MOLECULAR DISTANCES FROM DIPOLAR COUPLED SPIN-LABELS - THE GLOBAL ANALYSIS OF MULTIFREQUENCY CONTINUOUS-WAVE ELECTRON-PARAMAGNETIC-RESONANCE DATA

For immobilized nitroxide spin-labels with a well-defined interprobe g eometry, resolved dipolar splittings can be observed in continuous wav e electron paramagnetic resonance (CW-EPR) spectra for interelectron d istances as large as 30 Angstrom using perdeuterated probes. In this w ork, algorithms are developed for calculating CW-EPR spectra of immobi lized, dipolar coupled nitroxides, and then used to define the limits of sensitivity to the interelectron distance as a function of geometry and microwave frequency. Secondly, the CW-EPR spectra of N-6-spin-lab led coenzyme NAD(+) bound to microcrystalline, tetrameric glyceraldehy de-3-phosphate dehydrogenase (GAPDH) have been collected at 9.8, 34, a nd 94 GHz. These data have been analyzed, using a combination of simul ated annealing and global analysis, to obtain a unique fit to the data . The values of the internitroxide distance and the five angles defini ng the relative orientation of the two nitroxides are in reasonable ag reement with a molecular model built from the known crystal structure. Finally, the effect of rigid body isotropic rotational diffusion on t he CW-EPR spectra of dipolar coupled nitroxides has been investigated using an algorithm based on Brownian dynamics trajectories. These calc ulations demonstrate the sensitivity of CW-EPR spectra to dipolar coup ling in the presence of rigid body rotational diffusion.