Thermal unfolding of tetrameric melittin: Comparison with the molten globule state of cytochrome c

Abstract

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric a-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion-and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric a-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)" K-l at 30 "C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)" K"), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.

Effect of ionic strength on folding and aggregation of the hemolytic peptide melittin in solution

“…We carefully chose neutral quenchers so as to avoid any specific interaction with the quencher and melittin since this peptide possesses +6 and +24 charges in its monomer and tetramer forms, respectively. 47 Acrylamide is a widely used neutral aqueous quencher of tryptophan fluorescence. Acrylamide quenching of tryptophan fluorescence is widely used to monitor tryptophan environments in proteins and peptides.…”

Ultrafast Polarized Fluorescence Measurements on Monomeric and Self-Associated Melittin

“…Temperaturedependent NMR studies have shown that upon heating the positions of the melittin monomers inside the tetramer differ from those in the crystal structure 7 and that the hydrophobic residues of melittin in solution are probably more exposed than in the crystal. 8 The symmetry of the tetramer structure suggests that melittin can also form dimers in solution and that selfassociation into a tetrameric state is a two-step reaction. However, stable melittin dimers have never been identified, 9 and the self-association process is usually described as one process in which four melittin monomers associate toward one tetrameric state.…”

“…[1][2][3][4]8,9,10 The self-association process of melittin is accompanied by a blue shift of the Trp fluorescence maximum due to shielding of the Trp residues in a hydrophobic pocket and can be observed by steady-state fluorescence measurements. Time-resolved magic-angle fluorescence and anisotropy measurements provide information about the Trp environment and the mobility of the protein.…”

Intricate Interactions within the ccd Plasmid Addiction System

“…The ccd system was the first one to be identified (6) and remains the best studied. The ccd operon encodes a toxin (CcdB: 101 amino acids, 11.7 kDa) and its antidote (CcdA: 72 amino acids, 8.3 kDa).…”