Membrane protein folding and assembly

膜蛋白折叠和组装

• 批准号: 8197738
• 负责人: STEPHEN H. WHITE
• 金额: $ 35.37万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197738
• 关键词: Accounting; Affect; Algorithms; Amino Acid Sequence; Bacteria; Belief; Bioinformatics; Biological; Canis familiaris; Code; Collaborations; Complex; Defect; Development; Escherichia coli; Eukaryota; Goals; Hydrogen Bonding; Hydrophobicity; In Vitro; Laboratories; Lead; Lipid Bilayers; Lipids; Measurement; Mediating; Membrane; Membrane Proteins; Methanococcus; Molecular; Mutation; Organism; Pancreas; Pathway interactions; Peptide Hydrolases; Peptide Signal Sequences; Pharmaceutical Preparations; Protein Secretion; Proteins; Quality Control; Research; Signal Recognition Particle; Signal Transduction; System; base; biological research; design; genome-wide; in vivo; insight; molecular dynamics; mutant; protein folding; protein function; public health relevance; research study; therapeutic target; three dimensional structure

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. PUBLIC HEALTH RELEVANCE: An accurate first-principles algorithm for predicting the three-dimensional structure of membrane proteins from amino acid sequence would profoundly affect research on membrane proteins, which are major targets for therapeutic drugs. The project is guided by the idea that the keys to successful prediction are to understand the physical principles of membrane protein stability and the biological principles of membrane protein assembly. Our goal is to tighten the connection between the physical and biological principles as a basis for prediction algorithms.

描述（由申请人提供）：一种用于预测氨基酸序列1螺旋膜蛋白（MP）3D结构的准确第一原理算法将对MP功能的生物学研究进一步影响。这里提出的研究旨在使我们更接近该目标。该项目的指导是我们相信成功预测的关键是要了解脂质双层中MP稳定性的物理原理和MP组装的生物学原理，尤其是Translocon Complect的跨膜（TM）螺旋构成原理（真核生物中的SEC61123和细菌中的Secyeg）。该项目的“全局”目标是收紧以疏水性量表来预测TM螺旋的物理原理和生物学原理之间的联系。我们通过jannaschii的甲虫球菌对Secyeg易位的分子动力学模拟进行了发现，该甲壳虫的3D结构已知，易位能够通过复杂的氢键网络以其封闭状态稳定，该网络必须在信号序列引发的转运过程中进行重组。此外，我们发现该网络受到众所周知的大肠杆菌的PRLA突变的强烈扰动，这些突变引起蛋白质分泌和螺旋插入的缺陷。了解PRLA突变缺陷的分子基础将提供有关转运介导的MP组装机理的见解。因此，我们建议检查PRLA突变对MP组装过程中在体内选择跨膜螺旋的代码的影响。为了将PRLA突变与选择代码联系起来，我们将使用单SPAN MPS为大肠杆菌开发体内TM螺旋疏水性尺度，以利用沿两个不同途径插入TM螺旋的可能性：SECA后传输后途径或共同传播信号识别粒子（SRP）途径。这种方法将阐明物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令人困惑的结果。即使单个SPAN MP是所有生物中最丰富的MP，但它们从未在大肠杆菌中进行系统研究。初步结果表明，存在可能涉及FTSH MP质量控制蛋白酶的先前未识别的MP靶向信号。这些考虑因素导致了四个特定的目标：（1）在体内生物疏水性尺度上建立沿SECA和SRP途径插入单跨度MP的尺度。 （2）在分子动力学模拟的指导下，使用AIM 1的所得量表检查PRL突变体对TM螺旋的Secyeg选择的影响。 （3）为增强对MP的全基因组分析，对大肠杆菌单跨MPS进行了详细的生物信息学分析，并结合系统的实验研究，以表征和分类单跨度MPS。 （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。 公共卫生相关性：一种准确的第一原理算法，用于预测氨基酸序列的膜蛋白的三维结构，将深刻影响对膜蛋白的研究，膜蛋白是治疗药物的主要靶标。该项目的指导是成功预测的关键是了解膜蛋白稳定性的物理原理和膜蛋白质组装的生物学原理。我们的目标是收紧物理和生物学原理之间的联系，作为预测算法的基础。