In reconstituted vesicles above the lipid phase transition temperature, bacteriorhodopsin (BR) undergoes rotational diffusion about an axis perpendicular to the plane of the bilayer [Cherry, R. J., Muller, U., & Schneider, G. (1977) FEBS Lett. 80, 465]. This diffusion narrows the 13C NMR powder line shape of the BR peptide carbonyls. In contrast, BR in native purple membrane is relatively immobile and exhibits a rigid-lattice powder line shape. By use of the principal values of the rigid-lattice chemical shift tensor and the motionally narrowed line shape from the reconstituted system, the range of Euler angles of the leucine peptide groups relative to the diffusion axis has been calculated. The experimentally observed line shape is inconsistent with those expected for structures which consist entirely of either α helix or β sheet perpendicular to the membrane or β sheet tilted at angles up to about 60° from the membrane normal. However, for two more complex structural models, the predicted line shapes agree well with the experimental one. These are, first, a structure consisting entirely of α1 helices tilted at 20° from the membrane normal and, second, a combination of 60% an helix perpendicular to the membrane plane and 40% antiparallel β sheet tilted at 10-20° from the membrane normal. The results also indicate that the peptide backbone of bacteriorhodopsin in native purple membrane is extremely rigid even at 40°. The experiments presented here demonstrate a new approach, using solid-state nuclear magnetic resonance (NMR) methods, for structural studies of transmembrane proteins in fluid membrane environments, either natural or reconstituted. Analysis of NMR powder line shapes which are narrowed by anisotropic rotational diffusion can provide information not only on secondary structure but also, in general, on the orientation of labeled groups relative to the axis of rotational diffusion. Such information on the orientation of membrane proteins in the bilayer plane is difficult to obtain by more conventional structural methods.
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