ENDOR STUDIES OF ALLOSTERIC INTERACTIONS

该计划的总体论点是血红蛋白（Hb）提供了一个定义良好，易于处理的“分子工作台”，可用于开发低聚蛋白复合物进行变构调节的规则。一个中心目标是测试和进一步发展在上一个资助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诱导的血红素结构变化传播到亚基-亚基界面，从而促进从低亲和力的T型结构向高亲和力的T型结构转变，并最终转变为一系列R型四元结构。该项目的具体目标是利用电子-核双共振（ENDOR）光谱学的进步来解决两个基本问题。i)血红素上的关键界面如何耦合到配体结合事件：Hb连接中间体的配体血红素和配体血红素上诱导了哪些结构扰动，以及它们如何调节配体亲和力。ii)通过测量血红素铁和关键血红素袋残基之间的实际距离，我们将能够首次准确探测血红素连接途径的三级结构开关点，这样做将为测试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.