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位 在脂质双层中。大多数研究集中在膜蛋白(MP)和膜插入机械。 很少考虑脂质环境对跨膜区(TMD)组织的影响。长期的 这项建议的目的是了解脂-蛋白质相互作用在组装、结构和功能中的作用 下院议员。利用分子遗传学和生物化学相结合的方法，我们证实了脂质依赖 TMD取向在体内MP组装过程中和之后是动态的(即，可以可逆地改变)，并且是独立的 其他细胞因子的体外实验。我们提出了电荷平衡规则，它包含了对脂质的积极作用 环境，来解释MPS的动态行为。我们建立了一个大肠杆菌脂类突变体文库 其中脂质成分可以在MP膜插入期间或之后进行调节。生理学 膜脂双分子层不对称的意义是一个研究不足的领域。我们现在向该库添加一组E。 可控制膜脂双层不对称性的大肠杆菌菌株和蛋白脂质体系统 确定脂质双层不对称性在MP动态组织中的作用。使用这套脂类试剂和 无论是本地的还是工程的MPS，我们提出了三个综合的具体目标：目标1，我们将在热力学中量化 定义并确定分子驱动力背后的机械原理和结构原理，如 由电荷平衡规则提出，该规则根据以下条件管理动态TMD拓扑组织 膜脂成分和不对称性。目的2，CL在细胞功能中的作用还不是很清楚。电子邮件 跨越任何生物膜的跨双分子层不对称性是未知的，而且仍然没有可靠的方法来 估计它在任何膜上的分布。缺乏CL的大肠杆菌在好氧条件下表现出几种表型 以及厌氧条件，这将严重危及野外的生长。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个CL合成酶，这将是 适用于理解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(缺陷)相关的必需MP(FtsK) 在缺乏磷脂酰乙醇胺的细胞中)，在拓扑上对脂质成分的变化和 其膜外域的磷酸化。我们将讨论脂质成分/不对称性的变化 而磷酸化/去磷酸化循环影响FtsK的功能，作为生理学的一个例子 可能受电荷平衡规则支配的功能。通过重点研究蛋白质-脂质之间的界面相互作用 具有不同脂类组成和不对称性的脂双层，测量作用于新生MPS的电场力 在装配过程中和装配后，并表征CL在生理上的重要角色(如我们为 磷脂酰乙醇胺)，我们将加强我们对定义MP结构和 功能和膜脂在细胞过程中所起的作用。

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.