Journal Article

Surface Dynamics of Bacteriorhodopsin as Revealed by <sup>13</sup>C NMR Studies on [<sup>13</sup>C]Ala-Labeled Proteins: Detection of Millisecond or Microsecond Motions in Interhelical Loops and C-Terminal α-Helix

Satoru Yamaguchi, Satoru Tuzi, Koka Yomebayashi, Akira Naito, Richard Needleman, Janos K. Lanyi and Hazime Saitô

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 129, issue 3, pages 373-382
Published in print March 2001 | ISSN: 0021-924X
Published online March 2001 | e-ISSN: 1756-2651 | DOI: https://dx.doi.org/10.1093/oxfordjournals.jbchem.a002867
Surface Dynamics of Bacteriorhodopsin as Revealed by 13C NMR Studies on [13C]Ala-Labeled Proteins: Detection of Millisecond or Microsecond Motions in Interhelical Loops and C-Terminal α-Helix

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We have recorded 13C NMR spectra of [2-13C]-, [1-13C]-, [3-13C],- and [1,2,3-13C3]Ala-labeled bacteriorhodopsin (bR), and its mutants, A196G, A160G, and A103C, by means of cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques, to reveal the conformation and dynamics of bR, with emphasis on the loop and C-terminus structures. The 13C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-13C]-, [1-13C]Ala-, and [1-13C]Glylabeled bR, due to the presence of conformational fluctuation with correlation times of 10−4 s that interfered with the peak-narrowing by magic angle spinning. The observation of such suppressed peaks for specific residues provides a unique means of detecting intermediate frequency motions on the time scale of ms or (is in the surface loops of membrane proteins. Instead, the three well-resolved 13C CP-MAS NMR signals of [2-13C]Ala-bR, at 50.38, 49.90, and 47.96 ppm, were ascribed to the C-terminal a-helix previously proposed from the data for [3-13C]Ala-bR: the former two peaks were assigned to Ala 232 and 238, in view of the results of successive proteolysis experiments, while the highest-field peak was ascribed to Ala 235 prior to Pro 236. Even such 13C NMR signals were substantially broadened when 13C NMR spectra of fully labeled [1,2,3-13C]Ala-bR were recorded, because the broadening and splitting of peaks due to the accelerated transverse relaxation rate caused by the increased number of relaxation pathways through a number of 13C-13C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among 13C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal α-helix, loops, and transmembrane α-helices, were estimated by measurement of the spin-lattice relaxation times in the laboratory frame and spin-spin relaxation times under the conditions of cross-polarization-magic angle spinning, and comparative study of suppressed specific peaks between the CP-MAS and DD-MAS experiments.

Keywords: bacteriorhodopsin; C-terminal α-helix; interhelical loops; membrane proteins; surface dynamics

Journal Article.  0 words. 

Subjects: Biochemistry

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