STABILITY OF THE C-TERMINAL ALPHA-HELICAL DOMAIN OF BACTERIORHODOPSINTHAT PROTRUDES FROM THE MEMBRANE-SURFACE, AS STUDIED BY HIGH-RESOLUTION SOLID-STATE C-13 NMR

We have recorded C-13 NMR spectra of [1-C-13]Ala-and [3-C-13] Ala-bact eriorhodopsin (bR), [1-C-13]Ala-and [3-C-13]Ala-papain-cleaved bR, and [3-C-13]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 Gin (R227Q), in order to clarify their effects on the stability of the alpha-helical domain of the C-terminus that protrudes fi om the membrane surface, The comparative C-13 CP- and DD-MAS NMR study of [3 -C-13]Ala-bR, rather than [1-C-13]Ala-bR, turned out to be the best me ans to distinguish the C-13 NMR signals of the C-terminus from those o f 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 domai n (resonated at 15.9 ppm) either at neutral pH and/or at 10 to -10 deg rees C. The alpha-helical peak was not seen, however, after either cle avage of the C-terminus with papain or lowering the pH to 4.25, This a lpha-helical structure, and a part of the random coil which was produc ed from the helix at pH 4.25, were further converted to a low-temperat ure-type alpha-helix, as indicated by an upfield displacement of the C -13 NMR signal, when the temperature was lowered to 10-10;C. Surprisin gly, the corresponding helical structure in R227Q is more stable than in the wild type at the acidic pH, This alpha-helical peak was classif ied as an alpha(II)-helix from the C-13 chemical shifts of C beta carb on, although it was ascribed to an alpha(I)-helix on the basis of the carbonyl shifts, This is in contrast to Ala 53 which adopts the alpha( II)-helix as judged from the C-13 chemical shifts of C beta and the ca rbonyl carbons, Therefore, this discrepancy might be caused by differe ntial sensitivity of the two types of carbon signals to conformation a nd to modes of hydrogen bonding when motional fluctuation is involved, It is likely that the alpha(II)-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 a lpha-helix.