ENDOR STUDIES OF ALLOSTERIC INTERACTIONS

该计划的总体论点是，血红蛋白（Hb）提供了一个定义明确、易于处理的“分子工作台”，可用于开发寡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。该模型描述了特定的残基如何传播连接诱导的血红素结构的亚基-亚基界面的变化，从而促进从低亲和力的T结构的过渡到更高的亲和力T-样结构，并最终到一系列的R四级结构。该项目的具体目标是利用电子-核双共振（ENDOR）光谱学的进展来解决两个基本问题。i）关键界面如何与血红素处的配体结合事件偶联：在Hb连接中间体的配体和配体血红素处诱导什么结构扰动，以及它们如何起作用以调节配体亲和力。ii）通过测量血红素-铁和关键血红素口袋残基之间的实际距离，我们将能够首次准确地探测Hb连接途径的三级结构转换点，并且这样做将建议测试和改进立体化学变构模型的方法。

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.