C-13 NMR-STUDY ON CONFORMATION AND DYNAMICS OF THE TRANSMEMBRANE ALPHA-HELICES, LOOPS, AND C-TERMINUS OF [3-C-13]ALA-LABELED BACTERIORHODOPSIN

We have recorded C-13 CP-MAS and DD-MAS NMR spectra of untreated and deionized [3-C-13]Ala-labeled bacteriorhodopsin (bR) and those cleaved with carboxypeptidase A and papain to gain insight into the conformation and dynamics of the transmembrane alpha-helices, loops, and C-terminus. It turned out that the C-terminus does not contribute to the C-13 CP-MAS NMR spectra of [3-C-13]Ala-bR recorded at ambient temperature owing to its rapid reorientational motions, since the relative peak intensities were unchanged in spite of the enzymatic cleavages. Therefore, the C-13 CP-MAS NMR peaks-of bR should be ascribed both to the transmembrane alpha-helices and loops. We further distinguished the peaks of the alpha-helix form at 16.3 ppm (60%) from those of the alpha-helix form at 14.9 ppm (20%) by deconvolution of the respective peaks of the hydrated [3-C-13]Ala-bR, as referred to the C-13 chemical shift of polyalanine in hexafluoroisopropyl alcohol. The remaining CP-MAS NMR peak of [3-C-13]Ala-bR at 17.2 ppm was ascribed to the loops (20%) taking a variety of turn structures. In contrast, the C-13 NMR signals from the C-terminal residues were significantly enhanced by recording the dipolar-decoupled (DD)-MAS NMR spectra. Conformational features of the two different portions of the C-terminus, residues 245-248 and 231-244, were revealed by the conformation-dependent C-13 signals of bR successively cleaved by carboxypeptidase A and papain, respectively. The terminal end, residues 245-248, containing two Ala residues is virtually disordered and undergoing rapid motions. The inner segment (231-244) containing four Ala residues participates in an ordered conformation involving the alpha-helical conformation. This conformation is very stable with a lifetime much longer than 10(-2)s, as viewed from the observed separation of sharp peaks.