Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR

The structure of mastoparan-X (MP-X), a G-protein activating peptide from wasp venom, in the state tightly bound to anionic phospholipid bilayers was determined by solid-state NMR spectroscopy. Carbon-13 and nitrogen-15 NMR signals of uniformly labeled MP-X were completely assigned by multidimensional intraresidue C-C, N-(CC beta)-C-alpha, and N-C-alpha-C', and interresidue C-alpha-(CC beta)-C-alpha, N-(CC beta)-C-alpha, and N-C'-C-alpha correlation experiments. The backbone torsion angles were predicted from the chemical shifts of C-13', C-13(alpha), C-13(beta), and N-15 signals with the aid of protein NMR database programs. In addition, two C-13-C-13 and three C-13-N-15 distances between backbone nuclei were precisely measured by rotational resonance and REDOR experiments, respectively. The backbone structure of MP-X was determined from the 26 dihedral angle restraints and five distances with an average root-mean-square deviation of 0.6 angstrom. Peptide MP-X in the bilayer-bound state formed an amphiphilic alpha-helix for residues Trp(3) - Leu(14) and adopted an extended conformation for Asn(2). This membrane-bound conformation is discussed in relation to the peptide's activities to form pores in membranes and to activate G-proteins. This study demonstrates the power of multidimensional solid-state NMR of uniformly isotope-labeled molecules and distance measurements for determining the structures of peptides bound to lipid membranes.