Temperature-dependent conformational change of bacteriorhodopsin as studied by solid-state C-13 NMR

Cross-polarization and dipolar-decoupled magic-angle spinning C-13-NMR spectra of [3-C-13]Ala-labelled bacteriorhodopsin were obtained for hydrated purple membrane in the temperatures range 23 degrees C to -110 degrees C. Well-resolved C-13-NMR signals were observed either at ambient temperature or at -20 degrees C but were broadened considerably at lower temperature below -40 degrees C, This situation was interpreted in terms of the presence of exchange processes with a rate constant of 10(2) s(-1) at ambient temperature among several conformations slightly different from each other. We found that such an exchange process was strongly influenced by the manner of organization of the lipid bilayers depending upon the presence or absence of cations responsible for electric shielding of negative charge at the polar head groups. The manner of organization of the lipid bilayers was conveniently characterized by a characteristic temperature at which the methyl peaks of fatty acyl groups of lipids in the purple membrane were suppressed due to interference of motional frequency with the decoupling frequency (10-100 kHz) for preparations containing 10 mM NaCl or CaCl2. No such spectral change in the absence of these cations was noted even if a preparation was cooled to -110 degrees C. The secondary structures of [3-C-13]Ala-labelled bacteriorhodopsin was not always identical at temperatures between ambient and low temperatures, since the C-13 chemical shifts and relative peak intensities for purple membrane preparations containing these salts changed with temperature in the range -110 degrees C to 23 degrees C. In particular, we found that some residues involving Ala residues at the alpha(II)-helix and loop region were converted at temperatures below -60 degrees C to a conformation involving alpha(I)-helix. In other words, some portion of the alpha-helical conformation of bacteriorhodopsin proposed from results obtained by cryo-electron microscopy, at very low temperatures, is not always retained at ambient temperature.