Comparison of Electron Paramagnetic Resonance Methods to Determine Distances between Spin Labels on Human Carbonic Anhydrase II

Abstract

Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 Å. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the non-interacting component was compared.