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.

膜生物学的一个基本目标是理解和预测蛋白质序列如何折叠， 在脂质双层中定向。至少10%的膜蛋白（MP）致病性突变会导致错误- 它们的跨膜结构域（TMD）的方向。大多数研究集中在蛋白质和膜 很少考虑脂质环境如何影响TMD组织。长期 该提案的目标是了解脂质-蛋白质相互作用在组装，结构和 MP的功能。使用分子遗传学和生物化学相结合的方法，我们建立了脂质- 在体内MP组装期间和组装之后，依赖性TMD取向是动态的，并且独立于其他因素。 体外细胞因子。TMD拓扑结构仅依赖于MP及其脂质的内在性质 环境指示可以在任何时间在任何细胞膜中发生的生物驱动过程。 我们开发了一套大肠杆菌的脂质突变体，其中脂质组成可以在 暂时在MP插入后。这种突变体集和一种新的蛋白脂质体系统，其中脂质成分可以 MP复溶前后的控制将用于检测TMD的动力学和热力学限度 通过脂质-蛋白质相互转化，建立直接的脂质-蛋白质相互作用，作为体内观察到的表型的基础。 结合MP和脂质性质的研究，我们制定了电荷平衡规则， 假设脂质头部基团和MP膜外结构域（EMD）之间的电荷相互作用是一个 MP拓扑的行列式该规则解释了MP的稳定、动态和对偶拓扑组织， 提供了在更复杂的真核系统中MP的脂质依赖性组装的原理证明。目标1 我们将通过后组装来确定对MP拓扑中脂质诱导的后组装变化施加的限制， 翻译糖基化和EMD的磷酸化。我们将确定磷酸化的速率- 触发拓扑变化发生迅速，足以代表一种新的代谢调节机制 与MP动态组织相关联。我们将把我们的研究扩展到FtsK，它的磷酸化似乎改变了 TMD拓扑结构是E.杆菌在目标2中，我们将建立基于生物学的 膜插入尺度的个别氨基酸，并纳入脂质组成，这还没有 以前考虑过。了解脂质成分如何影响TMD插入和定向将提供 初始TMD取向的分子基础，可逆的组装后TMD重取向，MP拓扑 突变体或异源表达的MP的异质性和错误折叠。在目标3中，我们将分析脂质 组成、脂质跨双层不对称性和膜生物能参数调节有效网络 这将提供对电荷平衡规则的机械水平的理解。 将新的MP组装规则外推到真核细胞将为以下方面的分子基础提供深入了解： 由错误折叠的MP引起的疾病。