STATISTICAL THERMODYNAMIC STUDIES OF MEMBRANES

这个项目的主要目标是获得一个定量的了解 控制装配、结构稳定性的力和机制 和膜蛋白的功能特性。 因此，三大 膜蛋白和磷脂之间的相互作用 双层膜将详细研究使用实验 热力学方法：1）折叠的机制和能量学， 膜蛋白的组装和稳定; 2）脂质的作用 部分和物理化学环境对稳定性和功能的影响 膜蛋白的能量学; 3）蛋白质的能量学 插入膜中。 该研究计划的重点是两个明确的 膜系统：a）细胞色素c氧化酶和B）白喉毒素。 细胞色素c氧化酶是一种多亚基酶，位于细胞内， 线粒体膜 三个最大的亚基是在 线粒体和其余的亚基从线粒体中输入， 细胞质 我们有兴趣研究组装过程， 酶的结构稳定，磷脂双分子层的作用 矩阵在维持结构和功能的完整性， 酶，亚基之间的相互作用， 底物细胞色素c，以及与特定脂质成分的相互作用 线粒体内膜（如心磷脂）。 白喉毒素由两个片段A（MW= 21，000）和B组成 (MW= 37，200），通过二硫键连接。 毒素通过以下途径进入细胞 在胞吞作用中，其暴露于约5的pH。曝光 毒素释放到酸性环境中， 膜插入和易位的有毒A片段进入 胞质液 这个项目的目标之一是了解 导致蛋白质渗透的蛋白质-膜相互作用 进入双层。 在这种情况下，目标是检查 体外蛋白质解折叠-->穿透-->易位-->重折叠， 来提供一个完整的热力学机制 过程 该研究项目利用定义良好的重构系统和 生物化学和生物物理方法的组合，包括不同的 量热法的类型（高灵敏度差示扫描量热法， 高灵敏度等温滴定量热法 量热法），计算机辅助定量凝胶电泳，光学 光谱和其他光谱技术。

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.