1H‐15N‐NMR studies of bacteriorhodopsin Halobacterium halobium

Series of uniformly and selectively 15N-labeled bacteriorhodopsins of Halobacterium halobium (strain ET 1001) were obtained and a 1H-15N-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 15N-labeled bacteriorhodopsin were observed, using heteronuclear 1H-15N coherence spectroscopy. In order to assign the observed cross-peaks, a selective 15N-labeling of amino acid residues (Tyr, Phe, Trp, Lys, Gly, Leu, Val or Ile) was carried out and 1H-15N-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 1H-15N 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 (79-189 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.