Dynamic Lipid and Protein Organization in Cell Membranes

摘要 膜生物学的一个基本目标是了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， 很少考虑脂质环境如何影响跨膜结构域（TMD）组织。长期 该计划的目标是了解脂质-蛋白质相互作用在组装、结构和功能中的作用 的MP。使用分子遗传学和生物化学相结合的方法，我们建立了脂质依赖性 TMD取向是动态的（即，在体内MP组装期间和之后， 其他细胞因子的作用。我们提出了电荷平衡规则，其中包括脂质的活性作用， 环境，以解释MP的动态行为。我们建立了大肠杆菌脂质突变体库 其中脂质组成可以在MP膜插入期间或之后暂时调节。生理 膜脂双层不对称性的意义是一个未充分研究的领域。我们现在向这个库添加一组E。 大肠杆菌菌株和蛋白脂质体系统，其中膜脂质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， 天然或工程MP，我们提出了三个综合的具体目标：目标1，我们将量化在热力学 术语，并确定了分子驱动力背后的机械和结构原理，如 提出的电荷平衡规则，管理动态TMD拓扑结构的功能， 膜脂组成和不对称性。目的2，CL在细胞功能中的作用还不清楚。CL 跨任何生物膜的跨双层不对称性是未知的，并且仍然没有可靠的方法来测量跨双层不对称性。 估计其在任何膜上的分布。E.缺乏CL的大肠杆菌在有氧条件下表现出几种表型 和厌氧条件，这将严重损害在野外的生长。我们将讨论CL的作用， 它的跨膜不对称性和三种CL激酶在支持关键细胞功能，这将是 适用于了解CL和cls基因多样性在致病性革兰阴性菌中的重要性， 细菌目的3，我们发现了一种与细胞分裂晚期（一种缺陷）相关的必需MP（FtsK 在缺乏磷脂酰乙醇胺的细胞中），其在拓扑学上响应于脂质组成的变化， 其膜外结构域的磷酸化。我们将讨论脂质组成/不对称性的变化 和磷酸化/去磷酸化循环影响FtsK的功能，作为一个例子， 功能可能受费用余额规则控制。通过关注蛋白质-脂质界面相互作用， 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 在组装过程中和组装后，并表征CL的生理学重要作用（如我们对 磷脂酰乙醇胺），我们将提高我们的力量，定义MP结构的理解， 功能和膜脂在细胞过程中的作用。

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.