Conformational studies of anionic melittin analogues: effect of peptide concentration, pH, ionic strength, and temperature--models for protein folding and halophilic proteins.

Melittin (MLT), a 26-residue cationic (net charge +5 at pH 7.2) peptide from bee venom, is well known to be a monomeric, approximately random coil; but when its charges are reduced by titration, by acetylation (net charge +2) or succinylation (net charge -2), or by screening by salt, it goes over to tetrameric alpha-helix. The conversion is promoted by raising the peptide concentration. The tetramer is held together by hydrophobic forces. We have changed the net charge to -6 by acylation with acetylcitric anhydride (a new acylating agent); this anionic derivative forms tetrameric helix at neutral pH, without salt, and at relatively low concentration, conditions under which the cationic MLT does not become helical. Thus, a high net charge is not sufficient to prevent association and helix formation. We have synthesized an anionic melittin analogue of MLT (E-MLT; net charge -4) in which all five lysine and arginine residues are replaced with glutamate, and acetyl and succinyl derivatives of E-MLT (net charges -5 and -6). All three of these are resistant to helix formation. They require much higher NaCl or NaClO4 concentration for helix formation than does MLT. Even CaCl2, MgCl2, and spermine.4HCl are less effective in helicizing E-MLT than MLT. MLT, at pH 7.2, shows increasing helix as the peptide concentration increases (8-120 microM), but E-MLT and its acyl derivatives do not. MLT and acylated MLTs in the helical tetramer show both cold- and heat-induced unfolding, with maximum stability near room temperature. At high temperature, a significant amount of residual structure remains. Heating (to 100 degrees C) monomeric MLT (i.e., MLT at low concentration) or E-MLT results in a monotonic increase in negative ellipticity. In 1.0 M NaCl, E-MLT (at sufficiently high concentration) also shows cold and hot unfolding. The results are discussed in respect to charge-charge and charge-dipole interactions, and hydrophobic effects. E-MLT is also discussed in relation to proteins of halophilic bacteria, which have higher proportions of anionic residues than do corresponding proteins of nonhalophiles.