ENDOR STUDIES OF ALLOSTERIC INTERACTIONS

本计划的总体论点是，血红蛋白(Hb)提供了一个定义明确、易于处理的“分子工作台”，可用于制定寡聚蛋白质复合体经历变构调节的规则。一个中心目标是测试和进一步开发在上一个供资期间制定的预测性立体化学模型。该模型描述了特定残基如何将连接引起的血红素结构变化传播到亚单位-亚单位界面，从而促进从低亲和力T结构到高亲和力T类结构，最后到一系列R四元结构的转变。该项目的具体目标是利用电子-核双共振(Endor)光谱学的进展来解决两个基本问题。I)Hb连接中间体的关键界面是如何与配体结合事件耦合的：在Hb连接中间产物的连接和连接的HEME上诱导了什么结构扰动，以及它们如何作用于调节配体亲和力。Ii)通过测量血红素-铁和关键的血红素口袋残基之间的实际距离，我们将首次能够准确地探测Hb连接途径的三级结构切换点，从而为检验和完善立体化学变构模型提供方法。

The overall thesis of this Program is that hemoglobin (Hb) provides a well-defined, tractable "molecular workbench" which can be used to develop the rules by which oligomeric protein complexes undergo allosteric regulation. A central goal is to test and further develop a predictive stereochemical model that was formulated during the last funding period. The model describes how specific residues propagate ligation-induced changes in heme structure to the subunit-subunit interfaces and thereby promote the transitions from a low affinity T structure to higher affinity T-like structures, and finally to a range of R quaternary structures. The specific aims of this Project are to capitalize on advances in electron-nuclear double resonance (ENDOR) spectroscopy to address two basic issues. i) How are the critical interfaces coupled to ligand binding events at the hemes: What structural perturbations are induced at the liganded and liganded hemes of Hb ligation intermediates, and how do they function to modulate ligand affinity. ii) By measuring the actual distances between the heme-iron and critical heme-pocket residues, we will be able for the first time to accurately probe the tertiary-structure switch points of the Hb ligation pathway, and in so doing will suggest ways to test and refine the stereochemical allosteric model.