H-1-NMR N-15-NMR STUDIES OF BACTERIORHODOPSIN HALOBACTERIUM-HALOBIUM - CONFORMATIONAL DYNAMICS OF THE 4-HELICAL BUNDLE

Series of uniformly and selectively N-15-labeled bacteriorhodopsins of Halobacterium halobium (strain ET 1001) were obtained and a H-1-N-15-NMR study was performed in methanol/chloroform (1 : 1) and 0. 1 M NH4CHOO, medium which mimics that in the membrane in vivo. Less than half of the cross-peaks expected from the amino acid sequence of uniformly N-15-labeled bacteriorhodopsin were observed, using heteronuclear H-1-N-15 coherence spectroscopy. In order to assign the observed cross-peaks, a selective N-15-labeling of amino acid residues (Tyr, Phe, Trp, Lys, Gly, Leu, Val or Ile) was carried out and H-1-N-15-NMR spectra of bacteriorhodopsin and its fragments C1 (residues (72-231), C2 (residues 1 - 71), B1 (residues 1 - 155) and BP2 (residues 163 - 231) were investigated. By this procedure, all observed H-1-N-15 cross-peaks of the entire bacteriorhodopsin were found to belong to the transmembrane segments A, B. and G. The cross-peaks from four (C, D, E and F) helical bundles (79 - 1 89 residues) were missed. These results clearly indicate that dynamic processes occur in the four helice bundle. The significance of this, in respect to bacteriorhodopsin functioning, is discussed.