Proton-Coupling Strategies in Alkaliphilic Bacillus

描述(申请人提供)：这个项目将阐明嗜碱芽孢杆菌在高外部pH下将质子移入内部的两个过程所使用的机制：在化学渗透驱动力(负的和碱性的In)非常低的条件下，如何发生极其强劲的质子偶联氧化磷酸化(OXPHOS)；以及MRP钠/质子逆向转运体的新特性如何支持其支持显著的碱性pH动态平衡的能力，从而在外部pH值下导致“反向pH梯度”，从而使细胞质pH比最佳外部pH 10.5低2个单位以上。三个特定的目标将集中在以下问题上：(I)膜包埋的ATP合成酶a-和c-亚基的特定功能如何在通过ATP合成酶捕获和移动质子方面做出不同的关键贡献？这些方法将包括对改变质子路径中关键ATP合成酶残基性质的定点突变的分析，以及对合成酶c亚单位化学计量学的研究。(Ii)特定的呼吸链复合体，CTA细胞色素氧化酶，在高pH时与ATP合成酶配对，将隔离的质子转移到合成酶中，有什么特殊的参与？CTA和ATP合成酶之间的蛋白质-蛋白质相互作用以及参与这种相互作用的两个复合体的具体条件和特征将是重点，(Iii)七种MRP蛋白的作用和MRP反向转运体的性质是解释其异常复杂性和有效性的原因？一种多管齐下的生化、遗传学和生理学方法将检验以下假设：(A)这种次级钠/质子逆向转运蛋白作为一种新的异源低聚复合体发挥作用；以及(B)一些MRP蛋白具有明显的催化活性，与MRP依赖的反向转运蛋白具有正向协同作用。 研究了两个领域的细菌，它们在碱性环境中茁壮成长。它们是一个模型系统，用于解开氧气依赖细胞产生化学能量(如ATP)的细节。这些细菌被假设与线粒体共享策略，因此关于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。对MRP系统的研究表明，在许多细菌病原体中发现了一种新的蛋白质载体。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.