Structural Basis of Proton Transfer Pathways

描述（由申请人提供）：蛋白质介导的电子和质子跨细胞或细胞器膜转移的偶联对生命至关重要，因为它是生物系统建立电化学梯度的手段，可用于产生和储存ATP形式的能量。这项研究将发现跨膜蛋白中电子耦合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。该策略通过操纵红细菌的生理学以施加选择性压力来“修复”功能受损的工程化RC的能力来补充。 利用单晶的X射线衍射，我们将确定来自R的RC的结构。携带控制质子转移到仲醌QB的突变的类球蛋白。该项目的主要重点将是一组RC的结构表征，这些RC来源于光合能力不强的工程菌株的表型回复突变体。携带工程化突变的RC不能将第一和/或第二质子转移至还原的QB。生物物理学研究已经确定，表型回复突变体中的第二位点补偿突变-其中一些与QB或原始工程取代位点相当遥远-通过激活替代质子传递途径恢复RC的功能。在初步研究中，我们已经确定了一种功能受损的突变体RC的结构（Pokkuluri等人，Biochemistry 41：5998-6007，2002）。我们现在正在确定两个RC的结构，这两个RC来自于它的表型回复突变体，来自于2.7-2.8 A的晶体。远距离补偿突变通过相邻氨基酸侧链的相关运动影响Q8附近残基的位置和性质。我们已经观察到，主链位置和侧链方向的这些变化可以从补偿突变位点延伸到40埃。 从这些突变体和回复突变体RC晶体中收集的数据产生了极好的电子密度图，清楚地显示了突变的几个意想不到的影响。我们建议研究的约10个表型回复突变体的当前面板在接近和远离天然RC内使用的表观质子转移途径的位点处包含理化多样的补偿性氨基酸变化。通过将晶体学数据与光谱学数据相关联，我们将认识到构成功能性的、有效的质子转移途径的结构元件;因此，我们将能够辨别出跨膜质子转运是其共同特征的其他蛋白质中的类似元件。

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.