title: Molecular distances from dipolar coupled spin-labels: the global analysis of multifrequency continuous wave electron paramagnetic resonance data.
abstract: For immobilized nitroxide spin-labels with a well-defined interprobe geometry, resolved dipolar splittings can be observed in continuous wave electron paramagnetic resonance (CW-EPR) spectra for interelectron distances as large as 30 A using perdeuterated probes. In this work, algorithms are developed for calculating CW-EPR spectra of immobilized, 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 epsilon-spin-labeled coenzyme NAD+ bound to microcrystalline, tetrameric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been collected at 9.8, 34, and 94 GHz. These data have been analyzed, using a combination of simulated annealing and global analysis, to obtain a unique fit to the data. The values of the intermitroxide distance and the five angles defining the relative orientation of the two nitroxides are in reasonable agreement with a molecular model built from the known crystal structure. Finally, the effect of rigid body isotropic rotational diffusion on the CW-EPR spectra of dipolar coupled nitroxides has been investigated using an algorithm based on Brownian dynamics trajectories. These calculations demonstrate the sensitivity of CW-EPR spectra to dipolar coupling in the presence of rigid body rotational diffusion.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: E J
authors.initials: EJ
authors.lastname: Hustedt
authors.forename: A I
authors.initials: AI
authors.lastname: Smirnov
authors.forename: C F
authors.initials: CF
authors.lastname: Laub
authors.forename: C E
authors.initials: CE
authors.lastname: Cobb
authors.forename: A H
authors.initials: AH
authors.lastname: Beth
mesh_terms: D000465:Algorithms; D000818:Animals; D004058:Diffusion; D004578:Electron Spin Resonance Spectroscopy; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D008433:Mathematics; D015394:Molecular Structure; D018482:Muscle, Skeletal / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013113:Spin Labels
chemical_list: D013113:Spin Labels; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases