Structural Basis of Proton Transfer Pathways

质子传递途径的结构基础

• 批准号: 6743737
• 负责人: MARIANNE SCHIFFER
• 金额: $ 28.39万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6743737
• 关键词: Rhodospirillales; X ray crystallography; bacterial proteins; electron transport; gene expression; gene mutation; hydrogen ions; ion transport; membrane permeability; membrane transport proteins; photosynthesis; protein structure function

DESCRIPTION (provided by applicant): The coupling of protein-mediated electron and proton transfer across a membrane of a cell or organelle is essential for life as it is the means by which biological systems establish an electrochemical gradient that can be used for the generation and storage of energy in the form of ATP. The proposed research will discover fundamental structural factors that underlie the bioenergetics and efficiency of electron-coupled proton transfer in transmembrane proteins. The power of our approach comes from the ability to correlate high-resolution structures of the bacterial photosynthetic reaction center (RC) with a wealth of spectroscopic data characterizing function of native and mutant RC complexes. This strategy is complemented by the ability to manipulate the physiology of Rhodobacter to apply selective pressure for 'repair' of engineered RCs whose function is impaired. Using x-ray diffraction of single crystals, we will determine the structures of RCs from R. sphaeroides carrying mutations that control proton transfer to the secondary quinone QB. The main emphasis of this project will be structural characterization of a panel of RCs derived from phenotypic revertants of engineered strains that are photosynthetically incompetent. RCs carrying the engineered mutations are incapable of transferring the first and/or second proton to reduced QB. Biophysical studies have determined that second-site compensatory mutations - some of which are quite distant from QB or the site of the original engineered substitutions - in the phenotypic revertants restore function to the RCs by activating alternative proton delivery pathways. In preliminary studies, we have determined the structure of one functionally impaired mutant RC (Pokkuluri et al., Biochemistry 41: 5998-6007, 2002). We are now in the process of determining the structures of two RCs derived from phenotypic revertants of it, from crystals that diffract to 2.7-2.8 A. The distant compensatory mutations influence the positions and properties of residues near Q8 through correlated motions of neighboring amino acid side chains. We have observed that these changes in main chain positions and side chain orientations can extend up to 40 angstrom from the site of the compensatory mutation. Data collected from these crystals of mutant and revertant RCs resulted in excellent electron density maps that clearly showed several unexpected effects of the mutations. The current panel of about 10 phenotypic revertants that we propose to study contains physiochemically diverse compensatory amino acid changes at sites both close to and distant from the apparent proton transfer pathway used within the native RC. By correlating the crystallographic data with the spectroscopic data, we will recognize the structural elements that constitute a functional, efficient proton transfer pathway; therefore, we will be able to discern similar elements in other proteins for which transmembrane proton translocation is a common feature.

描述（由申请人提供）：蛋白质介导的电子和质子通过细胞膜或细胞器传递的耦合对生命至关重要，因为它是生物系统建立电化学梯度的手段，可用于以ATP形式产生和储存能量。提出的研究将发现跨膜蛋白中电子偶联质子转移的生物能量学和效率的基本结构因素。我们的方法的力量来自于将细菌光合反应中心（RC）的高分辨率结构与表征天然和突变RC复合物功能的丰富光谱数据相关联的能力。这一策略还可以通过操纵红杆菌的生理机能来对功能受损的工程RCs施加选择性压力来“修复”。