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)光谱将对铜掺杂的简单氨基酸晶体体系进行研究，作为铜金属蛋白活性中心的模型。由这些研究得出的张量参数提供了对这些系统中金属配位性质的结构和成键的洞察。这些研究具有非常重要的意义，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，并可用于分析和解释许多类似的冷冻溶液中金属蛋白研究的结果。EPR方法的实验进展使人们能够测量到未配对电子与邻近但遥远的磁核之间越来越弱的相互作用。因此，蛋白质活性部位受损的电子波功能正变得越来越详细。例如，这类研究在蓝色铜蛋白中检测到主链多肽氮偶联和谷氨酰胺侧链氮偶联，在铁硫蛋白中检测到从氮氢键到铁配体的ANC偶联。然而，由于没有一个好的模型系统来明确和准确地量化顺磁性离子和远距离非咪唑氮之间的耦合，因此仍然缺乏对测量到的相互作用的可靠解释。这项研究的目的是通过对掺入肌酸、甘氨酸、甘氨酸锌和甲硫氨酸的单晶进行CW-EPR和ESEEM测量来实现这一点。第二个重点是开发一种拟合方法，通过这种方法，有限但选择的单晶取向ESEEM数据可以准确地确定14N耦合张量参数。长期而言，将为实际金属蛋白单晶的类似EPR和ESEEM研究提供实验方法学和分析基础。

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.