TOWARD A PHYSICAL DESCRIPTION OF IMMUNOSUPPRESSION

这个应用程序是一个项目的延伸 赠款，其重点是机构的结构和职能作用， 亲环素（CyP）蛋白家族。 在这个快速发展的领域， 我们将继续我们的结构和动力学表征的 环孢菌素A（CsA）相关配体之间的相互作用， 与CyP家族成员和钙调磷酸酶相关，目前 被认为是各种细胞类型中的主要目标。 一个中心焦点 核磁共振方法将与动力学评估相协调， 这些与停流相互作用的热力学参数 荧光研究。 人CyP-18，主要的胞质受体， 以及新的CyP-40 c物种将通过从组织中分离来提供 并从E.杆菌 我们还将准备和研究 这些物种的定点突变形式。 此外，两名新 膜结合的CyP物质，CyP-22 m，与细胞膜结合的CyP-22 m， 内质网和CyP-20，将在其天然的 糖化状态 最近，已经表明配体- 亲免素复合物与钙调神经磷酸酶在Ca++依赖性 反应，因此各种多维金属NMR以及1H， 13 C和15 N技术是可能的。 具体地，113 Cd NMR方法 将被用于提供一个同构的，磁活性探头的 Ca++位点，因为它们参与了这些结构和动力学， 多聚体亲免蛋白复合物。 此外，多维NMR 利用1H、13 C和19 F同位素标记的CsA衍生物的方法 将有助于鉴定位于位点的特定氨基酸 多聚体复合物中的相互作用。 独立评估 这些相互作用的动力学方面将通过瞬态 使用CyP中的单个色氨酸进行荧光测量， 已经显示出灵敏地反映CyP与配体的相互作用， 作为CsA。 将通过停流荧光法评估这种相互作用 研究，以发展一个完整的动力学和热力学的理解， CsA-CyP相互作用以及为表征新的 CyP蛋白和定点突变形式。 这些研究也将 扩展到检查钙调神经磷酸酶与免疫亲和素的相互作用 复杂. 通过化学交联获得的初步信息 揭示了一个意想不到的拓扑关系的组成部分， CyP-calcineurin复合物。 这些结果将推广到基板 CyP-CsA复合物的位点和潜在的新细胞靶点也可能 用这些物理技术进行检查。 这些融合不同人才的独特机会令人期待 不仅要确定与之相互作用的特定氨基酸位点， 三维信息，但也作为新概念的指导， 药物合成和了解这一行动的机制 新型免疫抑制剂。

This application, an extension of research derived from a program project grant, has as its focus the structural and functional role of the cyclophilin (CyP) family of proteins. In this rapidly evolving field, we will continue our structural and kinetic characterization of the interaction between the Cyclosporin A (CsA) related ligands as they associate with the CyP family members and calcineurin, currently considered a major target within various cell types. A central focus will be NMR methods coordinated with an assessment of kinetic and thermodynamic parameters of these interactions with stopped-flow fluorescence studies. Human CyP-18, the predominant cytosolic receptor, as well as new CyP-40c species will be provided by isolation from tissues and in recombinant form from E. Coli. We will also prepare and study site-directed mutant forms of these species. In addition, two new membrane bound CyP species, CyP-22m, richly associated with the endoplasmic reticulum and CyP-20, will be available in their native glycosylated state. Recently, it has been shown that the ligand- immunophilin complex associates with calcineurin in a Ca++-dependent reaction, thus a variety of multidimensional metallo-NMR as well as 1H, 13C and 15N techniques are possible. Specifically, 113Cd NMR methods will be used to provide an isomorphic, magnetically active probe of the Ca++ sites as they are involved in the structure and dynamics of these multimeric immunophilin complexes. Additionally, multidimensional NMR methods utilizing 1H, 13C and 19F isotopically labeled CsA derivatives will aid in the identification of specific amino acids located at sites of interaction in the multimeric complex. Independent assessment of kinetic aspects of these interactions will be accomplished by transient state fluorescence measurements using the single tryptophan in CyP which has been shown to sensitively reflect CyP's interaction with ligands such as CsA. This interaction will be assessed by stopped-flow fluorescence studies to develop a complete kinetic and thermodynamic understanding of the CsA-CyP interaction as well as provide a basis for characterizing new CyP proteins and site-directed mutant forms. These studies will also be extended to examine the interaction of calcineurin with the immunophilin complex. Preliminary information obtained by chemical crosslinking has revealed an unexpected topological relationship of the components in the CyP-calcineurin complex. These results will be extended to the substrate site and potential new cellular targets for the CyP-CsA complex may also be examined with these physical techniques. These unique opportunities for integrating different talents are expected not only to define specific amino acid loci of interaction with dimensional information but also serve as a guide for new concepts in drug synthesis and an understanding of the mechanism of action of this new class of immunosuppressive agent.