TOWARD A PHYSICAL DESCRIPTION OF IMMUNOSUPPRESSION

此应用程序是源自计划项目的研究的延伸 格兰特，其重点是结构和功能作用的 亲环素(Cyp)家族的蛋白质。在这个快速发展的领域， 我们将继续我们的结构和动力学特征 环孢素A(CsA)相关配体的相互作用 与CYP家族成员和钙调神经磷酸酶关联，目前 被认为是各种细胞类型的主要目标。一个中心焦点 将是核磁共振方法与动力学和 这些与停流相互作用的热力学参数 荧光研究。人类主要的胞浆受体CYP-18， 以及新的CYP-40c物种将通过从组织中分离提供 和来自E.Coli的重组形式。我们还将准备和研究 这些物种的定点突变形式。此外，还有两个新的 膜结合的CYP物种，CYP-22M，与 内质网和CYP-20将在其原生版本中提供 糖基化状态。最近，有研究表明，配体- 免疫亲和素复合体与钙依赖的钙调神经磷酸酶相关 反应，从而得到各种多维金属核磁共振以及1H， 13C和15N技术是可能的。具体地说，~(113)Cd核磁共振方法 将被用来提供同构的、磁活性的探头 CA站点参与了这些站点的结构和动态 多聚免疫亲和素复合体。此外，多维核磁共振 方法利用~1H、~(13)C和~(19)F同位素标记的CsA衍生物 将有助于识别位于特定位置的氨基酸 多聚体复合体中的相互作用。独立评估 这些相互作用的动力学方面将通过瞬变来实现 用单一色氨酸在CYP中的状态荧光测量 已被证明能灵敏地反映CYP与配体的相互作用 作为CsA。这种相互作用将通过停流荧光法进行评估 研究发展一个完整的动力学和热力学的理解 CsA-CYP的相互作用，并为表征新的 Cyp蛋白和定点突变形式。这些研究也将是 钙调神经磷酸酶与免疫亲和素相互作用的研究 很复杂。通过化学交联法获得的初步信息 中组件的意外拓扑关系。 CYP-钙调神经磷酸酶复合体。这些结果将推广到衬底 CYP-CsA复合体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。 这些整合不同人才的独特机会是可以期待的 不仅要定义与之相互作用的特定氨基酸位点 中的新概念的指南。 药物合成及其作用机理的认识 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.