Membranes of Archaebacteria and Acidophilic Thiobacilli

本项目探讨了嗜热嗜酸古细菌和嗜酸硫杆菌的膜。这些细菌生活在恶劣的环境中，包括低pH值（2-4）和高温（50-70℃）。由于这种环境的不寻常方面，人们假设它们将具有不寻常的膜，使它们能够在极端的pH值和温度下生存。事实上，众所周知，古细菌作为一个群体不具有正常的酯类脂质，而是具有四醚类脂质。该建议的基础是这些细菌膜的不寻常性质将提供与真核生物膜不同的脂质和离子运输蛋白。这些差异将有助于了解这些细菌的细胞生物学以及真核脂质和离子转运蛋白的功能。本项目拟利用两个实验室的专业知识来研究这些细菌膜中的脂质和蛋白质。该项目包括两个子项目，一个是Smith博士实验室的离子转运蛋白，另一个是Chong博士实验室的脂质和脂质蛋白相互作用，如下所述。子项目1：鉴定和表征与嗜酸硫杆菌和嗜热嗜酸古细菌跨膜离子运输相关的膜atp酶。这个子项目的目标是：1。筛选各种菌株的古细菌和嗜酸硫杆菌的膜为离子易位atp酶使用免疫和酶的测定。2. 表征在特定目标1中发现的atp酶活性，关于抑制剂特异性，pH最优，阳离子运输和动力学性质。3. 纯化与特定目的1和2中描述的atp酶活性相关的一些蛋白质。特别强调的是p型atp酶。子项目2：从嗜热酸性古细菌中分离的四醚脂质组成的膜的物理性质的表征。在这个子项目中：1；稳态和动态荧光法将用于研究芘标记的脂类的横向运动和二苯己三烯（DPH）和苝在四醚脂质膜中的旋转运动。2. 四醚脂质膜中小分子的横向和旋转运动作为压力的函数将使用高压荧光法进行研究。3. 荧光法和常规生化方法将用于研究四醚脂质膜中古细菌（Na+,K+）型atp酶的拓扑结构。

This project explores the membranes of the thermoacidophilic archaebacteria and the acidophilic thiobacilli. These bacteria live in harsh environments including low pH (2-4) and high temperature (50-70C). Due to the unusual aspects of such environments it is hypothesized that they will have unusual membranes which allow them to survive at extremes of pH and temperature. In fact, it is known that the archaebacteria as a group do not have normal ester lipids but rather tetraether lipids. The foundation of the proposal is that the unusual nature of the membranes of these bacteria will provide lipids and ion transport proteins distinct from those in eukaryotic membranes. These differences will be informative in understanding both the cell biology of these bacteria and the function of eukaryotic lipids and ion transport proteins. This project proposes to use the expertise in two laboratories to study the lipids and proteins in the membranes of these bacteria. This project comprises two sub-projects, one on ion transport proteins in Dr. Smith's laboratory and the other on lipids and lipid protein interactions in Dr. Chong's laboratory as outlined below. Sub-project 1: Identification and characterization the membrane ATPases associated with transmembrane ion transport in acidophilic thiobacilli and thermoacidophilic archaebacteria. The goals of this sub- project are to: 1. Screen the membranes of various strains of archaebacteria and acidophilic thiobacilli for ion translocating ATPases using immunologic and enzymatic assays. 2. Characterize the ATPase activities found in specific aim 1, with regard to inhibitor specificity, pH optima, cation transport and kinetic properties. 3. Purify of some of the proteins associated with the ATPase activities described in specific aims 1 and 2. Particular emphasis will be placed on P-type ATPases. Sub-project 2: Characterization of the physical properties of membranes comprised of tetraether lipids isolated from thermoacidophilic archaebacteria. In this sub-project: 1. Steady-state and dynamic fluorometry will be employed to investigate the lateral motion of pyrene-labeled lipids and the rotational motion of diphenylhexatriene (DPH) and perylene in tetraether lipid membranes. 2. The lateral and rotational motions of small molecules in tetraether lipid membranes as a function of pressure will be studied using high pressure fluorometry. 3. Fluorometry and conventional biochemical methods will be used to study the topology of archaebacterial (Na+,K+)- type ATPase in tetraether lipid membranes.