A HIGH-RESOLUTION H-1-NMR APPROACH FOR STRUCTURE DETERMINATION OF MEMBRANE PEPTIDES AND PROTEINS IN NON-DEUTERATED DETERGENT - APPLICATION TO MASTOPARAN-X SOLUBILIZED IN N-OCTYLGLUCOSIDE

Application of H-1 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated det ergents. The unavailability of any of the common biochemical detergent s in deuterated form has therefore limited to some extent the scope of this approach. Here a H-1 NMR method is described which allows struct ure determination of membrane peptides and small membrane proteins by H-1 2D NMR in any type of non-deuterated detergent. The approach is ba sed on regioselective excitation of protein resonances with DANTE-Z or spin-pinging pulse trains. It is shown that regioselective excitation of the amide-aromatic region of solubilized membrane proteins and pep tides leads to an almost complete suppression of the two orders of mag nitude higher contribution of the protonated detergent to the H-1 NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating s uch regioselective excitation in the F2 dimension yield protein H-1 2D NMR spectra of quality comparable to those obtained in deuterated det ergents. Regioselective TOCSY and NOESY spectra display all through-bo nd and through-space correlations within amide-aromatic protons and be tween these protons and aliphatic and alpha-protons. Regioselective CO SY spectra provide scalar coupling constants between amide and alpha-p rotons. Application of the method to the membrane-active peptide masto paran X, solubilized in n-octylglucoside, yields complete sequence-spe cific assignments and extensive secondary structure-related spatial pr oximities and coupling constants. It is shown that mastoparan adopts a n alpha-helical conformation when bound to nonionic detergent micelles . The present method is expected to increase the applicability of H-1 solution NMR methods to membrane proteins and peptides.