High Resolution Membrane Structure From Fluorescence

描述(申请人提供)：该项目的目标将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的鞘磷脂和富含胆固醇的膜域。在其他功能中，木筏涉及膜之间的蛋白质和脂类的分类，信号转导，以及某些类型的细菌和病毒感染。该项目之前的研究是与黛博拉·布朗(Stony Brook)博士的实验室(继续合作)的一部分，建立了一些基本原理，解释鞘脂、类固醇和某些蛋白质是如何形成木筏的。新的荧光和荧光猝灭方法允许检测完全控制RAFT成分的纳米级筏子，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控制筏子中脂质和蛋白质参与的规则。我们将在模型膜系统中研究缺乏鞘磷脂的质膜内叶中的RAFT形成的基础。将研究胆固醇生物合成前体的筏子形成行为，以帮助确定阻碍胆固醇生物合成步骤的疾病(例如Smith-Lemli-Opitz病)可能与筏子行为的有害变化有关。神经酰胺取代了木筏上的甾醇。为此，将在模型系统和细胞中研究具有重要生物学意义的富含神经酰胺的木筏，以确定它们在特性和蛋白质相互作用方面与普通木筏的区别。为了更深入地了解浮筏的形成原理，将在模型膜和细胞中研究各种具有浮筏促进和浮筏破坏稳定行为的小分子。作为这些研究的一部分，将在模型膜和细胞中研究多烯类抗生素已知的RAFT破坏稳定作用的功能意义。我们将研究蛋白质与RAFT的相互作用，以确定蛋白质结构与RAFT亲和力之间的关系。我们将比较跨膜蛋白、脂类锚定蛋白和胆固醇结合蛋白。在最后一次授权期中发现的支持RAFT形成的不同程度的甾醇类似物将被用来确定蛋白质甾醇结合特异性的性质。蛋白质与普通和富含神经酰胺的木筏是否有不同的相互作用也将被确定。最后，我们将研究蛋白质对RAFT形成的调控程度。

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.