Regio-selective Detection of Dynamic Structure of Transmembrane α-Helices as Revealed from 13C NMR Spectra of [3- 13C]Ala-labeled Bacteriorhodopsin in the Presence of Mn 2+ Ion

Abstract

13C Nuclear magnetic resonance (NMR) spectra of [3- 13C]Ala-labeled bacteriorhodopsin (bR) were edited to give rise to regio-selective signals from hydrophobic transmembrane α-helices by using NMR relaxation reagent, Mn 2+ ion. As a result of selective suppression of 13C NMR signals from the surfaces in the presence of Mn 2+ ions, several 13C NMR signals of Ala residues in the transmembrane α-helices were identified on the basis of site-directed mutagenesis without overlaps from 13C NMR signals of residues located near the bilayer surfaces. The upper bound of the interatomic distances between 13C 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 Å to result in the signal broadening to 100 Hz and consistent with the data based on experimental finding. The Ala C β 13C 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 13C 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 α-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 α-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.