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通过细胞或细胞器的膜转移的耦合对生命是必不可少的，因为它是生物系统建立电化学梯度的手段，可用于以ATP形式产生和存储能量。这项拟议的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。我们方法的力量来自于将细菌光合作用反应中心(RC)的高分辨率结构与表征天然和突变RC复合体功能的丰富光谱数据相关联的能力。这一策略的补充是操纵红杆菌的生理学能力，以对功能受损的工程RCS施加选择性压力。 利用单晶的X射线衍射，我们将确定携带控制质子转移到次生对苯二酚QB的突变的球形红曲霉的RCS的结构。该项目的主要重点将是一组RCS的结构特征，这些RCS来自于不具有光合作用能力的工程菌株的表型回复突变体。携带工程突变的RCS不能将第一和/或第二质子转移到还原的QB。生物物理研究已经确定，表型反转剂中的第二位点补偿突变-其中一些距离QB或原始工程取代位相当远-通过激活替代的质子传递途径恢复RCS的功能。在初步研究中，我们确定了一个功能受损的突变体RC的结构(Pokkuluri等人，生物化学41：5998-6007,2002)。我们现在正在确定两个RCS的结构，这两个RCS来自它的表型逆变体，来自衍射到2.7-2.8A的晶体。远距离的补偿突变通过邻近氨基酸侧链的相关运动影响Q8附近的残基的位置和性质。我们观察到，这些主链位置和侧链方向的变化可以从补偿突变的位置延伸到40埃。 从这些突变和回复RCS晶体中收集的数据导致了出色的电子密度图，清楚地显示了突变的几个意想不到的影响。目前我们建议研究的大约10个表型逆转体包含不同的生理化学补偿氨基酸变化，这些变化发生在靠近和远离天然RC中所用的表观质子转移途径的位置。通过将结晶学数据与光谱数据相关联，我们将识别构成功能性、高效质子转移途径的结构元素；因此，我们将能够在其他蛋白质中区分类似的元素，其中跨膜质子转移是其共同特征。

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.