Surface dynamics of bacteriorhodopsin as revealed by (13)C NMR studies on [(13)C]Ala-labeled proteins: detection of millisecond or microsecond motions in interhelical loops and C-terminal alpha-helix.
We have recorded (13)C NMR spectra of [2-(13)C]-, [1-(13)C]-, [3-(13)C],- and [1,2,3-(13)C(3)]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 (13)C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-(13)C]-, [1-(13)C]Ala-, and [1-(13)C]Gly-labeled 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 micros in the surface loops of membrane proteins. Instead, the three well-resolved (13)C CP-MAS NMR signals of [2-(13)C]Ala-bR, at 50.38, 49.90, and 47.96 ppm, were ascribed to the C-terminal alpha-helix previously proposed from the data for [3-(13)C]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 (13)C NMR signals were substantially broadened when (13)C NMR spectra of fully labeled [1,2,3-(13)C]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 (13)C-(13)C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among (13)C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal alpha-helix, loops, and transmembrane alpha-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.
Yamaguchi
S
S
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Kouto 3-chome, Kamigori, Hyogo 678-1297, Japan.
Tuzi
S
S
Yonebayashi
K
K
Naito
A
A
Needleman
R
R
Lanyi
J K
JK
Saitô
H
H
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D019617:Halobacterium salinarum / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D001436:Bacteriorhodopsins; D000409:Alanine