Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II.
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 A. 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.
Persson
M
M
Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208, USA.
Harbridge
J R
JR
Hammarström
P
P
Mitri
R
R
Mårtensson
L G
LG
Carlsson
U
U
Eaton
G R
GR
Eaton
S S
SS
D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013113:Spin Labels
D013113:Spin Labels; D002256:Carbonic Anhydrases