STATISTICAL THERMODYNAMIC STUDIES OF MEMBRANES

膜的统计热力学研究

• 批准号: 3293743
• 负责人: Ernesto Freire
• 金额: $ 18.26万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3293743
• 关键词: calorimetry; computer simulation; cytochrome c; cytochrome oxidase; densitometry; diphtheria toxin; glycosphingolipids; lipid bilayer membrane; membrane lipids; membrane proteins; membrane reconstitution /synthesis; membrane structure; microcalorimetry; mitochondrial membrane; phosphatidylcholines; phosphatidylserines; protein folding; thermodynamics

The major goal of this project is to gain a quantitative understanding of the forces and mechanisms that control the assembly, structural stability and functional properties of membrane proteins. As such, three major aspects of the interactions between membrane proteins and phospholipid bilayer membranes will be studied in detail using an experimental thermodynamic approach: 1) The mechanisms and energetics of folding, assembly and stabilization of membrane proteins; 2) The role of the lipid moiety and physicochemical environment on the stability and functional energetics of membrane proteins; and, 3) The energetics of protein insertion into membranes. The research program focuses on two well defined membrane systems: a) Cytochrome c oxidase and b) Diphtheria Toxin. Cytochrome c oxidase is a multisubunit enzyme located in the inner mitochondrial membrane. The three largest subunits are synthesized inside the mitochondria and the remaining subunits are imported from the cytoplasm. We are interested in studying the assembly process and structural stability of the enzyme, the role of the phospholipid bilayer matrix in maintaining the structural and functional integrity of the enzyme, the interactions between subunits, the interactions with its substrate cytochrome c, and the interactions with specific lipid components of the inner mitochondrial membrane (e.g. cardiolipin). Diphtheria toxin is composed of two fragments, A (MW=21,000) and B (MW=37,200), joined by a disulfide bridge. The toxin enters the cell by endocytosis, where it is exposed to a pH of approximately 5. The exposure of the toxin to an acidic environment provides the driving force for the membrane insertion and translocation of the toxic A fragment into the cytosol. One of the goals of this project is to understand protein-membrane interactions that result in the penetration of the protein into the bilayer. The goal in this case is to examine the sequence of protein unfolding -->penetration -->translocation -->refolding in vitro and to provide a complete thermodynamic mechanism for this process. This research project utilizes well defined reconstituted systems and a combination of biochemical and biophysical approaches including different types of calorimetry (high sensitivity differential scanning calorimetry, high sensitivity isothermal] titration calorimetry and multifrequency calorimetry), computer assisted quantitative gel electrophoresis, optical spectroscopy, and other spectroscopic techniques.

这个项目的主要目标是对 控制组件的力和机制，结构稳定性 以及膜蛋白的功能特性。因此，有三大 膜蛋白与磷脂相互作用的研究进展 我们将使用一种实验装置对双层膜进行详细研究。 热力学方法：1)折叠的机制和能量， 膜蛋白的组装和稳定；2)脂类的作用 部分和物理化学环境对稳定性和功能性的影响 膜蛋白的能量学；3)蛋白质的能量学 插入到膜中。该研究计划侧重于两个明确定义 膜系统：a)细胞色素c氧化酶和b)白喉毒素。 细胞色素c氧化酶是一种位于体内的多亚单位酶。 线粒体膜。三个最大的亚基在内部合成 线粒体和剩余的亚基是从 细胞质。我们有兴趣研究组装过程和 酶的结构稳定性、磷脂双层的作用 矩阵在维护结构和功能的完整性方面 酶，亚基之间的相互作用，与其相互作用 底物细胞色素c及其与特定脂类成分的相互作用 线粒体内膜(如心磷脂)。 白喉毒素由两个片段组成，A(MW=21,000)和B (相对分子质量=37,200)，由二硫键连接。毒素通过以下途径进入细胞 内吞作用，即暴露在pH约为5的环境中。 这种毒素在酸性环境中的扩散为 毒素A片段的膜插入和移位 胞浆。这个项目的目标之一是了解 导致蛋白质穿透的蛋白质-膜相互作用 进入双层。本例中的目标是检查 蛋白质去折叠--&gt；穿透--&gt；易位--&gt；体外重折叠 为此提供一个完整的热力学机制 进程。 该研究项目利用定义良好的重组系统和 生化和生物物理方法的结合，包括不同的 量热类型(高灵敏度差示扫描量热仪， 高灵敏度等温滴定量热法与多频 量热法)，计算机辅助定量凝胶电泳法，光学 光谱学和其他光谱技术。