Role of electrostatic repulsion in the acidic molten globule of cytochrome c

Abstract

The molten globule has been assumed to be a major intermediate state of protein folding. To extend our understanding of protein folding it is important to elucidate the thermodynamic mechanism of conformational stability of the molten globule. To clarify the role of electrostatic charge repulsion in the stability of the acidic molten globule state, we prepared a series of acetylated horse ferricytochrome c species with various degrees of charge repulsion. On the basis of circular dichroism measurement, we show that the stability of the acidic molten globule is determined by a balance of electrostatic repulsions between positive residues, which favor the extended conformation, and the opposing forces, which stabilize the molten globule. These results provide a clear example of charge repulsions producing unfolding of the compact protein structure, and suggest that the reversibly denatured conformation of ferricytochrome c under physiological conditions (i.e. neutral pH, ambient temperature and no denaturant) is the molten globule.