BIOPHYSICAL MEASUREMENTS OF MEMBRANE STRUCTURE

我们的长期目标是确定脂质结构之间的关系 膜的功能特性，特别强调 理解能量转换积分的酶调节 膜蛋白 这项工作的一个重要方面是进一步 开发新的光谱探针来测量脂质结构 性质，以及使用互补的光谱技术（即， 荧光共振能量转移，荧光各向异性，和 自旋标记EPR方法），以消除关于 实验数据的解释。 作为第一步， 了解调节膜的基本物理力 结构，我们建议利用模型膜组成的良好定义的 脂质的单一或二元混合物。 这些简单的系统可以让我们 系统地研究潜在的分子关系， 影响膜的宏观性质。 我们将 调查i）构象之间的可能关系 单独的磷脂酰基链，ii）在 磷脂烃链上的限定位置，iii）结构 关于磷脂头基构象的变化，和iv） 双层的相变温度，其提供了 简单的物理测量，关于稳定的相互作用， 单个膜脂。 第二个主题和最终目标 项目，是应用这些方法，以获得洞察力， 脂质结构变化的基本调节机制 性质调节膜蛋白的功能。 我们将主要 利用肌浆网中的Ca-ATP酶 从骨骼肌中获得的膜，除了其 在调节细胞内钙水平中起重要作用， 直接评估基本机制的模型系统， 活性转运蛋白有效地将ATP水解偶联到 离子相对于浓度梯度的物理运动。 这将 涉及脂质和蛋白质结构特性的测量， 在将Ca-ATP酶掺入到确定的脂质中之后， 环境. 除了测量脂质结构外， 我们将测量蛋白质的构象和动力学， 优点i）固有荧光团（即，（图）位于 Ca-ATP酶的跨膜螺旋提供了直接测量 关于钙离子通道构象的变化，和ii） 与细胞质共价结合的定点外源性发色团 Ca-ATPase的一部分，以测量分子内 ATP酶的构象变化与酶的 在空间上远离的核苷酸和钙位点之间的偶联，以及 作为ATP酶多肽链之间的寡聚相互作用。 这 全面的方法将使我们能够确定功能 膜脂质和蛋白质结构之间的相关相互作用。 在 此外，了解调节这一点的物理机制， 相对简单的离子转运蛋白应该提供基本的 了解其他膜转运的功能特性 和通道蛋白的调节。

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.