NMR REPORTER PROBES FOR DYNAMIC METALLOPROTEIN STUDIES

我们建议开发一种实用方便的方法， 定量描述可访问性和电子状态， 金属蛋白活性位点。我们称之为PhoRE的方法（用于 磷松弛增强）依赖于相关的变化， 化学探针分子的31 P NMR谱线宽度随速率的变化 探针与顺磁性物质发生物理接触 离子在酶的活性部位。 它们的化学和磁性 这些特性使得磷酸二烷基酯探针特别适合于此 应用程序. 虽然这份手稿描述了一项研究， 抑制和调节蛋白磷酸酶1（PP 1）的方法， 不要求探针的化学结构类似于 酶的底物。 我们强调， 了解酶的调节和疾病机制，以及新的 筛选候选药物的方法。 的具体目标 研究是：（1）表征差异的可用性 PP1的不同稳定构象的活性位点，并确定 天然毒素、小离子抑制剂和调节剂的作用 活动现场可用性的要素。 我们将把这些 与已知酶活性变化的差异。(2)我们将 测量这些相互作用对环境变化的敏感性， 探针的空间体积、电子电荷和亲核性 分子，从而将反应性的变化与局部 活性位点的构象变化。 (3)我们将进一步 通过研究探针与以下物质的相互作用来表征该方法： 具有结构特征的合成金属络合物， 肌红蛋白以及锰和铁超氧化物歧化酶。 (4)我们 长期目标是将该方法应用于各种各样的 金属蛋白 我们将从结构上比较 类似金属酶和相关的活性差异， 结构性差异。 这种方法有可能提供 化学解释酶行为的变化， 与结合抑制剂，毒素， 和调节元件，以及致病突变。

We propose to develop a practical and convenient method to quantitatively characterize the accessibility and electronic stat of metalloprotein active sites. The method, which we call PhoRE (for Phosphorus Relaxation Enhancement) relies on correlating changes in the 31 P NMR line widths of chemical probe molecules with the rate at which the probes come into physical contact with paramagnetetic ion(s) at the enzyme~s active site. Their chemical and magnetic properties make dialkylphosphate probes uniquely suited for this application. While this manuscript describes a study of the inhibition and regulation of protein phosphatase 1 (PP1), the method does not require that the chemical structure of the probe resemble that of the enzyme~s substrate. We stress that the greater understanding of enzyme regulation and disease mechanisms, and to new methods for screening drug candidates. The specific aims of the study are: (1) to characterize differences in the availability of the active sites of different stable conformations of PP1 and determine the effects of natural toxins, small ionic inhibitors, and regulatory elements on active site availability. We will correlate these differences with known changes in enzymatic activity. (2) We will measure the sensitivities of these interactions to changes in the steric bulk, electronic charge, and nucleophilicity of the probe molecules, thereby correlating changes in reactivity with local conformational changes at the active site. (3) We will further characterize the method by studying interactions of the probes with structurally characterized synthetic metal complexes, and with myoglobin, and the manganese and iron superoxide dismutases. (4) Our long term objective is to apply the method to a wide variety of metalloproteins. We will compare the activities of structurally similar metalloenzyme and correlate differences in activities with structural differences. This method has the potential to provide chemical explanations for changes in enzyme behavior that accompany changes in conformations associated with binding inhibitors, toxins, and regulatory elements, and with disease causing mutations..