3-DIMENSIONAL STRUCTURE OF (1-71)BACTERIOOPSIN SOLUBILIZED IN METHANOL CHLOROFORM AND SDS MICELLES DETERMINED BY N-15-H-1 HETERONUCLEAR NMR-SPECTROSCOPY/

Spatial structures of a chymotryptic fragment C2 (residues 1-71) of ba cterioopsin from Halobacterium halobium, solubilized in a mixture of m ethanol/chloroform (1:1, by vol.) and 0.1M (HCO2NH4)-H-2, or in perdeu terated sodium (H-2)dodecyl sulfate (SDS) micelles in the presence of perdeuterated (2,2,2-H-2)trifluoroethanol, were determined by two-dime nsional and three-dimensional heteronuclear N-15-H-1 NMR techniques. T he influence of (2,2,2-H-2)trifluoroethanol on the conformational dyna mics of C2 in micelles and the effect of the salt (organic mixture) we re studied. Under the best conditions, H-1 and N-15 resonances of N-15 -uniformly enriched protein were assigned in both milieus by homonucle ar two-dimensional NOE (NOESY) and two-dimensional total-correlated (T OCSY) spectra and heteronuclear three-dimensional NOESY-multiple-quant um-correlation (HMQC) and TOCSY-HMQC spectra. 651 (organic mixture) an d 520 (micelles) interproton-distance constraints, derived from volume s of cross-peaks in two-dimensional NOESY and three-dimensional NOESY- HMQC spectra, along with deuterium exchange rates of amide groups meas ured in both milieus and 51 HN-C alpha H coupling constants obtained i n the case of the organic mixture, were used in the construction of C2 spatial structures. Obtained structures are similar in both milieus a nd have two right-handed alpha-helical regions stretching from Pro8 to Met32 and Phe42 to Tyr64 (organic mixture), and from Pro8 to Met32 an d Ala39 to Leu62 (micelles). In micelles, the second alpha helix is te rminated by C-cap Gly63, adopting a conformation characteristic of a l eft-handed helix. Residues Gly65 to Thr67 form the turn of a right-han ded helix. In the isotropic medium of the organic mixture, the C-termi nal region of residues 65-71 lacks an ordered structure. Torsion angle s kappa(1) were unequivocally determined for 18 alpha-helical residues in both milieus. In the isotropic organic mixture and anisotropic mic ellar system, C2 remains a compact structure with a characteristic siz e 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, E, Beckman, E. and Downing, K. H. (1990) J. Mal. Biol. 213, 899-929], we suggested a model for the conformation of C2 in this putative close-packed state. However, no NOE contact betwe en alpha helices was found in either milieu.