N-TERMINUS AND LYSINE SIDE-CHAIN PK(A) VALUES OF MELITTIN IN AQUEOUS-SOLUTIONS AND MICELLAR DISPERSIONS MEASURED BY N-15 NMR

Melittin (MLT) is a 26-amino acid cytolytic peptide from the Apis mell ifera honey bee. It is known to exist as an alpha-helical tetramer, as an alpha-helical monomer, or as a monomeric random coil depending on solvent conditions. The charge state of MLT is believed to be a major factor in determining its aggregation properties and its interaction w ith lipids. Several, contradictory, indirect measurements of the pK(a) values of the three lysine groups in MLT have been reported. In the p resent study, high-resolution N-15 NMR at 50.6 MHz was used to directl y measure the pK(a) values of the amino groups of the Gly-1, Lys-7, Ly s-21, and Lys-23 residues of MLT. Specifically, the pH dependence of M LT N-15 chemical shifts was measured separately for the isotopically e nriched backbone nitrogen of Gly-1 and the side chain nitrogen atoms o f Lys-7, Lys-21, and Lys-23 at a MLT concentration of 1.2 mM and a tem perature of 23 degrees C. Measurements were made for MLT in potassium phosphate buffer, in neat water, and in 1-myristoyl-2-hydroxy-sn-glyce ro-3-phosphocholine (MMPC) lipid micelles. The experiments showed for MLT tetramer in aqueous phosphate buffer that the amino nitrogen of Gl y-1 has a pK(a) of 8.15, and that the Lys-7, Lys-21, and Lys-23 side c hain nitrogen atoms have pK(a) values of 10.21, 10.03, and 10.24 respe ctively. The pK(a) values were somewhat lower for MLT in neat water wi th Gly-1 at 7.85 (in which case MLT is a monomer), and Lys-21 and Lys- 23 at 9.83 and 9.70, respectively (in which case MLT is a tetramer). S imilarly for MLT in 48 mM MMPC micelles and 50 mM potassium phosphate, the pK(a) values are 7.90, 10.09, 9.87, and 10.10 for Gly-1, Lys-7, L ys-21, and Lys-23, respectively. These pK(a) values indicate that the lysine residues are positively charged when MLT forms a tetramer at pH values between 8.5 and 10.