Regio-selective detection of dynamic structure of transmembrane alpha-helices as revealed from (13)C NMR spectra of [3-13C]Ala-labeled bacteriorhodopsin in the presence of Mn2+ ion.
13C Nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala-labeled bacteriorhodopsin (bR) were edited to give rise to regio-selective signals from hydrophobic transmembrane alpha-helices by using NMR relaxation reagent, Mn(2+) ion. As a result of selective suppression of (13)C NMR signals from the surfaces in the presence of Mn(2+) ions, several (13)C NMR signals of Ala residues in the transmembrane alpha-helices were identified on the basis of site-directed mutagenesis without overlaps from (13)C NMR signals of residues located near the bilayer surfaces. The upper bound of the interatomic distances between (13)C nucleus in bR and Mn(2+) ions bound to the hydrophilic surface to cause suppressed peaks by the presence of Mn(2+) ion was estimated as 8.7 A to result in the signal broadening to 100 Hz and consistent with the data based on experimental finding. The Ala C(beta) (13)C NMR peaks corresponding to Ala-51, Ala-53, Ala-81, Ala-84, and Ala-215 located around the extracellular half of the proton channel and Ala-184 located at the kink in the helix F were successfully identified on the basis of (13)C NMR spectra of bR in the presence of Mn(2+) ion and site-directed replacement of Ala by Gly or Val. Utilizing these peaks as probes to observe local structure in the transmembrane alpha-helices, dynamic conformation of the extracellular half of bR at ambient temperature was examined, and the local structures of Ala-215 and 184 were compared with those elucidated at low temperature. Conformational changes in the transmembrane alpha-helices induced in D85N and E204Q and its long-range transmission from the proton release site to the site around the Schiff base in E204Q were also examined.
Tuzi
S
S
Department of Life Science, Himeji Institute of Technology, Harima Science Garden City, Kouto 3-chome, Kamigori, Hyogo 678-1297, Japan.
Hasegawa
J
J
Kawaminami
R
R
Naito
A
A
Saitô
H
H
D000409:Alanine / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011522:Protons; D013696:Temperature; D013997:Time Factors
D008565:Membrane Proteins; D011522:Protons; D008345:Manganese; D001436:Bacteriorhodopsins; D000409:Alanine