INTERACTION OF SPIN LABELS WITH TRANSITION METALS

这项拟议研究的总体目标是开发、验证和应用电子顺磁共振(EPR)方法来测量生物系统中的距离。EPR研究可以在无序固体或溶液中进行，甚至可以在整个细胞中进行。EPR对于表征难以结晶的膜结合蛋白特别重要。EPR要研究的位点可以是自然产生的金属离子或自由基，也可以是自旋标记或通过定点突变引入的附着在选定位置的金属。即使是几个更长距离的测量，也可以提供关键信息来定义大型蛋白质或蛋白质组装的三维结构。未配对电子的磁矩比核自旋的磁矩大，因此EPR可以比核磁共振测量更长的距离。所提出的研究重点是脉冲EPR方法，以确定大于约20埃的距离，这大约是基于连续波线型的EPR方法的上限。我们建议使用电子自旋驰豫速率随距离的变化来确定快速驰豫的电子自旋(如铁(III))和氮氧基自旋标记之间的距离。这些方法将用已知结构的高铁肌红蛋白的自旋标记变体进行校准。铁运输蛋白铁蛋白A的X射线晶体结构没有定义铁铁载体铁蛋白的结合部位。我们建议使用我们的脉冲技术来确定铁结合位点的位置。我们的模型预测，在较低微波频率下的实验将允许比9.2 GHz下可访问的距离更长的测量，我们建议通过在2.5 GHz下进行测量来验证这一预测。电流脉冲EPR距离测量是在低温下进行的。我们将测试在液体溶液中测量的可行性。对于双自旋标记的人碳酸氢酶II、T4溶菌酶和铁蛋白A的样品，我们建议开发和测试脉冲方法来确定大于约20埃的自旋-自旋距离。

The overall goal of the proposed research is to develop, validate, and apply electron paramagnetic resonance (EPR) methodology for measuring distances in biological systems. EPR studies can be performed in disordered solids or in solutions and even in whole cells. EPR is particularly important for characterizing membrane-bound proteins that are difficult to crystallize. The sites to be studied by EPR can be naturally-occurring metal ions or radicals, or spin labels or metals attached at selected locations introduced by site-directed mutagenesis. Even a few measurements of longer distances can provide key information to define the three-dimensional structure of a large protein or assembly of proteins. The larger magnetic moment of an unpaired electron than of a nuclear spin permits measurement of longer distances by EPR than by nuclear magnetic resonance (NMR). The emphasis of the proposed research is on pulsed EPR methods to determine distances longer than about 20 Angstroms, which is approximately the upper limit for EPR methods based on continuous wave lineshapes. We propose to use the distance-dependent changes in electron spin relaxation rates to determine the distance between a rapidly relaxing electron spin such as iron(III) and a nitroxyl spin label. These methods will be calibrated with spin-labeled variants of metmyoglobin of known structure. The X-ray crystal structure of the iron transport protein, iron protein A, did not define the binding site of the iron siderophore, iron enterobactin. We propose to use our pulse techniques to determine the location of the iron binding site. Our model predicts that experiments at lower microwave frequency will permit measurements of longer distances than are accessible at 9.2 GHz and we propose to test that prediction by performing measurements at 2.5 GHz. Current pulsed EPR measurements of distances are performed at cryogenic temperatures. We will test the feasibility of measurements in fluid solution. For doubly spin-labeled samples of human carbonic anhydrase II, T4 lysozyme, and iron protein A we propose to develop and test pulsed methods to determine spin-spin distances greater than about 20 Angstroms.