Membrane protein folding and assembly

描述（由申请人提供）：用于从氨基酸序列预测1-螺旋膜蛋白（MP）的3D结构的准确的第一原理算法将深刻影响MP功能的生物学研究过程。这里提出的研究旨在使我们更接近这一目标。该项目是由我们的信念，成功的预测的关键是了解MP稳定性的脂质双层的物理原理和MP组装的生物学原理，特别是跨膜（TM）螺旋选择的原则，由translocon复合物（Sec 61123真核生物和SecYEG在细菌）。该项目的“大局”目标是加强物理和生物学原理之间的联系，以预测TM螺旋的疏水性尺度为代表。我们已经发现，通过分子动力学模拟的SecYEG translocon从詹氏甲烷球菌，其三维结构是已知的，该translocon是稳定在其封闭状态下的一个复杂的氢键网络，必须在translocon开放的信号序列启动重建。此外，我们已经发现，这个网络是强烈扰动的大肠杆菌，引起蛋白质分泌和螺旋插入缺陷的众所周知的prlA突变。了解prlA突变缺陷的分子基础将提供对translocon介导的MP组装机制的见解。因此，我们建议检查prlA突变对E. coli translocons在MP组装过程中选择跨膜螺旋。为了将prlA突变与选择密码联系起来，我们将开发E.利用TM螺旋沿着两条不同的途径插入的可能性：SecA翻译后途径或共翻译信号识别颗粒（SRP）途径，使用单跨MP的大肠杆菌。这种方法将阐明物理学原理和生物学原理之间的关系。我们开发单跨MP系统的第一步产生了意想不到的和令人困惑的结果。尽管单跨膜蛋白是所有生物体中含量最丰富的膜蛋白，但在大肠杆菌中还没有对其进行系统的研究。杆菌初步结果表明，存在以前未识别的MP靶向信号，可能涉及FtsH MP质量控制蛋白酶。这些考虑导致了四个具体的目标：（1）建立体内生物疏水性尺度的插入单跨度MP沿着SecA和SRP途径。(2)在分子动力学模拟的指导下，使用Aim 1的所得尺度来检查prl突变体对TM螺旋的SecYEG选择的影响。(3)为了加强对MP的全基因组分析，对E.大肠杆菌单跨膜蛋白结合系统的实验研究，对单跨膜蛋白进行表征和分类。(4)表征新的靶向信号和可能涉及FtsH MP质量控制蛋白酶的潜在单跨MP插入途径。

DESCRIPTION (provided by applicant): An accurate first-principles algorithm for predicting the 3D structure of 1-helical membrane proteins (MPs) from amino acid sequence would profoundly affect the course of biological research on MP function. The research proposed here is designed to bring us closer to that goal. The project is guided by our belief that the keys to successful prediction are to understand the physical principles of MP stability in lipid bilayers and the biological principles of MP assembly, especially the principles of transmembrane (TM) helix selection by the translocon complex (Sec61123 in eukaryotes and SecYEG in bacteria). The "Big Picture" goal of this project is to tighten the connection between the physical and biological principles as represented by hydrophobicity scales for predicting TM helices. We have discovered through molecular dynamics simulations of the SecYEG translocon from Methanococcus jannaschii, whose 3D structure is known, that the translocon is stabilized in its closed state by an intricate hydrogen-bond network that must be restructured during translocon opening initiated by signal sequences. Furthermore, we have found that this network is strongly perturbed by the well known prlA mutations of Escherichia coli that cause defects in protein secretion and helix insertion. Understanding the molecular basis for prlA-mutation defects will provide insights into the mechanism of translocon-mediated MP assembly. We thus propose to examine the effect of prlA mutations on the code used by E. coli translocons in vivo to select transmembrane helices during MP assembly. To connect prlA mutations to the selection code, we will develop in vivo TM-helix hydrophobicity scales for E. coli using single-span MPs to take advantage of the possibility of inserting TM helices along two different pathways: the SecA post-translational pathway or the co-translational signal recognition particle (SRP) pathway. This approach will clarify the relation between physical and biological principles. Our first steps in the development of a single-span MP system yielded unexpected and puzzling results. Even though single- span MPs are the most abundant MPs in all organisms, they have never been subjected to systematic study in E. coli. Preliminary results suggest the existence of previously unrecognized MP targeting signals that may involve the FtsH MP quality-control protease. These considerations lead to four specific aims: (1) Establish in vivo biological hydrophobicity scales for the insertion of single-span MPs along the SecA and SRP pathways. (2) Guided by molecular dynamics simulations, use the resulting scales of Aim 1 to examine the effects of prl mutants on SecYEG selection of TM helices. (3) To enhance genome-wide analyses of MPs, carry out a detailed bioinformatics analysis of E. coli single-span MPs in combination with systematic experimental studies to characterize and classify single-span MPs. (4) Characterize new targeting signals and a potential single- span MP insertion pathway that may involve the FtsH MP quality-control protease.