Influence of Cholesterol on Phase Behavior and Nonrandom Mixing of Membrane Components

该奖项是根据2009年美国复苏和再投资法案(公法111-5)资助的。胆固醇是哺乳动物质膜中含量最丰富的单一脂质物种。尽管如此，它与邻近的膜分子相互作用的潜在性质仍然不清楚，它对膜生物化学的影响也是如此。众所周知，胆固醇与其他膜脂的混合是非随机的，但在大多数情况下，对胆固醇的横向分布以及胆固醇影响其他膜组分的横向分布的方式缺乏明确的了解。该项目有四个主要概念要素：(I)三组分双层脂质混合物作为哺乳动物质膜外叶和内叶的模型。这些三组分混合物足够复杂，可以模拟真实的生物膜，但在化学上定义得很好；(Ii)使用几种独立的、互补的方法来彻底绘制和表征这些模型膜的组成相行为；(Iii)一些最真实的模型膜，也许是所有的，都具有具有相分离的“纳米域”的组成区域。这些微小的结构域，不同于在普通的非理想混合中发现的簇，必须从大小上进行表征，并且它们的化学性质与研究得很好的宏观相相关；(Iv)膜蛋白的结合影响纳米结构域的大小，反过来，这些纳米结构域的存在影响蛋白质的结合。在实验上，这项工作涉及到大量的荧光共振能量转移测量数据集，以找到脂质混合物的相边界和荧光探针的分配行为，包括膜结合肽。共聚焦荧光显微镜被用来观察和识别共存相。康奈尔高能同步加速器源的X射线衍射实验可以在不使用荧光探针的情况下检测纳米域。该项目的一个长期目标是发现脂类、极性和碳氢化合物结构中包含的信息的基本性质，从而服务于更广泛的科学界。通过这种方式，膜脂应该变得和核酸和氨基酸一样被理解。以前的工作表明，脂质结构信息部分地体现在脂质混合物的相行为中，本项目旨在阐明这种混合物行为。这项研究还将产生有关膜探针性质的系统信息，这将对使用荧光显微镜的细胞生物学家社区广泛使用。作为该项目的一个组成部分，为了促进科学教育和培训，一组本科生将接受脂类物理化学和荧光光谱学的讲座，随后将接受脂类分析化学、脂类有机合成以及光谱和显微镜技术的系统培训，然后他们将在指定的独立研究中使用这些培训。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Cholesterol is the single most abundant lipid species in mammalian plasma membranes. Nonetheless, the underlying nature of its interactions with neighboring membrane molecules has remained obscure, as have its effects on membrane biochemistry. Cholesterol is known to mix nonrandomly with other membrane lipids, and yet in most cases a clear understanding is missing for the lateral distribution of cholesterol, and the way cholesterol influences the lateral distribution of other membrane components. This project has four main conceptual elements: (i) 3-component bilayer lipid mixtures serve as models for the outer and the inner leaflets of mammalian plasma membranes. These 3-component mixtures are sufficiently complex to model real biomembranes, yet are chemically well-defined; (ii) Several independent, complementary methods are used to thoroughly map and characterize the compositional phase behavior of these model membranes; (iii) Some, perhaps all, of the most realistic model membranes have compositional regions with phase separated "nanodomains". These tiny domains, not the same as the clusters found in ordinary nonideal mixing, must be characterized as to size, and their chemical properties correlated with the well-studied macroscopic phases; (iv) Binding of membrane proteins influences the size of nanodomains, and conversely the presence of these nanodomains influences protein binding. Experimentally, this work involves large data sets of fluorescence resonance energy transfer measurements to find the lipid mixture phase boundaries and the partition behavior of fluorescent probes, including membrane-bound peptides. Confocal fluorescence microscopy is used to visualize and identify coexisting phases. X-ray diffraction experiments at the Cornell High Energy Synchrotron Source can detect nanodomains without the use of fluorescent probes. A long-range objective of this project is to discover the fundamental nature of the information that is contained in the structure of lipid polar and hydrocarbon moieties, thereby serving the broader scientific community. In this way, membrane lipids should become as well understood as are nucleic acids and amino acids. Previous work shows that lipid structural information, in part, is manifested in the phase behavior of lipid mixtures, and this project is designed to elucidate this mixture behavior. This research also will yield systematic information about properties of membrane probes that will be of wide use to the community of cell biologists who use fluorescence microscopy. As an integral feature of this project, in order to promote scientific education and training, a group of undergraduate students will receive lectures in lipid physical chemistry and fluorescence spectroscopy, followed by systematic training in lipid analytical chemistry, lipid organic synthesis, and spectroscopic and microscope techniques, which they will then use in their assigned independent research.