HIGH RESOLUTION MEMBRANE STRUCTURE FROM FLUORESCENCE

我们团队已经开发出一种光谱方法，可以 膜中位置横向位置(深度)的测量 在埃的分辨率级别。它涉及到荧光猝灭 使用携带氮氧化物猝灭剂的磷脂进行测量 不同的深度。然后使用简单的代数表达式来 计算深度。现在已经开发出程序，允许 在广泛的深度范围内进行精确测量。在下一个项目中 期间，该方法将被用于调查一个基本的问题 膜结构，即支配关系的规则 膜相互作用分子的化学结构与其相互作用 深度。作为这一目标的一部分，将为S系统地测量深度 一系列疏水和脂质连接的探针，我们将准备这样的 附着点、探针极性和反应的性质 探头上的组是不同的。胆固醇对深度意志的影响 也要有决心。考虑到最近的情况，这一点特别有趣 研究表明，富含胆固醇和鞘脂的结构域可能 存在于细胞膜中。在合作研究中，对 这种结构域的形成可能存在于细胞膜中。在……里面 合作研究，这种领域的形成的规则 通过脂类组成，它们与蛋白质的相互作用 检查过了。 猝灭法也将被用来确定氨基酸如何 成分调节所插入的膜的结构 多肽。这将通过深度测量进行检查 已知跨膜插入多肽的衍生物。这将是 通过对已知导数的深度测量进行检查 跨膜α-螺旋疏水肽不同于 极性残留物的类型和数量已在特定情况下介绍 各就各位。在这些样品中单个色氨酸残留物的深度测量 与圆二色和能量转移相结合的多肽 测量，将允许方便地分析影响 多肽结构上的取代，以及相关规则的制定 跨记忆定位到序列。该方法也将是 应用于膜蛋白的高分辨率形貌研究。这 将通过测量单个荧光基团的深度来完成 通过定点突变引入一系列残基。第一, 方法的标定及最佳荧光剂的确定 此类研究的标签将通过更全面的比较来完成 单点标记的理论深度和实验深度 跨膜多肽。然后，该方法将应用于 一系列单一半胱氨酸突变体的膜蛋白 有，金黄色葡萄球菌的a-溶血素。

Our group has developed a spectroscopic method allowing measurement of the transverse location (depth) of sites in a membrane at the angstrom level of resolution. It involves fluorescence quenching measurements using phospholipids carrying nitroxide quenchers at different depths. Simple algebraic expressions are then used to calculate depth. Procedures have now been developed which allow accurate measurements over wide range of depths. In the next project period, the method will be used to investigate a fundamental issue of membrane structure, namely, the rules governing the relationship of the chemical structure of membrane-interacting molecules to their depth. As part of this aim depth will be measured for s systematic series of hydrophobic and lipid-linked probes we will prepare such that the attachment site, probe polarity, and the nature of the reactive group on the probe is varied. The effect of cholesterol on depth will also be determined. This is of particular interest in view of recent studies showing that cholesterol and sphingolipid-rich domains may exist in cellular membranes. In collaborative studies, the regulation of the formation of such domains may exit in cellular membranes. In collaborative studies, the regulation of the formation of such domains by lipid composition, and their interactions with proteins will be examined. The quenching method will also be used to determine how amino acid composition regulates the structure of membrane inserted polypeptides. This will be examined by depth measurements on derivatives of known transmembrane inserted polypetides. This will be examined by depth measurements on derivatives of known transmembrane a-helical hydrophobic peptides into which different types and numbers of polar residues have been introduced at specific positions. Measurement of the depth of a single Trp residue in these peptides, in conjunction with circular dichroism and energy transfer measurements, will allow facile analysis of the effects of the substitutions on peptide structure, and the formulation of rules relating transmembrance orientation to sequence. The method will also be applied to the high resolution topography of membrane proteins. This will be done by measuring the depth of single fluorescent groups introduce at a series of residues by site-directed mutagenesis. First, the calibration of the method and identification of the best fluorescent labels for such studies will be completed by a fuller comparison of the theoretical and experimental depths for single site-labeled transmembrane peptides. Then, the method will be applied to a membrane protein for which a series of single Cys mutants are now available, the a-hemolysin of S. Aureus.