THERMAL UNFOLDING OF TETRAMERIC MELITTIN - COMPARISON WITH THE MOLTENGLOBULE STATE OF CYTOCHROME-C

Whereas melittin at micromolar concentrations is unfolded under condit ions of low salt at neutral pH, it transforms to a tetrameric ct-helic al structure under several conditions, such as high peptide concentrat ion, high anion concentration, or alkaline pH. The anion- and pH-depen dent stabilization of the tetrameric structure is similar to that of t he molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globu le state. To test this possibility, we studied the thermal unfolding o f tetrameric melittin using far-UV CD and differential scanning calori metry. The latter technique revealed a broad but distinct heat absorpt ion peak. The heat absorption curves were consistent with the unfoldin g transition observed by CD and were explainable by a 2-state transiti on mechanism between the tetrameric alpha-helical state and the monome ric 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)(-1) K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 k J (mol of tetramer)(-1) K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that t he structure is more ordered than that of a typical molten globule sta te. We consider that the conformation is intermediate between the molt en globule state and the native state of globular proteins.