Sequence‐specific ¹H‐NMR assignment and conformation of proteolytic fragment 163–231 of bacterioopsin

Abstract

Proteolytic fragment 163 -231 of bacterioopsin was isolated from Halobacterium halobium purple membrane treated with NaBH, and papain under nondenaturing conditions. Two-dimensional 'H-NMR spectra of (163 -231)-bacterioopsin solubilized in chloroform/methanol (1 : I), 0.1 M LiC104 indicated the existence of one predominant conformation. Most of the resonances in the 'H-NMR spectra of (1 63 -231)-bacterioopsin were assigned by two-dimensional techniques. Two extended right-handed a-helical regions Ala168 -Ilel91 and Am202 -Arg227 were identified on the basis of NOE connectivities and deuterium exchange rates. The N-terminal part of the peptide is flexible and the region of Gly192-Leu201 adopts a specific conformation. The protons of OH groups of Thr178, Ser183 and Ser214 slowly exchange with solvent, and side-chain conformations of these residues, as evaluated by NOE connectivities of OH protons, are optimal for the formation of hydrogen bonds between OH and backbone carbonyl groups.Bacteriorhodopsin, a light-driven proton pump, is the main component of the purple membrane of Halobacterium halobium (reviewed in [l -31). It consists of 248 amino acid residues, most of which are embedded into the membrane. The light absorption by chromophore retinal bound to Lys216 [4] triggers the photocycle that leads to proton translocation across the membrane. Site-directed mutagenesis revealed some aromatic and charged amino acid residues thus affecting the proton translocation efficiency and/or the chromophore absorption maximum [5 -71. The detailed three-dimensional structure of bacteriorhodopsin will undoubtedly promote a better understanding of the mechanism of vectorial proton translocation.Bacteriorhodopsin readily forms stable and highly ordered two-dimensional crystals. Electron microscopy of such crystals revealed seven electron-dense membrane-spanning segments, probably being the seven a-helices A-G [8]. Proteolytic digestion, immunochemical methods and theoretical considerations [l -31 resulted in a rough location of transmembrane segments in the bacteriorhodopsin amino acid sequence.Two-dimensional (2D) 'H-NMR is an elaborate method for conformational analysis of polypeptides in solution [9]. Unfortunately, the proton resonances of bacteriorhodopsin in bilayer membranes or small liposomes are too broad to be resolved. Thus an artificial medium conserving the protein structure and providing high-resolution NMR spectra is the main hindrance when using this method to study membrane proteins. According to CD and "F-NMR spectroscopy, a chloroform/methanol mixture is a suitable medium [lo]. Bacteriorhodopsin and its proteolytic fragments, being This paper deals with the conformation of (163 -231)-bacterioopsin obtained by NaBH, and papain treatment of the purple membrane and then solubilized in the chloroform/ methanol mixture. Most of the resonance in the 'H-NMR spectra of (163 -231)-bacterioopsin were assigned. NOE connectivities and deuterium exchange rates underlay identification of two right-handed a-helical regions...

Secondary solute transport in bacteria

“…In principle, it should be possible to determine the structure of parts of the transport proteins (a few transmembrane segments) by two-dimensional or multidimensional NMR [137], provided these segments can be expressed and purified to homogeneity in sufficient quantities and retain their native configuration. Structure information on overlapping parts of these proteins, from which possibly significant information on the three-dimensonal structure of the entire protein can be obtained, could lead to an enormous advancement of our understanding of secondary solute transport mechanisms.…”

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

“…BR fragments solubilized in methanol/chloroform solution (Arseniev et al, 1988;Barsukov et al, 1990;Maslennikov et al, 1991;Maslennikov et al, 1992;Sobol et al, 1992). Thus, the conclusion is the following: conformational mobility detected in HMQC spectra of BR is due to specific interactions between helices in the four-(C, D, E and F)-helical bundle.…”

“…BR fragments (30 -70 residues) were obtained by peptide synthesis or selective cleavage of the protein and solubilized in this solvent. They were then examined by two-dimensional 'H-'H-NMR spectroscopy in order to localize a-helical stretches within the amino acid sequence (Arseniev et al, 1988;Barsukov et al, 1990;Maslennikov et al, 1991;Maslennikov et al, 1992;Sobol et al, 1992).…”

“…Secondary-structure model of BR. a-helical segments A -G were located according to electron cryomicroscopy (Henderson et al, 1990), NMR (Arseniev et al, 1988;Barsukov et al, 1990;Pervushin et al, 1991 ;Maslennikov et al, 1991 ;Sobol et al, 1992;Maslennikov et al, 1992) and site-directed mutagenesis (Gilles-Gonzalez et al, 1991;Altenbach et al, 1990) data. Grey and white squares indicate amino acid residues where cross-peaks are present and absent, respectively, in HMQC spectra of des-(232 -248)-BR.…”

“…The B1 (residues 1-155) and BP2 (residues 163-231) fragments were produced by NaBH4 treatment of the corresponding I5N-labeled BR, which was followed by papain digestion (Barsukov et al, 1990). Digested BR was washed by centrifugation in water and lyophilized.…”