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Biophys J, June 2001, p. 2886-2897, Vol. 80, No. 6

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

Malin Persson,* James R. Harbridge,* Per Hammarström,dagger Ragheed Mitri,* Lars-Göran Mårtensson,dagger Uno Carlsson,dagger Gareth R. Eaton,* and Sandra S. Eaton*

 *Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208 USA; and  dagger IFM-Department of Chemistry, Linköping University, Linköping SE-581 83, Sweden

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.

Biophys J, June 2001, p. 2886-2897, Vol. 80, No. 6
© 2001 by the Biophysical Society   0006-3495/01/06/2886/12  $2.00


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