High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin.
Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.
Steinhoff
H
H
Lehrstuhl für Biophysik, Ruhr-Universität Bochum, 44780, Bochum, Germany. hjs@bph.ruhr-uni-biochum.de
Savitsky
A
A
Wegener
C
C
Pfeiffer
M
M
Plato
M
M
Möbius
K
K
D001436:Bacteriorhodopsins / Q000737:chemistry*; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006217:Halobacterium; D008698:Mesylates; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013113:Spin Labels
D003497:Cyclic N-Oxides; D008698:Mesylates; D013113:Spin Labels; D001436:Bacteriorhodopsins; C034248:(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate