Stability of the C-terminal alpha-helical domain of bacteriorhodopsin that protrudes from the membrane surface, as studied by high-resolution solid-state 13C NMR.
We have recorded 13C NMR spectra of [1-(13)C]Ala- and [3-(13)C]Ala-bacteriorhodopsin (bR), [1-(13)C]Ala- and [3-(13)C]Ala-papain-cleaved bR, and [3-(13)C]Ala-labeled R227Q bR mutant by cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) methods. The pH and temperature were varied, and Arg 227 was replaced with Gln (R227Q), in order to clarify their effects on the stability of the alpha-helical domain of the C-terminus that protrudes from the membrane surface. The comparative 13C CP- and DD-MAS NMR study of [3-(13)C]Ala-bR, rather than [1-(13)C]Ala-bR, turned out to be the best means to distinguish the 13C NMR signals of the C-terminus from those of the rest of the transmembrane helices or loops. The inner segment of the C-terminus, from Ala 228 to Ala 235, forms an alpha-helical domain (resonated at 15.9 ppm) either at neutral pH and/or at 10 to -10 degrees C. The alpha-helical peak was not seen, however, after either cleavage of the C-terminus with papain or lowering the pH to 4.25. This alpha-helical structure, and a part of the random coil which was produced from the helix at pH 4.25, were further converted to a low-temperature-type alpha-helix, as indicated by an upfield displacement of the 13C NMR signal, when the temperature was lowered to 10- -10 degrees C. Surprisingly, the corresponding helical structure in R227Q is more stable than in the wild type at the acidic pH. This alpha-helical peak was classified as an alphaII-helix from the 13C chemical shifts of Cbeta carbon, although it was ascribed to an alphaI-helix on the basis of the carbonyl shifts. This is in contrast to Ala 53 which adopts the alphaII-helix as judged from the 13C chemical shifts of Cbeta and the carbonyl carbons. Therefore, this discrepancy might be caused by differential sensitivity of the two types of carbon signals to conformation and to modes of hydrogen bonding when motional fluctuation is involved. It is likely that the alphaII-helix form present at the C-terminus is not always the type originally proposed but should be considered as a form undergoing large-amplitude conformational fluctuation around alpha-helix.
Yamaguchi
S
S
Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo.
Tuzi
S
S
Seki
T
T
Tanio
M
M
Needleman
R
R
Lanyi
J K
JK
Naito
A
A
Saitô
H
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001436:Bacteriorhodopsins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D019617:Halobacterium salinarum / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013696:Temperature
D001436:Bacteriorhodopsins