INTERACTION OF SPIN LABELS WITH TRANSITION METALS

生物大分子各部分之间距离的测量， 这种蛋白质，是一个重大的挑战，特别是对膜结合的 proteins.我们的初步结果表明，测量的可行性 两个顺磁中心之间的距离，尤指在 范围为10-25埃，这填补了范围之间的差距， NMR和荧光能量转移是最有用的。一些生物分子 在其天然形式下具有两个顺磁中心。一个或多个非天然 顺磁中心可以通过金属置换引入蛋白质， 通过自旋标记，或通过定点诱变来产生新的位点， 金属和自旋标签。因此，用于测量距离的EPR方法是 具有普遍适用性。我们开发了新的理论模型， 通过对自旋标记的低自旋 高铁血红蛋白，用于测量金属和自旋之间的距离 标签我们已经从理论和实验上证明， 了解顺磁中心弛豫时间的重要性 没有相互作用，以及弛豫时间对 在EPR谱中的位置。我们建议扩展该理论， 实验到其他金属自旋状态，测试金属的影响， 超精细、g-各向异性和零场分裂（对于自旋> 1/2）， 验证模型并找出其适用性的局限性。本 最后，我们将在选定的距离处制造带有自旋标记的突变肌红蛋白， 以及相对于血红素环的角度取向，并使用高自旋 铁（III）和其他金属的自旋标记血红蛋白，以提炼 计算。我们预测，更长的距离可以在 微波频率低于正常的X波段，我们将测试这个 预测.这项研究的结果将是一个经过验证的模型， 向其他工作人员提供具体的指导方针，告诉他们什么范围的距离可以 可以测量各种对顺磁中心，以及 不确定性是，作为一个函数，有多少信息是可用的， 每个中心。

The measurement of distances between parts of biological macromolecules, such a proteins, is a major challenge, especially for membrane-bound proteins. Our preliminary results demonstrate the feasibility of measuring the distance between two paramagnetic centers, especially in the distance range of 10-25 Angstroms, which fills the gap between the ranges in which NMR and fluorescence energy transfer are most useful. Some biomolecules have two paramagnetic centers in their native form. One or more non-native paramagnetic centers can be introduced into proteins by metal replacement, by spin labeling, or by site-directed mutagenesis to create new sites for metals and for spin labels. Thus, an EPR method for measuring distance is of general applicability. We have developed new theoretical models, confirmed by preliminary experiments on spin-labeled low-spin methemoglobin, for the measurement of distances between metals and spin labels. We have demonstrated, both theoretically and experimentally, the importance of knowing the relaxation times of the paramagnetic centers in the absence of interaction, and the dependence of the relaxation times on the position in the EPR Spectrum. We propose to extend the theory and experiments to other metal-spin states, testing the effects of metal hyperfine, g-anisotropy, and zero-field splitting (for spin> 1/2), to validate the models and find the limits of their applicability. To this end we will make mutant myoglobins with spin labels at selected distances and angular orientations relative to the heme ring, and use high-spin iron(III) and other metals in spin-labeled hemoglobin to refine the calculations. We predict that longer distances can be measured at microwave frequencies lower than the normal X-band, and we will test this prediction. The result of this research will be a validated model, and specific guidelines to other workers telling what range of distances can be measured for various pairs of paramagnetic centers, and what the uncertainties are, as a function of how much information is available for each center.