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 膜生物学的一个基本目标是理解和预测蛋白质序列如何折叠和定向 在脂质双层中。大多数研究集中在膜蛋白（MP）和膜插入机制上 很少考虑脂质环境如何影响跨膜结构域（TMD）组织。长期来看 该提案的目标是了解脂质-蛋白质相互作用在组装、结构和功能中的作用 议员。使用分子遗传学和生化相结合的方法，我们建立了脂质依赖性 在 MP 体内组装期间和之后，TMD 方向是动态的（即可以可逆地改变）并且是独立的 体外其他细胞因子。我们提出了电荷平衡规则，其中包括脂质的积极作用 环境，解释议员的动态行为。我们开发了大肠杆菌脂质突变体库 其中脂质成分可以在MP膜插入期间或之后暂时进行调节。生理学 膜脂双层不对称性的意义是一个尚未研究的领域。我们现在向该库添加一组 E. 大肠杆菌菌株和蛋白脂质体系统，其中膜脂双层不对称性可以被控制 确定脂双层不对称性在 MP 动态组织中的作用。使用这套脂质试剂和 对于原生或工程 MP，我们提出了三个综合的具体目标： 目标 1，我们将在热力学方面进行量化 术语并确定分子驱动力背后的机械和结构原理，如 由电荷平衡规则提出，该规则将动态 TMD 拓扑组织作为以下函数进行管理 膜脂组成和不对称性。目标 2，CL 在细胞功能中的作用尚不清楚。化学发光 跨任何生物膜的跨双层不对称性都是未知的，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 的大肠杆菌在有氧条件下表现出多种表型 和厌氧条件，这会严重损害野生生长。我们将讨论 CL 的角色， 它的跨膜不对称性和支持关键细胞功能的三种 CL 合酶，这将是 适用于理解 CL 和 cls 基因多重性在致病性革兰氏阴性菌中的重要性 细菌。目标 3，我们鉴定出与细胞分裂后期（一种缺陷）相关的重要 MP（FtsK） 在缺乏磷脂酰乙醇胺的细胞中），它对脂质成分的变化有拓扑反应，并且 其膜外结构域的磷酸化。我们将讨论脂质成分/不对称性如何变化 磷酸化/去磷酸化循环影响 FtsK 的功能，作为生理学的一个例子 功能可能受费用平衡规则管辖。通过关注界面蛋白质-脂质相互作用 具有不同脂质成分和不对称性的脂质双层，测量作用于新生 MP 的电力 组装过程中和组装后，并表征 CL 的重要生理作用（正如我们所研究的那样） 磷脂酰乙醇胺），我们将增强对定义 MP 结构和 功能和膜脂在细胞过程中发挥的作用。