Backbone dynamics of bacteriorhodopsin as studied by (13)C solid-state NMR spectroscopy.
The surface dynamics of bacteriorhodopsin was examined by measurements of site-specific (13)C-(1)H dipolar couplings in [3-(13)C]Ala-labeled bacteriorhodopsin. Motions of slow or intermediate frequency (correlation time <50 micro s) scale down (13)C-(1)H dipolar couplings according to the motional amplitude. The two-dimensional dipolar and chemical shift (DIPSHIFT) correlation technique was utilized to obtain the dipolar coupling strength for each resolved peak in the (13)C MAS solid-state NMR spectrum, providing the molecular order parameter of the respective site. In addition to the rotation of the Ala methyl group, which scales the dipolar coupling to 1/3 of the rigid limit value, fluctuations of the Calpha-Cbeta vector result in additional motional averaging. Typical order parameters measured for mobile sites in bacteriorhodopsin are between 0.25 and 0.29. These can be assigned to Ala103 of the C-D loop and Ala235 at the C-terminal alpha-helix protruded from the membrane surface, and Ala196 of the F-G loop, as well as to Ala228 and Ala233 of the C-terminal alpha-helix and Ala51 from the transmembrane alpha-helix. Such order parameters departing significantly from the value of 0.33 for rotating methyl groups are obviously direct evidence for the presence of fluctuation motions of the Ala Calpha-Cbeta vectors of intact preparations of fully hydrated, wild-type bacteriorhodopsin at ambient temperature. The order parameter for Ala160 from the expectantly more flexible E-F loop, however, is unavailable under highest-field NMR conditions, probably because increased chemical shift anisotropy together with intrinsic fluctuation motions result in an unresolved (13)C NMR signal.
Barré
Patrick
P
Institute of Medical Physics and Biophysics, University of Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany.
Yamaguchi
Satoru
S
Saitô
Hazime
H
Huster
Daniel
D
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000032:analysis / Q000737:chemistry* / Q000528:radiation effects*; D003198:Computer Simulation; D008027:Light; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D011487:Protein Conformation / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D001436:Bacteriorhodopsins