Folding and degradation of membrane proteins

摘要 (项目名称：膜蛋白的折叠和降解) 我的研究计划的目标是阐明潜在的化学和物理原理 膜蛋白的折叠和降解。单元格应该 保持生理上的最佳水平 有功能的蛋白质。这是通过分子伴侣的平衡作用来实现的，以促进 折叠，降解错误折叠和多余蛋白质的蛋白酶，以及压力反应信号 调节伴侣和蛋白水解酶水平的途径。这台蛋白质质量控制机 使用多层机制来监控蛋白质组的折叠状态。因此，一个人的命运 给定蛋白质，它是否会折叠或被降解，很大程度上取决于其固有的折叠特性 蛋白质的含量。虽然大多数折叠和降解研究都集中在水溶性 蛋白质，目前还不清楚膜蛋白质的内在折叠特性是如何影响的 他们的堕落。知识鸿沟主要源于研究膜的固有困难 蛋白质在天然脂双层环境中的折叠以及对 降解的折叠和序列决定因素。我们使用组合模型，该模型采用 以大肠杆菌膜整合三磷酸腺苷依赖的蛋白酶FtsH为模型降解机， 以大肠杆菌的膜内蛋白酶GlpG为模型底物，两者都是广泛存在的 在原核和真核细胞中保守。我们开发了一系列方法来研究 基于新的空间捕获原理的双层膜蛋白折叠。我们也 建立了一个体外双层系统来研究FtsH介导的膜蛋白的降解。 使用这些方法论创新，我对未来研究的愿景是学习可概括的 细胞内膜蛋白的折叠、蛋白质相互作用和质量控制课程 膜。我们将深入研究三个悬而未决的问题：1)详细的分子是什么 FtsH介导的膜蛋白降解机制？2)脂双层是很好的溶剂吗 对于膜蛋白的变性状态或促进其非特异性的不良溶剂 崩溃？3)嗜热生物的膜蛋白是如何实现其不同寻常的 热稳定性和活性？如果成功，这项研究的结果将推动我们的根本 了解膜蛋白降解的机理和能量学，确定新的 控制膜蛋白折叠和相互作用的脂双层的物理性质， 并发现膜蛋白的新的稳定基序，这将提供有用的设计和 工程学原理。

ABSTRACT (Project Title: Folding and Degradation of Membrane Proteins) The goal of my research program is to elucidate the chemical and physical principles underlying the folding and degradation of membrane proteins. Cells should maintain physiologically optimal levels of functional proteins. This is achieved by the balanced actions of molecular chaperones to facilitate folding, proteases to degrade misfolded and superfluous proteins, and stress-response signaling pathways to regulate levels of chaperones and proteases. This protein quality control machinery uses multiple layers of mechanisms to monitor the folding status of a proteome. Thus, the fate of a given protein, whether it will fold or be degraded, strongly depends on the intrinsic folding properties of the protein. While a majority of folding and degradation studies have focused on water-soluble proteins, it is not well understood how the intrinsic folding properties of membrane proteins affect their degradation. The knowledge gap mainly stems from inherent difficulties in studying membrane protein folding in their native lipid bilayer environment as well as an insufficient understanding of folding and sequence determinants for degradation. We use a combined model employing the membrane-integrated ATP-dependent protease FtsH of E. coli as a model degradation machine, and the intramembrane protease GlpG of E. coli as a model substrate, both of which are widely conserved in prokaryotic and eukaryotic cells. We developed an array of methods to study membrane protein folding in the bilayers based on the novel steric-trapping principle. We also developed an in vitro bilayer system to study FtsH-mediated degradation of a membrane protein. Using these methodological innovations, my vision for future research is to learn the generalizable lessons of folding, protein-protein interactions and quality control of membrane proteins in the cell membranes. We will delve into three unanswered problems: 1) What is the detailed molecular mechanisms of FtsH-mediated membrane protein degradation? 2) Is the lipid bilayer a good solvent for the denatured states of membrane protein or a poor solvent that promotes their nonspecific collapse? 3) How do membrane proteins from thermophilic organisms achieve their unusual thermostability and activity? If successful, the outcome of this study will advance our fundamental understanding of mechanisms and energetics of membrane protein degradation, identify new physical properties of the lipid bilayer that control folding and interactions of membrane proteins, and discover new stabilizing motifs for membrane proteins that will provide a useful design and engineering principle.