Protein sequence determinants and properties of the lipid bilayer that govern membrane protein dynamic organization

控制膜蛋白动态组织的脂质双层的蛋白质序列决定因素和特性

• 批准号: 9381548
• 负责人: WILLIAM DOWHAN
• 金额: $ 45.84万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9381548
• 关键词: Acidic Amino Acids; Affect; Amino Acid Sequence; Amino Acids; Attenuated; Biochemical; Bioenergetics; Biological; Biophysics; Cell division; Cell membrane; Cells; Charge; Complex; Crystallography; Cystic Fibrosis; Cytoplasm; Dementia; Dependence; Diabetes Mellitus; Disease; Endoplasmic Reticulum; Energy Transfer; Engineering; Environment; Equilibrium; Escherichia coli; Eukaryotic Cell; Event; Formulation; Goals; Head; Heterogeneity; In Vitro; Individual; Kinetics; Link; Lipid Bilayers; Lipids; Liposomes; Measures; Membrane; Membrane Biology; Membrane Lipids; Membrane Potentials; Membrane Proteins; Membrane Structure and Function; Metabolic; Methods; Mitochondria; Modeling; Molecular; Molecular Genetics; Mutation; Organelles; Organism; Pathogenicity; Peptides; Phenotype; Phosphatidylethanolamine; Phosphorylation; Physiological; Play; Positioning Attribute; Post-Translational Protein Processing; Prions; Process; Property; Proteins; Regulation; Role; Scabies; Signal Transduction; System; Testing; Thermodynamics; Time; Translations; Transmembrane Domain; base; glycosylation; in vivo; insight; membrane assembly; mutant; novel; pH gradient; protein folding; protein misfolding; protein reconstitution; proteoliposomes; response

A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. At least 10% of pathogenic mutations in membrane proteins (MPs) result in mis- orientation of their transmembrane domains (TMDs). Most studies focus on the protein and the membrane insertion machinery with little consideration of how lipid environment affects TMD organization. The long-term goal of this proposal is to understand the role of lipid-protein interactions in the assembly, structure and function of MPs. Using a combined molecular genetic and biochemical approach, we established that lipid- dependent TMD orientation is dynamic during and after MP assembly in vivo and is independent of other cellular factors in vitro. Dependence of TMD topology solely on the intrinsic properties of a MP and its lipid environment indicates a thermodynamically driven process that can occur in any cell membrane at any time. We developed a set of lipid mutants of Escherichia coli in which lipid composition can be regulated during or temporally after MP insertion. This mutant set and a new proteoliposome system in which lipid composition can be controlled before and after MP reconstitution will be used to test kinetic and thermodynamic limits of TMD interconversions and establish direct lipid-protein interactions as the basis for in vivo observed phenotypes. Combining studies of MP and lipid properties led to our formulation of the Charge Balance Rule, which postulates that charge interactions between lipid head groups and MP extramembrane domains (EMDs) are a determinant of MP topology. This rule explains stable, dynamic and dual topological organization of a MP and provides a proof of principle for lipid-dependent assembly of MPs in more complex eukaryotic systems. In Aim1 we will determine the limits imposed on lipid-induced post-assembly changes in MP topology by post- translation glycosylation and phosphorylation of EMDs. We will determine whether the rate of phosphorylation- triggered topological changes occur rapidly enough to represent a novel mechanism for metabolic regulation linked to MP dynamic organization. We will extend our studies to FtsK whose phosphorylation appears to alter TMD topology as a regulatory mechanism during cell division of E. coli. In Aim 2 we will build biologically based membrane insertion scales for individual amino acids that incorporate lipid composition, which has not been previously considered. Understanding how lipid composition affects TMD insertion and orientation will provide a molecular basis for initial TMD orientation, reversible post-assembly TMD reorientation, MP topological heterogeneity, and misfolding of mutant or heterologously expressed MPs. In Aim 3 we will analyze how lipid composition, lipid transbilayer asymmetry and membrane bioenergetic parameters modulate the effective net charge of EMDs, which will provide an understanding at the mechanistic level of the Charge Balance Rule. Extrapolating new MP assembly rules to eukaryotic cells will provide insight into the molecular basis for diseases resulting from misfolded MPs.

膜生物学的基本目标是了解和预测蛋白质序列如何折叠和如何折叠 脂质双层中的正面。膜蛋白中至少10％的致病突变（MPS）导致错误 其跨膜结构域（TMD）的方向。大多数研究侧重于蛋白质和膜 插入机械几乎没有考虑脂质环境如何影响TMD组织。长期 该建议的目标是了解脂质 - 蛋白质相互作用在组装，结构和 MPS的功能。使用分子遗传和生化方法，我们确定了脂质 在体内MP组装过程中和之后，依赖性TMD方向是动态的，并且与其他 体外细胞因子。 TMD拓扑的依赖性仅对MP及其脂质的内在特性 环境表明在任何时候都可以在任何细胞膜中发生热力学驱动的过程。 我们开发了一组大肠杆菌的脂质突变体，其中可以调节脂质成分 MP插入后的时间。这个突变体集和一个新的蛋白质脂体系统，其中脂质组成可以 在MP重建之前和之后，将使用用于测试TMD的动力学和热力学极限 相互转换并建立直接脂质 - 蛋白相互作用作为体内观察到的表型的基础。 结合MP和脂质特性的研究导致我们制定了电荷平衡规则，这 假设脂质头组和MP脑膜外域（EMD）之间的电荷相互作用是A MP拓扑决定因素。该规则解释了国会议员的稳定，动态和双重拓扑组织 在更复杂的真核系统中，MPS的脂质依赖性组装提供了原理证明。在AIM1中 我们将确定对脂质诱导的MP拓扑后组装后变化的限制 EMD的翻译糖基化和磷酸化。我们将确定磷酸化的速率是否 触发的拓扑变化发生得足够快，可以代表一种新型的代谢调节机制 链接到MP动态组织。我们将把研究扩展到FTSK，其磷酸化似乎改变了 TMD拓扑作为大肠杆菌细胞分裂期间的调节机制。在AIM 2中，我们将基于生物学建立 掺入脂质组成的单个氨基酸的膜插入量表，尚未是 先前考虑的。了解脂质成分如何影响TMD插入和方向将提供 初始TMD取向，可逆后TMD重新定位，MP拓扑的分子基础 突变体或异源表达MP的异质性和错误折叠。在AIM 3中，我们将分析如何脂质 组成，脂质跨贝贝不对称和膜生物能参数调节有效净 EMD的费用，这将在电荷余额规则的机械水平上提供理解。 将新的MP组装规则推送到真核细胞将提供有关分子基础的见解 由错误折叠的MPS产生的疾病。