Membrane protein folding and assembly

描述(申请人提供)：从氨基酸序列预测1-螺旋膜蛋白(MPS)三维结构的准确第一性原理算法将深刻影响MP功能的生物学研究进程。这里提出的研究旨在让我们更接近这一目标。该项目的指导思想是，成功预测的关键是了解MP在脂质双层中稳定性的物理原理和MP组装的生物学原理，特别是转位复合体(真核生物中的Sec61123和细菌中的SecYEG)选择跨膜(TM)螺旋的原理。该项目的“大图景”目标是加强物理和生物原理之间的联系，如用于预测TM螺旋的疏水性标尺所代表的那样。我们通过对已知三维结构的简氏甲烷球菌SecYEG易位子的分子动力学模拟发现，该易位子是由一个错综复杂的氢键网络稳定在其闭合状态的，在信号序列启动的转位子打开过程中必须对该网络进行重构。此外，我们还发现，这个网络受到众所周知的大肠杆菌prlA突变的强烈干扰，这些突变导致蛋白质分泌和螺旋插入缺陷。了解PrlA突变缺陷的分子基础将有助于深入了解转运子介导的MP组装的机制。因此，我们建议研究PrlA突变对体内大肠杆菌转座子在MP组装过程中选择跨膜螺旋所使用的编码的影响。为了将prlA突变与选择密码联系起来，我们将使用单跨MPS在体内开发大肠杆菌TM-螺旋疏水性标尺，以利用沿着两种不同途径插入TM螺旋的可能性：SecA翻译后途径或共翻译信号识别粒子(SRP)途径。这种方法将澄清物理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费解的结果。尽管单跨MPS是所有生物体中含量最丰富的MPS，但它们从未在大肠杆菌中进行过系统的研究。初步结果表明，存在以前未被识别的MP靶向信号，可能涉及FtsH MP质控蛋白酶。这些考虑导致了四个具体的目标：(1)建立体内生物疏水性标准，用于沿着SecA和SRP途径插入单跨MPS。(2)在分子动力学模拟的指导下，使用目标1的结果尺度来检验PRL突变体对TM螺旋的SecYEG选择的影响。(3)为了加强MPS的全基因组分析，结合系统的实验研究，对大肠杆菌单跨MPS进行了详细的生物信息学分析，对单跨MPS进行了表征和分类。(4)鉴定可能涉及FtsH MP质控酶的新靶向信号和潜在的单跨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.