Membrane Protein Folding and Assembly

摘要 膜蛋白折叠和组装(GM74637) 许多人类疾病，如囊性纤维化，都是由膜蛋白的错误折叠引起的。 (MPS)在合成和靶向过程中。因此，理解这些原则是很重要的 以及MP折叠和组装的机理。这个问题的一个很大程度上未被探索的部分是 在细胞环境的背景下理解折叠。为了实现这个目标，我们正在研究 膜蛋白的靶向、分泌和插入沿所谓的SECA后- 活体大肠杆菌的翻译途径。我们早些时候展示了赛卡发动机 ATPase是一个重要的药物靶点，可以跨膜插入单跨膜蛋白(S-SMPS) 大肠埃希氏菌内膜。这种简化的体内模型系统消除了许多 关于多跨MPS沿信号识别粒子折叠的未解问题 (SRP)途径，因为我们可以直接进入转运子-双层分配过程。 我们已经设计了两个不同的嵌合蛋白家族来系统地探测S- 使用GGPG-H-GPGG形式的TM片段的SMP稳定性(在早期的研究中使用 使用无细胞真核系统确定生物疏水性等级)。要确定 稳定性，我们已经开发出在体内通过天然切割TM片段的方法 膜内蛋白酶。我们发现，许多S-SMP在全国范围内是稳定的 仅因为它们的周质和细胞质区域不能穿过膜。 我们还发现，转位子到膜的转移能不等于 膜到细胞质转移的能量学及其稳定性依赖于生长 温度。尽管有数百篇论文，但人们对原子水平上的Seca函数知之甚少 在这个问题上。借助我们实验室在脂质-蛋白质相互作用方面的专业知识，我们已经开始 结合在脂质纳米盘上的SecA结构的电子冷冻显微镜(Cryo-EM)研究。 我们提出的研究的具体目标如下：(1)确定体内的 膜-细胞质疏水性标尺。(2)测定体内转位到- 膜疏水性标尺。(3)确定为什么刻度取决于温度， 我们推测这是由于温度相关的内膜脂组成所致。(4) 我们实验室开发的低温电子显微镜工具，用于测定赛卡在溶液和 当绑定到纳米盘上时，遥远的目标是描述分泌过程的每一步 在原子水平上。

ABSTRACT Membrane Protein Folding and Assembly (GM74637) Many human diseases, such as cystic fibrosis, result from misfolding of membrane proteins (MPs) during their synthesis and targeting. It is therefore important to understand the principles and mechanism of MP folding and assembly. A largely unexplored part of the problem is to understand folding in the context of the cellular milieu. Toward that goal, we are studying the targeting, secretion, and insertion of membrane proteins along the so-called SecA post- translational pathway of living Escherichia coli. We showed earlier that the SecA motor ATPase, a significant drug target, can insert single-span membrane proteins (S-SMPs) across the E. coli inner membrane. This simplified in vivo model system eliminates the many unanswered questions about the folding of multi-span MPs along the signal recognition particle (SRP) pathway, because we gain direct access to the translocon-bilayer partitioning process. We have engineered two different chimeric protein families for probing systematically S- SMP stability using TM segments of the form GGPG-H-GPGG (used in an earlier study to determine a biological hydrophobicity scale using a cell-free eukaryotic system). To determine stabilities, we have developed methods for cleaving TM segments in vivo via native intramembrane proteases. We have discovered that many S-SMPs are stable across the membrane only because their periplasmic & cytoplasmic domains cannot cross the membrane. We have also discovered that translocon-to-membrane transfer energetics are not equal to membrane-to-cytoplasm transfer energetics and that stability depends upon growth temperature. Little is known about SecA function at the atomic level despite hundreds of papers on the subject. Calling upon our lab’s expertise in lipid-protein interactions, we have begun electron cryomicroscopic (cryo-EM) studies of the structure of SecA bound to lipid nanodiscs. Our specific aims for the proposed research are the following: (1) Determine an in vivo membrane-to-cytoplasm hydrophobicity scale. (2) Determine an in vivo translocon-to- membrane hydrophobicity scale. (3) Determine why the scales depend upon temperature, which we hypothesize is due to temperature-dependent inner membrane lipid composition. (4) Develop in our laboratory cryo-EM tools for structural determinations of SecA in solution and when bound to nanodiscs with the distant goal of describing each step of the secretion process at the atomic level.