LIPID CONTROL OF MEMBRANE PROTEIN ORGANIZATION

这项工作的长期目标是确定 蛋白质和磷脂中发生的构象变化 在他们的相互作用。 所涉及的原则将 为理解分子组织提供了基础 生物膜以及组织如何被改变 在病理条件下。 为实现这一目标，将探讨三个具体目标： (1)为了确定膜整合蛋白 优先分配到特定化学结构的区域 或物理秩序在复杂的磷脂环境中， 确定任何观察到的分区与 膜结合酶的功能。 (2)为了确定相互作用的位置和大小 由膜蛋白和膜活性肽产生， 磷脂分子 (3)确定构象和取向顺序的变化 在磷脂和蛋白质成分中， 它们在模型囊泡和平面双层中的相互作用 系统，并在一个合理简单的天然准备（肺 表面活性剂）进行体外研究。 研究非物质文化遗产的主要物理方法是 脂质构型和蛋白质的微扰方式 二级结构是傅里叶变换红外（FT-IR） 谱 脂质-蛋白质相互作用的热力学是 采用高灵敏度差示扫描量热法进行评估 （DSC）。 为了达到目的1， 被分离、纯化并重组成磷脂 环境选择模仿那些在体内。 的相容性和 系统的分区特性由 FT-IR和DSC。 目的2是通过重组 所需的膜蛋白或肽与磷脂 在酰基链中的特定位置被氘代。 扰动 在后者的特定地点将受到衰减的监测 全反射光谱学 目标3由以下方面实现： 确定蛋白质的类型和几何取向，或 通过构象敏感酰胺的肽二级结构 I或II光谱区。 通过以上推导出的组织原则 实验将扩展到一个简单的天然组织，肺， 表面活性剂，在体外进行研究。 的分子基础 表面活性剂在空气-水界面上的稳定性和铺展性 将被探测。

The long term objective of this work is to determine the conformational changes that occur in proteins and phospholipids upon their mutual interaction. The principles involved will provide a basis for understanding the molecular organization of biological membranes and how the organization may be altered during pathological conditions. Three specific aims will be addressed to achieve this objective: (1) To determine whether integral membrane proteins preferentially partition into regions of specific chemical structure or physical order in complex phospholipid environments, and to determine the relationship of any observed partitioning to the function of membrane-bound enzymes. (2) To determine the site and magnitude of the interactions produced by membrane proteins and membrane-active peptides on phospholipid molecules. (3) To determine changes in conformation and orientational order that occur in both the phospholipid and protein components upon their mutual interaction in model vesicle and planar bilayer systems, and in a reasonably simple native preparation (lung surfactant) to be studied in vitro. The main physical method to be employed to study in non- perturbative fashion both lipid configuration and protein secondary structure is Fourier Transform Infrared (FT-IR) spectroscopy. The thermodynamics of lipid-protein interactions is evaluated with high sensitivity Differential Scanning Calorimetry (DSC). To achieve Aim 1, CaATPase from rabbit sarcoplasmic reticulum is isolated, purified, and reconstituted into phospholipid environments selected to mimic those in vivo. The miscibility and partitioning characteristics of the system are determined with FT-IR and DSC. Aim 2 is achieved by reconstitution of the desired membrane protein or peptide with phospholipids deuterated at specific positions in the acyl chains. Perturbations at the specific site in the latter will be monitored with attenuated total reflectance spectroscopy. Aim 3 is addressed by determination of the type and geometric orientation of protein or peptide secondary structure via the conformation-sensitive Amide I or II spectral regions. The organizational principles deduced through the above experiments will be extended to a simple native tissue, lung surfactant, to be studied in vitro. The molecular basis of the stability and spreading of surfactant at the air-water interface will be probed.