Membrane Protein Folding and Assembly

摘要 膜蛋白折叠和组装（GM 74637） 许多人类疾病，如囊性纤维化，是由膜蛋白的错误折叠引起的 (MPs)在合成和定位过程中。因此，理解这些原则非常重要。 以及MP折叠和组装的机构。问题的一个基本上未被探索的部分是 在细胞环境中理解折叠。为了实现这一目标，我们正在研究 靶向，分泌，和插入膜蛋白沿着所谓的SecA后， 活大肠杆菌的翻译途径。我们之前展示过SecA电机 ATP酶是一个重要的药物靶点，可以将单跨膜蛋白（S-SMPs）插入到细胞膜上， 急诊coli内膜。这种简化的体内模型系统消除了许多 关于沿着信号识别颗粒的多跨度MP折叠的未回答的问题 (SRP)途径，因为我们可以直接进入translocon-双层分配过程。 我们设计了两个不同的嵌合蛋白家族，用于系统地探测S- 使用GGPG-H-GPGG形式的TM片段的SMP稳定性（用于早期研究， 使用无细胞真核系统测定生物疏水性等级）。以确定 为了提高稳定性，我们已经开发了通过天然的方法在体内切割TM片段的方法。 膜内蛋白酶我们已经发现，许多S-SMP在整个 它们的细胞质和周质结构域不能穿过细胞膜。 我们还发现，translocon-膜转移能量不等于 膜到细胞质传递能量，稳定性取决于生长 温度尽管有数百篇论文，但在原子水平上对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， 结合到脂质纳米盘的SecA的结构的电子低温显微镜（cryo-EM）研究。 本研究的具体目标如下：（1）确定一种体内 膜-细胞质疏水性标度。(2)确定体内translocon-to- 膜疏水性标度(3)确定为什么刻度取决于温度， 我们假设这是由于温度依赖性内膜脂质组成。（四） 在我们的实验室中开发cryo-EM工具，用于溶液中SecA的结构测定， 当与纳米盘结合时，其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.