Evidence of local conformational fluctuations and changes in bacteriorhodopsin, dependent on lipids, detergents and trimeric structure, as studied by 13C NMR.
We examined how the local conformation and dynamics of [3-13C]Ala-labeled bacteriorhodopsin (bR) are altered as viewed from 13C NMR spectra when the natural membrane lipids are partly or completely replaced with detergents. It turned out that the major conformational features of bR, the alphaII-helices, are generally unchanged in the delipidated or solubilized preparations. Upon partial delipidation or detergent solubilization, however, a significant conformational change occurs, ascribed to local conversion of alphaII-->alphaI-helix (one Ala residue involved), evident from the upfield displacement of the transmembrane helical peak from 16.4 ppm to 14.5 ppm, conformational change (one or two Ala residues) within alphaII-helices from 16.4 to 16.0 ppm, and acquired flexibility in the loop region (especially at the F-G loop) as manifested from suppressed peak-intensities in cross-polarization magic angle spinning (CP-MAS) NMR spectra. On the other hand, formation of monomers as solubilized by Triton X-100, Triton N-101 and n-dodecylmaltoside is characterized by the presence of a peak at 15.5 ppm and a shifted absorption maximum (550 nm). The size of micelles under the first two conditions was small enough to yield 13C NMR signals observable by a solution NMR spectrometer, although 13C CP-MAS NMR signals were also visible from a fraction of large-sized micelles. We found that the 16.9 ppm peak (three Ala residues involved), visible by CP-MAS NMR, was displaced upfield when Schiff base was removed by solubilization with sodium dodecyl sulfate, consistent with our previous finding of bleaching to yield bacterioopsin.
Department of Life Science, Himeji Institute of Technology, Hyogo 678-1297, Japan.
M
M
Tanio
S
S
Tuzi
S
S
Yamaguchi
H
H
Konishi
A
A
Naito
R
R
Needleman
J K
JK
Lanyi
H
H
Saitô
D001436:Bacteriorhodopsins / Q000737:chemistry*; D002247:Carbon Isotopes; D003902:Detergents / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation
D002247:Carbon Isotopes; D003902:Detergents; D008055:Lipids; D001436:Bacteriorhodopsins