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

Abstract

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.

Three‐dimensional structure of (1–71)bacterioopsin solubilized in methanol/chloroform and SDS micelles determined by 15N‐1H heteronuclear NMR spectroscopy

“…Seigneuret et al (1991 ) proposed n-dodecyl maltoside detergent micelles as a membrane-mimicking milieu suitable for high-resolution NMR investigations, where the BR monomeric molecule keeps many spectral as well as structural features of the native chromoprotein. In this medium, BR had intermediaterate conformational dynamics that could be inferred from the absence of about 40% of the expected cross-peaks in the ',N-'H heteronuclear chemical shift correlation spectra (Seigneuret et al, 1992), which was also seen in the organic mixture (Orekhov et al, 1992). These results indicate a dynamic process that occurs in the BR molecule solubilized in artificial media.…”

“…halobium, strain ET1001, was cultured according to the published procedure (Crespi, 1982) in a medium containing the hydrolysate of "N-labeled Chlorellu vulgaris (95 % uniform enriched species). The uniformly "N-labeled C2 was obtained as described (Orekhov et al, 1992). Samples for NMR were prepared by dissolving dried C2 in C2H30W C2HC1, (1 : 1, by vol.)…”

“…A series of uniformly and selectively ''N-labeled BR was obtained and an '%-IH-NMR study was performed in methanolkhloroform (1 : 1, by vol. ), 0.1 M 'HCO,NH, (Orekhov et al, 1992). Most of the observed cross-peaks in heteronuclear 15N-'H correlation spectra of BR assigned by selective 15N labeling of amino acids and fragmentation of the entire protein stemmed from transmembrane segments A, B and G only.…”

“…Thus, the internal dynamics of BR could be considered as a combination of the dynamics of individual a helices and dynamics associated with helixhelix interactions, under which helices may bend, tilt, slide past each other, extend or shorten. We investigated the structure and conformational dynamics of fragment C2 (residues 1-71) of BR, which could be regarded as a minimal structural unit of two interacting transmembrane a helices, taking into account that C2 has the same structural properties as in the whole molecule of BR (Arseniev et al, 1987;Abdulaeva et a]., 1991;Orekhov et al, 1992). Here we present 15N and 'H resonance assignments and spatial-structure calculations of C2 in methanolkhloroform (1 : 1, by vol.…”

“…A series of uniformly and selectively ''N-labeled BR was obtained and an '%-IH-NMR study was performed in methanolkhloroform (1 : 1, by vol. ), 0.1 M 'HCO,NH, (Orekhov et al, 1992). Most of the Abbreviarions.…”