N-Terminus and Lysine Side Chain pK_a Values of Melittin in Aqueous Solutions and Micellar Dispersions Measured by ¹⁵N NMR

Melittin (MLT) is a 26-amino acid cytolytic peptide from the Apis mellifera honey bee. It is known to exist as an α-helical tetramer, as an α-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 with lipids. Several, contradictory, indirect measurements of the pK_a values of the three lysine groups in MLT have been reported. In the present study, high-resolution ¹⁵N NMR at 50.6 MHz was used to directly measure the pK_a values of the amino groups of the Gly-1, Lys-7, Lys-21, and Lys-23 residues of MLT. Specifically, the pH dependence of MLT ¹⁵N chemical shifts was measured separately for the isotopically enriched backbone nitrogen of Gly-1 and the side chain nitrogen atoms of Lys-7, Lys-21, and Lys-23 at a MLT concentration of 1.2 mM and a temperature of 23 °C. Measurements were made for MLT in potassium phosphate buffer, in neat water, and in l-myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) lipid micelles. The experiments showed for MLT tetramer in aqueous phosphate buffer that the amino nitrogen of Gly-1 has a pK_a of 8.15, and that the Lys-7, Lys-21, and Lys-23 side chain nitrogen atoms have pK_a values of 10.21, 10.03, and 10.24 respectively. The pK_a values were somewhat lower for MLT in neat water with 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). Similarly for MLT in 48 mM MMPC micelles and 50 mM potassium phosphate, the values are 7.90, 10.09, 9.87, and 10.10 for Gly-1, Lys-7, Lys-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.