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

Abstract

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.

The Effects of Interhelical Electrostatic Repulsions between Glutamic Acid Residues in Controlling the Dimerization and Stability of Two-stranded α-Helical Coiled-coils

“…The dramatic effects of pH and salt on protein folding observed in this study and previously in model coiled-coils (Zhou et al, 1994b) and other model helical proteins (Ramalingam et al, 1992) have suggested major implications for the potential of de novo design of environmentally sensitive proteins. One of the best examples of salt effects on protein folding in native proteins is the case of extreme halophilic bacteria, which are adapted to living in high salt concentration environments and whose cytoplasm is close to saturated in KCl.…”

“…This stabilization may be due to the combined effects of the salt stabilizing the hydrophobic core as well as interactions of hydrated salt ions with the surface of the folded protein (Zaccai and Eisenberg, 1990). It has been suggested that clustering of negatively charged residues on the surface of halophilic proteins may cause structural instability, possibly due to charge repulsion, that is removed by the effects of salts (Ramalingam, et al, 1992). pH sensitivity of coiled-coil formation has recently been illustrated for the influenza virus hemagglutinin protein, which is required for fusion of the viral and cellular membranes.…”

NMR studies on the monomer–tetramer transition of melittin in an aqueous solution at high and low temperatures

“…Monomeric melittin having the trans and cis proline, if it is necessary to make a clear distinction, will be referred to as the trans-and cis-monomer, respectively. By variation in pH or salt concentration or temperature of an aqueous solution, a conformation transition between the tetramer and the monomer is induced (Bello et al 1982;Faucon et al 1979;Goto and Hagihara 1992;Hagihara et al 1992Hagihara et al , 1994Iwadate et al 1998;Miura 2011;Quay and Condie 1983;Ramalingam et al 1991Ramalingam et al , 1992Wilcox and Eisenberg 1992). Interestingly, melittin undergoes the transition not only by heating but also by cooling from room temperature like a globular protein such as myoglobin or staphylococcal nuclease (Privalov 1990).…”

Purification, Characterization, and Structural Analysis of a Plant Low-Temperature-Induced Protein

“…Thus, BN28 may exist in a helical conformation in vivo given the proper cellular environment. High ionic concentrations can stabilize helices in peptides that are unstructured in low-ionicstrength environments (Ramalingam et al, 1992). It is believed that this stabilization is accomplished through either charge screening or enhancement of weak hydrophobic interactions.…”