High Resolution Membrane Structure From Fluorescence

描述（由申请人提供）：本项目的目标是继续研究脂筏的结构和功能：在哺乳动物和其他真核细胞中发现的有序鞘脂和胆固醇丰富的膜结构域。在其他功能中，筏涉及膜之间蛋白质和脂质的分选，信号转导以及某些类型的细菌和病毒感染。该项目的先前研究，作为与Deborah Brown博士（斯托尼布鲁克）实验室（持续）合作的一部分，建立了一些基本原理，解释了鞘脂，甾醇和某些蛋白质如何形成筏。新的荧光和荧光猝灭方法，允许检测的纳米级筏完全控制筏组成，结合以前开发的光谱和生物化学技术，将被用来定义几个仍然神秘的规则控制脂质和蛋白质参与筏。将在模型膜系统中研究鞘脂贫乏质膜内小叶中筏形成的基础。将研究胆固醇生物合成前体的筏形成行为，以帮助确定胆固醇生物合成中的疾病阻断步骤（例如Smith-Lemli-Opitz病）可能与筏行为的有害变化有关。神经酰胺取代了筏中的甾醇。因此，将在模型系统和细胞中研究生物学上重要的富含神经酰胺的筏，以确定它们在性质和蛋白质相互作用方面与普通筏的区别。为了进一步了解筏形成的原理，将在模型膜和细胞中研究具有筏促进和筏失稳行为的各种小分子。作为这些研究的一部分，将在模型膜和细胞中研究多烯抗生素的已知筏不稳定作用的功能意义。蛋白质与筏的相互作用将被研究，以确定蛋白质结构和筏亲和力之间的关系。将比较跨膜、脂质锚定和胆固醇结合蛋白。在最后一个资助期内发现的支持筏形成不同程度的甾醇类似物将用于定义蛋白甾醇结合特异性的性质。蛋白质与普通筏和富含神经酰胺的筏的相互作用是否不同也将被确定。最后，将研究蛋白质可以调节筏形成的程度。

DESCRIPTION (provided by applicant): The goal of this project will be to continue studies of the structure and function of lipid rafts: ordered sphingolipid and cholesterol-rich membrane domains found in mammalian and other eukaryotic cells. Among other functions, rafts are implicated in sorting of proteins and lipids between membranes, signal transduction, and some types of bacterial and viral infections. Previous studies in this project, as part of a (continuing) collaboration with the lab of Dr. Deborah Brown (Stony Brook), established some of the basic principles explaining how sphingolipids, sterols and certain proteins form rafts. New fluorescence and fluorescence quenching methods allowing detection of nanoscale rafts with full control over raft composition, combined with previously developed spectroscopic and biochemical techniques, will be used to define several of the still mysterious rules controlling lipid and protein participation in rafts. The basis of raft formation in the sphingolipid-poor plasma membrane inner leaflet will be studied in model membrane systems. Raft-forming behavior of biosynthetic precursors of cholesterol will be studied to help define how diseases blocking steps in cholesterol biosynthesis (e.g. Smith-Lemli-Opitz disease) may be related to deleterious changes in raft behavior. Ceramide displaces sterols from rafts. For this reason, biologically important ceramide-rich rafts will be studied in model systems and cells to define how they differ from ordinary rafts in terms of properties and protein interactions. To gain additional insights into the principles of raft formation, various small molecules with raft-promoting and raft-destabilizing behaviors will be studied in model membranes and cells. As part of these studies, the functional significance of the known raft-destabilizing effects of polyene antibiotics will be studied in both model membranes and cells. The interaction of proteins with rafts will be studied to define the relationship between protein structure and raft affinity. Transmembrane, lipid-anchored and cholesterol-binding proteins will be compared. Sterol analogs found to support raft formation to different degrees in the last grant period will be used to define the nature of protein sterol binding specificity. Whether proteins interact differently with ordinary and ceramide-rich rafts will also be determined. Finally, the degree to which proteins can regulate raft formation will be studied.