Alteration of conformation and dynamics of bacteriorhodopsin induced by protonation of Asp 85 and deprotonation of Schiff base as studied by C-13 NMR

According to previous X-ray diffraction studies, the D85N mutant of bacteri orhodopsin (bR) with unprotonated Schiff base assumes a protein conformatio n similar to that in the M photointermediate. We recorded C-13 NMR spectra of [3-C-13]Ala- and [1-C-13]Val-labeled D85N and D85N/D96N mutants at ambie nt temperature to examine how conformation and dynamics of the protein back bone are altered when the Schiff base is protonated (at pH 7) and unprotona ted (at pH 10). Most notably, we found that the peak intensities of three t o four [3-C-13]Ala-labeled residues from the transmembrane cl-helices, incl uding Ala 39, 51, and 53 (helix B) and 215 (helix G), were suppressed in D8 5N and D85N/D96N both from CP-MAS (cross polarization-magic angle spinning) and DD-MAS (dipolar decoupled-magic angle spinning) spectra, irrespective of the pH. This is due to conformational change and subsequent acquisition of intermediate time-range motions, with correlation times in the order of 10(-5) or 10(-4) s, which interferes with proton decoupling frequency or fr equency of magic angle spinning, respectively, essential for an attempted p eak-narrowing to achieve high-resolution NMR signals. Greater changes were achieved, however, at pH 10, which indicate large-amplitude motions of tran smembrane helices upon deprotonation of Schiff base and the formation of th e M-like state in the absence of illumination. The spectra detected more ra pid motions in the extracellular and/or cytoplasmic loops, with correlation times increasing from 10(-4) to 10(-5) s. Conformational changes in the tr ansmembrane helices were located at helices B, G, and D as viewed from the above-mentioned spectral changes, as well as at 1-C-13-labeled Val 49 (heli x B), 69 (B-C loop), and [3-C-13]Ala-labeled Ala 126 (D-helix) signals, in addition to the cytoplasmic and extracellular loops. Further, we found that in the M-like state the charged state of Asp 96 at the cytoplasmic side su bstantially modulated the conformation and dynamics of the extracellular re gion through long-distance interaction.