INTERACTION OF SPIN LABELS WITH TRANSITION METALS

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，甚至在整个细胞中进行。EPR对于表征难以结晶的膜结合蛋白质特别重要。通过EPR研究的位点可以是天然存在的金属离子或自由基，或通过定点诱变引入的附着在选定位置的自旋标记或金属。即使是一些较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。未成对电子的磁矩比核自旋的磁矩大，这使得EPR可以比核磁共振（NMR）测量更长的距离。拟议的研究的重点是脉冲EPR方法，以确定超过约20埃的距离，这大约是上限的EPR方法的基础上连续波线型。我们建议使用电子自旋弛豫速率的距离依赖性变化来确定快速弛豫的电子自旋，如铁（III）和硝酰基自旋标签之间的距离。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。铁转运蛋白（铁蛋白A）的X射线晶体结构没有确定铁载体（铁肠杆菌素）的结合位点。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，在较低的微波频率的实验将允许测量更长的距离比在9.2 GHz的访问，我们建议通过在2.5 GHz的测量进行测试，预测。电流脉冲EPR距离测量在低温下进行。我们将测试在流体溶液中测量的可行性。对于双自旋标记的人类碳酸酐酶II，T4溶菌酶，和铁蛋白A的样品，我们建议开发和测试脉冲的方法，以确定自旋-自旋距离大于约20埃。

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.