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.
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