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）光谱研究将在简单氨基酸的铜掺杂晶体系统上进行，这些简单氨基酸晶体系统可作为铜金属蛋白活性位点的模型。从这些研究中得出的张量参数为了解这些系统中金属配位的性质提供了结构和键合的见解。这些研究非常重要，因为它们能够在结构明确的模型系统中提供明确且准确的光谱参数，并且可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 方法的实验进展使得能够测量不成对电子（主要位于过渡金属上）与邻近但距离较远的磁核之间越来越弱的相互作用。因此，蛋白质活性位点受损的电子波功能被越来越详细地定义。例如，此类研究已检测到蓝铜蛋白中的主链肽氮偶联和谷氨酰胺侧链氮偶联，以及铁硫蛋白中与铁配体氢键结合的氮的偶联。然而，由于缺乏良好的模型系统，仍然缺乏对测量到的相互作用的可靠解释，在该模型系统中，顺磁性离子和遥远的非咪唑氮之间的耦合已被明确且准确地量化。拟议的研究旨在通过对掺杂有肌酸、甘氨酸、甘氨酰甘氨酸和 Zn(I 蛋氨酸)2 的 Cu(II) 单晶进行 CW-EPR 和 ESEEM 测量来实现这一目标。第二个重点是开发一种拟合方法，通过该方法可以使用一组有限但选定的单晶取向 ESEEM 数据来准确确定 14N 耦合张量参数。长期目标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.