STRUCTURE AND DYNAMICS OF HEME PROTEIN ACTIVE SITES

这项建议的目的是确定特定的血红素口袋 在CcP上传递过氧化物酶行为的氨基酸，等等 通常对所有过氧化物酶。 这将通过平行动力学来实现 和光谱研究的合理设计的突变体的CcP。 NMR 将用于监测H52、N82、R48的pH行为， 最有可能的“远端”血红素口袋氨基酸作为主要功能 反应性调节器 CcP的突变体来源于这些位点的改变， 位置将通过动力学筛选，并且它们的溶液化学将 通过NMR表征。 这将需要扩大应用 核磁共振波谱顺磁性蛋白质通过确定最佳 顺磁性蛋白的蛋白大小/场强组合， 通过扩展核磁共振顺磁共振应用程序的剧目， 蛋白质到异核（13 C和15 N）。 均匀同位素标记 这项工作所需的蛋白质将使用以下表达式来创建： 在过去的赠款期间开发的CcP系统。 将发生CcP的广泛蛋白质工程。 其目的是 产生CcP的远端氨基酸突变体， H2 O2活化，从而指示哪些对于H2 O2活化是关键的。 activation. 筛选这些突变体的主要方法将是 双分子初始速率常数的动力学测量 Ccp和H2 O2的反应（在CcP循环中）。 筛选将 在詹姆斯·厄曼教授的实验室里进行 一个工作 根据有限的公开信息，假设CcP 远端组氨酸（H52）是血红素配体结合的主要调节剂 化学和H2 O2活化。 我们还建议完成X射线 三种主要单体的晶体结构测定 从二鳃单胞藻中分离得到血红蛋白（GMH 2、GMH 3、GMH 4）。 这些蛋白质都没有末端组氨酸;都有末端亮氨酸 和高度改变的配体结合动力学和反应性相比， MB. 对于GMH 4，我们计划创建具有远端连接的Mb模拟蛋白。 组氨酸：（LE 7 H）GMH 4。 将对天然和突变体进行动力学研究 以确定它们与H2 O2反应的双分子速率常数。 如果我们对CcP的理解得到证实，那么我们相信， 通过PheB 10 His增强该蛋白中H2 O2活化的速率 突变体，从而迈出了第一步，工程师大量的非天然 酶活性转化为配体结合蛋白。

The objective of this proposal is to identify the specific heme pocket amino acids that convey peroxidase behavior upon CcP, and more generally to all peroxidases. This will be achieved by parallel kinetic and spectroscopic studies on rationally designed mutants of CcP. NMR will be used to monitor the pH behavior of H52, N82, R48 which are the most likely 'distal' heme pocket amino acids to function as primary reactivity regulators. Mutants of CcP derived from alterations at these positions will be screened by kinetics and their solution chemistry will be characterized by NMR. This will require expanding applications of NMR spectroscopy to paramagnetic proteins by identifying optimal protein size/field strength combinations for paramagnetic proteins and by expanding the repertoire of NMR applications for paramagnetic proteins to heteronuclei (13C and 15N). Uniformly isotope-labelled proteins necessary for this work will be created using the expression system for CcP that has been developed during the past grant period. Extensive protein engineering of CcP will occur. Its aim will be to create distal amino acid mutants of CcP that show altered reactivity towards H202 activation, thereby indicating which are critical for H202 activation. The primary method for screening these mutants will be kinetic measurement of the bimolecular rate constant for the initial reaction (in the CcP cycle) of Ccp and H202. Screening will be carried out in Professor James Erman's laboratory. A working hypothesis, based on limited published information, is that the CcP distal histidine (H52) is the primary regulator of heme ligand binding chemistry and H202 activation. We also propose to complete x-ray crystallographic structure determinations of the three major monomer hemoglobins (GMH2, GMH3, GMH4) from Glycera dibranchiata. None of these proteins have distal histidines; all have distal leucines and highly altered ligand binding dynamics and reactivity compared to Mb. For GMH4 we plan to create the Mb-mimic protein with a distal histidine: (LE7H)GMH4. Native and mutant will be kinetically studied to determine their bimolecular rate constants for reaction with H202. If our understanding of CcP bears out then we believe that we can enhance the rate of H202 activation in this protein via the PheB10His mutant, thereby taking the first step to engineer substantial non-natural enzymic activity into this ligand binding protein.