INTERACTION OF SPIN LABELS WITH TRANSITION METALS

生物大分子各部分之间距离的测量， 这样的蛋白质，是一个重大的挑战，特别是对于膜结合 蛋白质。我们的初步结果证明了测量的可行性。 两个顺磁中心之间的距离，尤指在 10-25埃的范围，填补了以下范围之间的空白 核磁共振和荧光能量转移是最有用的。一些生物分子 有两个自然形式的顺磁中心。一个或多个非本地 顺磁中心可以通过金属置换引入蛋白质中， 通过自旋标记或通过定点突变来创建新的位点 金属和自旋标签。因此，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， 自旋标记低自旋化合物的初步实验证实 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 知道顺磁中心驰豫时间的重要性 没有相互作用，以及弛豫时间依赖于 在EPR频谱中的位置。我们建议将这一理论推广到 其他金属自旋态的实验，测试金属的影响 超精细、g-各向异性和零场分裂(对于自旋&gt；1/2)，到 对模型进行验证，找出其适用范围。对这件事 最后，我们将在选定的距离制造带有自旋标记的突变肌红蛋白 和相对于血红素环的角度方向，并使用高自旋 自旋标记血红蛋白中的铁(III)和其他金属 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，我们将对此进行测试 预测。这项研究的结果将是一个经过验证的模型，以及 提供给其他工作人员的具体指南，告诉他们距离可以 对不同的顺磁中心进行测量，以及 作为可获得多少信息的函数，不确定性 每个中锋。

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.