CONFORMATION AND SELF-ASSOCIATION OF A HYBRID PEPTIDE OF CECROPIN-A AND MELITTIN WITH IMPROVED ANTIBIOTIC-ACTIVITY

A 15-residue hybrid peptide containing residues 1-7 from cecropin A an d residues 2-9 from melittin, (1-7)M(2-9), is a potent antibiotic with broader activity than cecropin A, bur without the cytotoxic character of melittin. The conformational behaviour of CA(1-7)M(2-9) including the formation of multimeric species in solution has been investigated by circular dichroism, ultracentrifugation, electrospray mass spectrom etry, NMR and energy calculations. Addition of hexafluoroisopropanol o r liposomes causes the appearance of a CD spectrum characteristic of a helical structure that changes with pH, buffer and peptide concentrat ion. The concentration dependence is atypical, as the ellipticity at 2 22 nm decreases with peptide concentration and is not correlated with a corresponding decrease in helix content as measured from the NMR spe ctra. The presence of aggregated structures is demonstrated by ultrace ntrifugation and ES-MS experiments, which also provide an indication o f the stoichiometry. Long-range NOEs suggest a model of aggregation wi th neighbouring molecules packed antiparallel. Aggregation causes very slow proton-deuterium exchange in some amide protons in the C-termina l region and provides a method for estimating a very large association constant (ca. 10(6) M(-1)) as well as the stoichiometry of the aggreg ates. The tendency to aggregate seems to be an inherited feature from melittin and may enhance the antibiotic activity either by facilitatin g the incorporation of the peptide into the membrane in large quantiti es or by promoting the disruption of the membrane.