Stability of the C-Terminal α-Helical Domain of Bacteriorhodopsin That Protrudes from the Membrane Surface, as Studied by High-Resolution Solid-State ¹³C NMR

Abstract

We have recorded ¹³C NMR spectra of [1-¹³C]Ala- and [3-¹³C] Ala-bacteriorhodopsin (bR), [1-¹³C] Ala- and [3-¹³C] Ala-papain-cleaved bR, and [3-¹³C]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 α-helical domain of the C-terminus that protrudes from the membrane surface. The comparative ¹³C CP- and DD-MAS NMR study of [3-¹³C] Ala-bR, rather than [1-¹³C] 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 α-helical domain (resonated at 15.9 ppm) either at neutral pH and/or at 10 to -10°C. The α-helical peak was not seen, however, after either cleavage of the C-terminus with papain or lowering the pH to 4.25. This a-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 α-helix, as indicated by an upfield displacement of the ¹³C NMR signal, when the temperature was lowered to 10--10°C. Surprisingly, the corresponding helical structure in R227Q is more stable than in the wild type at the acidic pH. This α-helical peak was classified as an α_{_??_}-helix from the ¹³C chemical shifts of Cβ carbon, although it was ascribed to an α₁-helix on the basis of the carbonyl shifts. This is in contrast to Ala 53 which adopts the α₁₁helix as judged from the ¹³C chemical shifts of Cβ 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 α₁₁-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 α-helix.