Dynamic Lipid and Protein Organization in Cell Membranes

细胞膜中的动态脂质和蛋白质组织

• 批准号: 10443292
• 负责人: Mikhail V. Bogdanov
• 金额: $ 52.45万
• 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 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10443292
• 关键词: Address; Aerobic; Affect; Amino Acid Sequence; Amino Acids; Anaerobic Bacteria; Area; Behavior; Biochemical; Biological; Cardiolipins; Cations; Cell division; Cell membrane; Cell physiology; Cells; Charge; Chemicals; Cystic Fibrosis; Cytoplasm; Defect; Dementia; Diabetes Mellitus; Disease; Engineering; Environment; Enzymes; Equilibrium; Escherichia coli; Eubacterium; Event; Genes; Genetic Complementation Test; Goals; Gram-Negative Bacteria; Growth; Hydrophobicity; In Vitro; Integral Membrane Protein; Laboratories; Libraries; Lipid Bilayers; Lipids; Measures; Mechanics; Membrane; Membrane Biology; Membrane Lipids; Membrane Proteins; Membrane Structure and Function; Methods; Modeling; Molecular; Molecular Genetics; Mutation; Organelles; Organism; Pathogenicity; Pattern; Peptides; Phenotype; Phosphatidylethanolamine; Phosphatidylglycerols; Phospholipids; Phosphorylation; Physiological; Play; Prions; Process; Protein Dephosphorylation; Protein Engineering; Proteins; Reagent; Role; Scabies; Septal Region; Signal Transduction; System; Testing; Thermodynamics; Transmembrane Domain; cardiolipin synthase; cell growth; driving force; experimental study; in vivo; insight; interfacial; membrane assembly; method development; misfolded protein; mutant; new growth; novel; protein misfolding; protein structure function; proteoliposomes; reconstitution; response; scaffold; temperature sensitive mutant

Abstract A fundamental objective in membrane biology is to understand and predict how protein sequences fold and orient in a lipid bilayer. Most studies focus on the membrane protein (MP) and the membrane insertion machinery with little consideration of how lipid environment affects transmembrane domain (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 (i.e., can reversibly change) during and after MP assembly in vivo and is independent of other cellular factors in vitro. We proposed the Charge Balance Rule, which includes an acitve role for lipid environment, to explain the dynamic behavior of MPs. We developed a library of lipid mutants of Escherichia coli in which lipid composition can be regulated during or temporally after MP membrane insertion. The physiological significance of membrane lipid bilayer asymmetry is an understudied area. We now add to this library a set of E. coli strains and proteoliposome systems in which membrane lipid bilayer asymmetry can be controlled to determine the role of lipid bilayer asymmetry in MP dynamic organization. Using this set of lipid reagents and native or engineered MPs, we propose three integrated Specific Aims: Aim 1, We will quantify in thermodynamic terms and determine the mechanistic and structural principles underlying the molecular driving forces, as proposed by the Charge Balance Rule, that govern dynamic TMD topology organization as a function of membrane lipid composition and asymmetry. Aim 2, The role of CL in cell functions is not well understood. CL transbilayer asymmetry across any biological membrane is unknown, and there is still no reliable method for estimating its distribution across any membrane. E. coli lacking CL display several phenotypes under aerobic and anaerobic conditions, which would seriously compromise growth in the wild. We will address the role of CL, its transmembrane asymmetry and the three CL synthases in supporting critical cell functions, which will be applicable to the understanding of the importance of CL and cls gene multiplicity in pathogenic Gram-negative bacteria. Aim 3, An essential MP (FtsK) was identified by us associated with a late stage of cell division (a defect in phosphatidylethanolamine-lacking cells) that is topologically responsive to changes in lipid composition and phosphorylation of its extramembrane domains. We will address how changes in lipid composition/asymmetry and phosphorylation/dephosphorylation cycles affect the function of FtsK as an example of a physiological function potentially governed by the Charge Balance Rule. By focusing on interfacial protein-lipid interactions in lipid bilayers with different lipid compositions and asymmetry, measuring electric forces acting on nascent MPs during and after assembly, and characterizing physiologically important roles for CL (as we have for phosphatidylethanolamine), we will enhance our understanding of the forces that define MP structure and function and the role membrane lipids play in cell processes.

抽象的 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方向 在脂质双层中。大多数研究侧重于膜蛋白（MP）和带有膜插入机械 几乎没有考虑脂质环境如何影响跨膜域（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，结构和功能中的作用 国会议员。使用分子遗传和生化方法，我们确定了脂质依赖性 TMD方向是动态的（即可以在体内和之后组装后和之后的动态变化），并且是独立的 其他细胞因子体外。我们提出了收费余额规则，其中包括脂质的敏捷角色 环境，解释MPS的动态行为。我们开发了大肠杆菌的脂质突变体库 其中在MP膜插入后或时间上可以调节脂质成分。生理 膜脂质双层不对称的重要性是研究的区域。现在，我们将其添加到此库中。 大肠杆菌菌株和蛋白质脂质体系统，其中膜脂质双层不对称可以控制为 确定脂质双层不对称在MP动态组织中的作用。使用这组脂质试剂和 本地或工程MPS，我们提出了三个集成的特定目的：AIM 1，我们将在热力学中进行量化 术语并确定分子驱动力的基础机械和结构原理，如 收费余额规则提出的，管理动态TMD拓扑组织 膜脂质组成和不对称性。 AIM 2，CL在细胞功能中的作用尚不清楚。 Cl 跨任何生物膜的跨贝伊尔不对称尚不清楚，并且仍然没有可靠的方法 估计其在任何膜上的分布。缺乏CL的大肠杆菌在有氧运动下显示几种表型 和厌氧条件，这将严重损害野外的生长。我们将解决CL的角色， 它的跨膜不对称和支持关键细胞功能的三个CL合酶，这将是 适用于理解CL和CLS基因多样性在致病性革兰氏阴性阴性中的重要性 细菌。 AIM 3，与细胞分裂后期相关的我们确定了必不可少的MP（FTSK）（缺陷 在磷脂酰乙醇胺的细胞中），在拓扑上，对脂质组成的变化和 其外膜外域的磷酸化。我们将解决脂质组成/不对称的变化 和磷酸化/去磷酸化周期影响FTSK的功能作为生理的一个例子 功能可能受电荷余额规则支配。通过专注于界面蛋白脂质相互作用 具有不同脂质组成和不对称性的脂质双层，测量作用于新生MP的电力 在组装过程中和之后，并表征了CL的生理上重要的作用（如我们所具有的 磷脂酰乙醇胺），我们将增强对定义MP结构和的力的理解 功能和膜脂质在细胞过程中起着作用。