BIOPHYSICAL MEASUREMENTS OF MEMBRANE STRUCTURE

我们的长期目标是确定脂质结构之间的关系 和膜的功能特性，特别强调 理解能量传递积分的酶调节 膜蛋白。这项工作的一个重要方面是进一步 用于测量脂质结构的新型光谱探针的研制 性质，以及使用免费的光谱技术(即， 荧光共振能量转移、荧光各向异性以及 自旋标记EPR方法)，以消除关于 实验数据的解释。作为……的第一步 理解调节膜的基本物理力 结构，我们建议利用由定义良好的 单脂或双脂混合物。这些简单的系统将允许我们 为了系统地研究潜在的分子关系 影响膜的宏观性能。我们会 研究i)构象之间的可能关系 单独的磷脂酰链，II)旋转动力学 磷脂碳氢链上的确定位置，III)结构 关于磷脂头基构象的变化，和iv) 双层的相变温度(S)，它提供了一个 关于稳定相互作用的简单物理测量 个别膜脂。第二个主题，也是 项目，就是应用这些方法，以便深入了解 脂类结构变化的基本调控机制 性质调节膜蛋白的功能。我们将主要 利用肌浆网中发现的具有良好特性的Ca-ATPase 从骨骼肌获得的膜，除了它的 在调节细胞内钙水平方面发挥重要作用，作为一种 直接评估基本机制的模式系统，其中 活性转运蛋白有效地将ATP水解酶与 离子相对于浓度梯度的物理运动。这将是 包括测量脂肪和蛋白质的结构性质， 在将Ca-ATPase掺入到定义的脂质中之后 环境。除了已有的脂类结构的测量 提到，我们将测量蛋白质的构象和动力学 优势：i)位于 Ca-ATPase的跨膜螺旋提供了一种直接测量 关于钙离子通道构象的变化，以及ii) 与细胞质共价结合的定点外源发色团 部分Ca-ATPase，以测量分子内 与酶相关的ATPase的构象变化 空间距离的核苷酸和钙位点之间的偶联也是如此 作为ATPase多肽链之间的低聚相互作用。这 全面的方法将使我们能够从功能上确定 膜脂与蛋白质结构之间的相关相互作用。在……里面 此外，对调节这一现象的物理机制的理解 相对简单的离子转运蛋白应该提供基本的 对其他膜转运功能特性的洞察 而通道蛋白则受到调控。

Our long-term goal is to identify relationships between lipid structure and functional properties of membranes, with a particular emphasis upon understanding the enzymatic regulation of energy transducing integral membrane proteins. An important aspect of this work is the further development of new spectroscopic probes to measure lipid structural properties, and the use of complimentary spectroscopic techniques (i.e., fluorescence resonance energy transfer, fluorescence anisotropy, and spin-label EPR methods) to eliminate ambiguities regarding the interpretation of experimental data. As a.first step aimed at understanding the fundamental physical forces that modulate membrane structure, we propose to utilize model membranes composed of well defined single or binary mixtures of lipids. These simple systems will allow us to systematically investigate the underlying molecular relationships that influence the macroscopic properties of the membrane. We will Investigate possible relationships between i) the conformation of individual phospholipid acyl chains, ii) the rotational dynamics at defined positions on the phospholipid hydrocarbon chain, iii) structural changes regarding the conformation of phospholipid headgroups, and iv) the phase transition temperature(s) of the bilayer, which provide a simple physical measure regarding the stabilizing interactions between individual membrane lipids. A second theme, and ultimate goal of the project, is to apply these methods in order to gain insight into fundamental regulatory mechanisms whereby changes in lipid structural properties modulate the function of membrane proteins. We will primarily utilize the well characterized Ca-ATPase found in sarcoplasmic reticulum membranes obtained from skeletal muscle that, in addition to its important role in regulating intracellular calcium levels, serves as a model system in which to directly assess fundamental mechanisms whereby active transport proteins efficiently couple ATP hydrolysis to the physical movement of ions against a concentration gradient. This will involve measurements of both the lipid and protein structural properties, subsequent to the incorporation of the Ca-ATPase into defined lipid environments. In addition to the measurements of lipid structure already mentioned, we will measure the protein's conformation and dynamics taking advantage of i) intrinsic fluorophores (i.e., tryptophans) located on the transmembrane helices of the Ca-ATPase that provide a direct measure regarding changes in the conformation of the calcium ion channel, and ii) site-directed extrinsic chromophores covalently bound to the cytoplasmic portion of the Ca-ATPase in order to measure both Intramolecular conformational changes of the ATPase associated with the enzymatic coupling between spatially distant nucleotide and calcium sites, as well as oligomeric interactions between ATPase polypeptide chains. This comprehensive approach will allow us to identify the functionally relevant interplay between membrane lipid and protein structure. In addition, an understanding of the physical mechanism regulating this relatively simple ion transport protein should provide fundamental insights into how the functional properties of other membrane transport and channel proteins are regulated.