A Pro --> Ala substitution in melittin affects self-association, membrane binding and pore-formation kinetics due to changes in structural and electrostatic properties
Melittin, the main component of bee venom of Apis mellifera, contains a proline at position 14, which is highly conserved in related peptides of various bee venoms. To investigate the structural and functional role of Pro14 a melittin analogue was studied where proline is substituted by an alanine residue (P14A). The investigations were focussed on: (i) the secondary structure in aqueous solution and membranes; (ii) the self-association in solution; (iii) the binding to POPC membranes; and (iv) the P14A-induced leakage and pore formation in membrane vesicles. Circular dichroism and gel filtration experiments showed that P14A exists at concentrations < 12 microM in monomeric form with an alpha-helicity of 28 +/- 7%. A further increase in peptide concentration leads to the formation of large aggregates consisting of 9 +/- 1 monomers. While binding studies with POPC vesicles revealed for P14A a stronger binding affinity towards membranes than for melittin, the peptide-induced leakage of fluorescent markers from vesicles was less efficient for P14A than for melittin. Furthermore, an unexpected efflux behaviour at high values of bound P14A was observed which indicated that the pore formation kinetics for P14A is more complex than it was reported for melittin. The different features of P14A in aggregation, binding and efflux compared to melittin are mainly ascribable directly to structural changes caused by the proline --> alanine substitution. Furthermore, the results indicate an improved screening of the positively charged residues of P14A by counterions which contributes additionally to the observed differences in peptide activities. It is suggested that the presence of proline in melittin is not only of structural importance but also influences indirectly the electrostatic properties of the native peptide.
Rex
S
Department of Biophysical Chemistry, Biocenter of the University of Basel, Switzerland. srex@bu.edu