanisms in a newly discovered family of proton pumps that deviate from those in a model system employed earlier to generate general principles for active ion transport. The aims of this proposal focus on the functional implications of our recently reported x-ray diffraction structure of xanthorhodopsin. We expect to uncover novel means of proton transfer in proteins, with insights into how energy input is used to move protons uphill, across the membrane. We hope that the findings will enrich the general field of membrane proteins, and that of transport proteins in particular. We will explore the consequences of three main differences from the x-ray structure of bacteriorhodopsin that we had reported earlier. a) The critical proton transfer, which in these proteins drives the transport, is from the retinal Schiff base to an aspartate connected via a water molecule at the active site. We now know that the proton acceptor can be also an aspartate-histidine complex with very likely shared protons. b) Release of protons to the bulk can be via an extended hydrogen-bonded network of polar residues and bound water, but now we know that an alternative exists that utilizes a deep cleft that extends into the proteins. c) Uptake of protons and reprotonation of the Schiff base by an internal acidic residue is via a transient chain of water molecules that spans an otherwise hydrophobic domain, during the transport cycle. We now know that this domain may contain water molecules that connect the proton donor to the Schiff base region. The experimental approaches to uncover the changed functions include the battery of methods we and our collaborators had developed over the past decades: construction of site-specific mutants expressed in E. coli, static and transient optical spectroscopy (absorption and fluorescence), site-specific spin labeling, Fourier Transform Infrared spectroscopy, crystallization and x-ray diffraction, and solution NMR. PUBLIC HEALTH RELEVANCE: Life and health depend on maintaining the correct ionic balance between the interior of cells and their surroundings. The proposed work will contribute to the fundamental understanding of how pumps transport ions like protons across cell membranes. It will take advantage of novel features in a newly discovered family of pumps, and extend the range of possible mechanistic elements that add up to the translocation of the ion in these membrane devices.

描述（由申请人提供）：跨细胞和细胞器膜的离子和电梯度由泵产生，其正确的功能对于所有生物学和生物医学过程至关重要。这种类型的功能失调的膜蛋白是许多心脏，神经系统和消化疾病的基础，它们一直是药物干预的重要靶标。我们将研究和描述新发现的质子泵家族中的质子转移机制，这些质子泵家族偏离了较早的模型系统中用于生成主动离子运输的一般原理的模型系统中的质子转移机制。该提案的目的集中于我们最近报道的黄摩淡鱼的X射线衍射结构的功能含义。我们期望揭示蛋白质中质子转移的新型手段，并了解如何使用能量输入将质子移动到整个膜上。我们希望这些发现将丰富膜蛋白的一般领域，尤其是运输蛋白的蛋白质。我们将探讨我们之前报道的细菌紫红质X射线结构的三个主要差异。 a）临界质子转移在这些蛋白质中驱动运输的临界质子转移是从视网膜席夫底座到通过活性位点的水分子连接的天冬氨酸。我们现在知道，质子受体也可以是具有很可能共享质子的天冬氨酸 - 希斯丁碱复合物。 b）将质子释放到散装物可以是通过延长的极性残基和结合水的氢键网络，但是现在我们知道存在一种替代性，它利用了延伸到蛋白质的深层裂口。 c）在运输周期内，内部酸性残基对质子的摄取和通过内部酸性残基对质子碱的反应是通过瞬时跨越原本疏水结构域的水分子链。我们现在知道，该域可能包含将质子供体连接到Schiff基地区域的水分子。发现变化功能的实验方法包括我们和我们的合作者在过去几十年中开发的一系列方法：在大肠杆菌中表达的现场特异性突变体的构建，静态和瞬态光谱（吸收和荧光），位点特异性的自旋标记，傅里叶特异性旋转标签，傅里叶傅立叶转换光谱谱图，结晶和X-ray diffractir和x-ray diffraction n nmr nmr nmr nmr nmr nmr nmr nmr。 公共卫生相关性：生活和健康取决于保持细胞内部及其周围环境之间的正确离子平衡。拟议的工作将有助于对泵如何运输质子等细胞膜等泵离子的基本理解。它将利用新发现的泵系列中的新功能，并扩展到这些膜设备中离子易位的可能机械元素的范围。