EPR/ESEEM SPECTROMETERY OF SINGLE CRYSTAL MODELS FOR PROTEIN ACTIVE SITES

电子顺磁共振（EPR）和电子自旋生态包络调制（ESEEM）光谱研究将对铜掺杂的简单氨基酸晶体系统进行研究，这些晶体系统可作为铜金属蛋白活性位点的模型。从这些研究中得到的张量参数为这些体系中金属配位的性质提供了结构和键合的见解。这些研究具有很大的重要性，因为它们能够在结构明确的模型系统中提供明确和准确的光谱参数，并且可以用于分析和解释冷冻溶液样品中金属蛋白的许多类似研究的结果。EPR方法的实验进展已经允许测量未配对电子（主要定位在过渡金属上）与邻近但较远的磁性原子核之间越来越弱的相互作用。因此，蛋白质活性位点上受损的电子波函数正变得越来越精细。例如，这些研究在蓝铜蛋白中检测到主肽氮偶联和谷氨酰胺侧链氮偶联，在铁硫蛋白中检测到氮氢键与铁配体的偶联。然而，由于缺乏一个良好的模型系统，顺磁离子和远端非咪唑氮之间的耦合已经明确和准确地量化，因此仍然缺乏对测量到的相互作用的可靠解释。提出的研究旨在通过对Cu（II）掺杂到肌酸、甘氨酸、甘氨酸和锌（I蛋氨酸）2中的单晶进行CW-EPR和ESEEM测量来实现这一目标。第二个重点将放在拟合方法的开发上，该方法可以使用有限但选择的单晶定向ESEEM数据集来准确确定14N耦合张量参数。长期目的是为实际金属蛋白单晶的类似EPR和ESEEM研究提供实验方法和分析基础。

Electron paramagnetic resonance (EPR) and electron spin eco envelope modulation (ESEEM) spectroscopic studies will be performed on copper doped crystal systems of simple amino acids that serve as models for active sites in copper metalloproteins. The tensor parameters derived from these studies provide structural and bonding insight into the nature of the metal coordination in these systems. These investigations have a great deal of importance because of their ability to provide unambiguous and accurate spectral parameters in model systems of well defined structure and can be utilized to analyze and interpret results of many similar studies of metalloproteins in frozen solution samples. Experimental advances in EPR methods have allowed the measurement of increasingly weaker interactions between an unpaired electron, mostly localized on a transition metal, and neighboring but distant magnetic nuclei. Thus, the impaired electron wave- function at the proteins active sites are becoming defined to an increasingly finer detail. For example, such studies have detected both backbone peptide nitrogen couplings and glutamine side chain nitrogen couplings in blue copper proteins, anc couplings from nitrogens hydrogen bonded to iron ligands in iron-sulfur proteins. However, solid interpretation of the measured interactions are still lacking due to the absence of a good model systems where the coupling between the paramagnetic ion and distant non- imidazole nitrogens have been unambiguously and accurately quantified. The proposed research aims to accomplish this by performing CW-EPR and ESEEM measurements on single crystals on Cu(II)-doped into creatine, glycine, glycylglycine and Zn(I methionine)2. A second focus will be on the development of a fitting method whereby a limited but select set of single crystal orientational ESEEM data can be used to accurately determine 14N coupling tensor parameters. The long term are to provide a basis both in terms of experimental methodology and analysis for similar EPR and ESEEM studies on single crystals on the actual metalloproteins.