Proton-Coupling Strategies in Alkaliphilic Bacillus

描述（由申请人提供）：本项目将阐明嗜碱芽孢杆菌在高外部pH值下将质子向内移动的两个过程所使用的机制：在化学渗透驱动力（负碱性）很低;以及Mrp钠/质子反向转运蛋白的新特性如何支持其支持显著的碱性pH稳态的能力，导致在外部pH值下的“反向pH梯度”，使得细胞质pH比最佳外部pH 10.5低2个单位以上。三个具体的目标将集中在以下问题，（一）如何具体功能的膜嵌入的a-和c-亚基的ATP合酶的质子捕获和运动通过ATP合酶作出不同的关键贡献？这些方法将包括分析定点突变体，改变质子路径中的关键ATP合酶残基的特性，并研究该合酶的c亚基化学计量。(ii)在高pH值条件下，一种特殊的呼吸链复合物--Cta细胞色素氧化酶与ATP合酶合作，将螯合质子转移到合酶中，它的特殊参与是什么？Cta和ATP合酶之间的蛋白质-蛋白质相互作用以及特定的条件和功能的两个复杂的参与这种相互作用将是重点，（iii）什么是七个MRP蛋白质的作用和MRP反向转运蛋白的属性，占其不寻常的复杂性和功效？多管齐下的生物化学，遗传学和生理学的方法将测试的假设：（a）这种二级钠/质子反向转运蛋白的功能作为一种新的，异源寡聚复合物;和（B）一些Mrp蛋白具有独特的催化活性，具有积极的协同作用与Mrp依赖性反向转运。 研究了两个区域在碱中繁衍生息的细菌。它们是一个模型系统，用于解开氧依赖性细胞产生化学能（如ATP）的细节。假设这些细菌与线粒体共享策略，因此ATP合成的发现对细胞呼吸的正常和病理状态具有药理学意义。Mrp系统的研究表征了在许多细菌病原体中发现的新型蛋白质载体。Mrp是开发新抗生素的潜在靶点。

DESCRIPTION (provided by applicant): This project will clarify the mechanisms used by alkaliphilic Bacillus for the two processes that move protons inward at high external pH: how extremely robust proton-coupled oxidative phosphorylation (OXPHOS) occurs under conditions in which the chemiosmotic driving force (negative and alkaline in) is very low; and how the novel properties of the Mrp sodium/proton antiporter underpin its capacity to support remarkable alkaline pH homeostasis, resulting in a "reversed pH gradient" at external pH values such that the cytoplasmic pH is more than 2 units below an optimal external pH of 10.5. Three specific aims will focus on the following questions, (i) How do specific features of the membrane-embedded a- and c-subunits of ATP synthase make distinct critical contributions to proton capture and movement through the ATP synthase? The approaches will include analyses of site-directed mutants that alter properties of critical ATP synthase residues in the proton path and studies of the c-subunit stoichiometry of the synthase. (ii) What is the special involvement of a particular respiratory chain complex, the Cta cytochrome oxidase, in partnering with the ATP synthase at high pH for sequestered proton transfers to the synthase? Protein-protein interactions between Cta and ATP synthase as well as specific conditions and features of the two complexes involved in such interactions will be the focus, (iii) What are the roles of the seven Mrp proteins and the properties of the Mrp antiporter that account for its unusual complexity and efficacy? A multi-pronged biochemical, genetic and physiological approach will test the hypotheses that: (a) this secondary sodium/proton antiporter functions as a novel, hetero-oligomeric complex; and (b) some Mrp proteins have distinct catalytic activities that have positive synergy with Mrp-dependent antiport. Two areas are studied bacteria that thrive in alkali. They are a model system for unraveling details of chemical energy production (as ATP) by oxygen-dependent cells. These bacteria are hypothesized to share strategies with mitochondria so the findings on ATP synthesis have pharmacalogical implications for normal and pathological states of cellular respiration. The studies of the Mrp system characterize a novel protein carrier found in many bacterial pathogens. Mrp is a potential target for development of new antibiotics.