Membrane fusion activity of succinylated melittin is triggered by protonation of its carboxyl groups

Publisher Website
Google Scholar
Abstract
The membrane fusion activity of melittin and its succinylated derivative was studied as a function of pH by the transfer of spin-labeled phosphatidylcholine as well as by internal content mixing and electron microscopy. The protonation process of the carboxyl groups introduced into melittin was studied by 13C NMR spectroscopy using the derivative prepared with [1,4-13C]succinic anhydride. Mellitin causes fusion of sonicated phosphatidylcholine vesicles in a wide range of pH. In marked contrast, melittin with all four amino groups succinylated induces fusion only at acidic pH lower than 5.2, with the maximum at pH 5.1. The fusion reactions are very rapid, reaching a saturation level within 1 min. The fusion efficiency depends on the peptide-to-phospholipid ratio in the reaction mixture. Trypsinized succinylated melittin, which has lost the four positively charged C-terminal residues, causes aggregation of vesicles at acidic pH but cannot induce fusion. The 13C NMR peaks for the carboxyl and carbonyl groups of succinylated melittin shifted to higher field as the pH was lowered. The pKa value of the four carboxyl groups was obtained as 5.19 and 4.83 in the presence and absence of vesicles, respectively. The pKa value in the presence of vesicles agrees quite well with the half-maximal pH for fusion of 5.15, indicating that the fusion activity is triggered by protonation of the carboxyl groups in the hydrophobic segment of the peptide. The higher shift of pKa value in the presence of vesicles can be due to stabilization of the protonated form by entrance into lipid bilayer hydrocarbon layer. Only a single peak was observed for each carboxyl and carbonyl group at various pH values, indicating fast exchange between the protonated and deprotonated forms of the segment, faster than the NMR time scale of 3 ms. If the protonated segment entered the lipid bilayer, the entrance and return to the surface of bilayer membrane should also be fast.