A high-resolution 1H NMR approach for structure determination of membrane peptides and proteins in non-deuterated detergent: application to mastoparan X solubilized in n-octylglucoside.
Application of 1H 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated detergents. The unavailability of any of the common biochemical detergents in deuterated form has therefore limited to some extent the scope of this approach. Here a 1H NMR method is described which allows structure determination of membrane peptides and small membrane proteins by 1H 2D NMR in any type of non-deuterated detergent. The approach is based 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 peptides leads to an almost complete suppression of the two orders of magnitude higher contribution of the protonated detergent to the 1H NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating such regioselective excitation in the F2 dimension yield protein 1H 2D NMR spectra of quality comparable to those obtained in deuterated detergents. Regioselective TOCSY and NOESY spectra display all through-bond and through-space correlations within amide-aromatic protons and between these protons and aliphatic and alpha-protons. Regioselective COSY spectra provide scalar coupling constants between amide and alpha-protons. Application of the method to the membrane-active peptide mastoparan X, solubilized in n-octylglucoside, yields complete sequence-specific assignments and extensive secondary structure-related spatial proximities and coupling constants.(ABSTRACT TRUNCATED AT 250 WORDS)
Seigneuret
M
M
Laboratoire de Biophysique Cellulaire, URA 526, Université Paris, France.
Lévy
D
D
D000595:Amino Acid Sequence; D003902:Detergents; D005960:Glucosides; D006859:Hydrogen / Q000737:chemistry; D036341:Intercellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D011522:Protons; D012995:Solubility
D003902:Detergents; D005960:Glucosides; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; D011522:Protons; C018619:octyl-beta-D-glucoside; C071123:mastoparan X; D006859:Hydrogen