Mixed micelles of the 26-residue, lytic peptide melittin (MLT) and 1- myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) in aqueous solution at 25°C were investigated by 13C- and 31P-NMR spectroscopy. 13Cα chemical shifts of isotopically labeled synthetic MLT revealed that MLT in the micelle is predominantly α-helical and that the peptide secondary structure is stable from pH 4 to pH 11. Although the helical transformation of MLT as determined from NMR is evident at lipid:peptide molar ratios as low as 1:2, tryptophan fluorescence measurements demonstrate that well-defined micellar complexes do not predominate until lipid:peptide ratios exceed 30:1. 31P linewidth measurements indicate that the interaction between phosphate ions in solution and cationic groups on MLT is pH dependent, and that the phosphoryl group of MMPC senses a constant charge, most likely +2, on MLT from pH 4 to pH 10. 13C-NMR relaxation data, analyzed using the model-free formalism, show that the peptide backbone of MLT is partially, but not completely, immobilized in the mixed micelles. Specifically, order parameters (S2) of Cα-H vectors averaged ~0.7 and were somewhat larger for residues in the N-terminal half of the molecule. The amino terminal glycine had essentially the same range of motion as the backbone carbons. Likewise, order parameters for the trp side chain were similar to those found for the peptide Cα moieties, as was verified by trp fluorescence anisotropy decay data. In contrast, the motion of the lysine side chains was less restricted, the average S2 values for the Cε-H vectors being 0.19, 0.30, and 0.44 for lys- 7, 21, and 23, respectively, for MLT in the mixed micelles. Values of the effective correlation time of the local motion τ(o) were in the motional narrowing limit and usually longer for side-chain atoms than for those in the backbone. The dynamics were independent of pH from pH 4 to pH 9, but at pH 11 the correlation time for the rotational motion of the mixed micelles as a whole increased from 10 ns to 16 ns, and S2 for the lys side chains increased. Overall it appears that the MLT helix lies near the surface of the micelle at low to neutral pH, but at higher pH its orientation changes, accompanied by deeper penetration of the lys side chains into the micelle interior. It is apparent, however, that the MLT-lipid interaction is not dependent on deprotonation of any of the titratable cationic groups in the peptide in the pH 4-10 range, and that there is substantial backbone and side-chain mobility in micelle-bound MLT.
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