Spatial structures of a chymotryptic fragment C2 (residues 1-71) of bacterioopsin from HaEobacterium halobium, solubilized in a mixture of methanolkhloroform (1: 1, by vol.) and 0.1 M 'HCO,N&, or in perdeuterated sodium (2H)dodecyl sulfate (SDS) micelles in the presence of perdeuterated (2,2,2-2H)trifluoroethanol, were determined by two-dimensional and three-dimensional heteronuclear "N-lH NMR techniques. The influence of (2,2,2-'H)trifluoroethanol on the conformational dynamics of C2 in micelles and the effect of the salt (organic mixture) were studied. Under the best conditions, 'H and I5N resonances of 15N-uniformly enriched protein were assigned in both milieus by homonuclear two-dimensional NOE (NOESY) and two-dimensional total-correlated (TOCSY) spectra and heteronuclear three-dimensional NOESY-multiple-quantum-correlation (HMQC) and TOCSY-HMQC spectra. 651 (organic mixture) and 520 (micelles) interproton-distance constraints, derived from volumes of cross-peaks in two-dimensional NOESY and three-dimensional NOESY-HMQC spectra, along with deuterium exchange rates of amide groups measured in both milieus and 51 HN-CaH coupling constants obtained in the case of the organic mixture, were used in the construction of C2 spatial structures. Obtained structures are similar in both milieus and have two right-handed a-helical regions stretching from Pro8 to Met32 and Phe42 to Tyr64 (organic mixture), and from Pro8 to Met32 and Ala39 to Leu62 (micelles). In micelles, the second a helix is terminated by C-cap Gly63, adopting a conformation characteristic of a left-handed helix. Residues Gly65 to Thr67 form the turn of a right-handed helix. In the isotropic medium of the organic mixture, the C-terminal region of residues 65-71 lacks an ordered structure. Torsion angles x' were unequivocally determined for 18 a-helical residues in both milieus. In the isotropic organic mixture and anisotropic micellar system, C2 remains a compact structure with a characteristic size of 3.0-3.5 nm. C2 seems to be present in at least two conformational states, packed and unpacked. Using NMR data, along with the electron cryomicroscopy model of bacteriorhodopsin [Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckman, E. & Downing, K. H. (1990) J. Mol. Biol. 213, 899-9291, we suggested a model for the conformation of C2 in this putative close-packed state. However, no NOE contact between a helices was found in either milieu.Bacteriorhodopsin (BR) is a transmembrane protein acting as a light-dependent proton pump in the purple membrane of Halobacterium halobium (for a review see Ovchinnikov, 1982). Electron cryomicroscopy (ECM) data suggested a seven-a-helix structural motif for BR (Henderson et al., 1990). Arseniev et al. (1987) and Abdulaeva et al. (1991) used methanolkhloroform (1 : 1, by vol.), 0.1 M LiClO, for the I9F-NMR study of BR. The individual signals in the IT-NMR spectra of "F-Trp-labeled and IgF-Phe-labeled BR species were assigned and several long-range contacts between Correspondence to A. S. Arseniev, Shemyakin ...

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.

The conformation of chymotryptic fragment C2 of bacteriohodopsin (residues 1-71) was studied by 2D 1H NMR. The fragment was solubilized in a mixture of chloroform/methanol (1:1), 0.1 M LiClO4. Most of the resonances in 1H NMR spectra of fragment C2 were assigned using phase-sensitive DQF-COSY, TOCSY, and NOESY techniques. To simplify the assignment procedure for overlapping regions of NMR spectra, an analog of fragment C2 with leucines deuterated in beta-positions was used. Deuterium exchange rates for amide protons were measured in a series of TOCSY spectra. Two right-handed alpha-helical regions Pro8-Lys30 and Lys41-Leu62 were identified on the basis of NOE connectivities and deuterium exchange rates. The N-terminal part of the fragment (Ala2-Gly6) adopts the helical conformation stabilized by 3 hydrogen bonds.

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