Thermal and Chemical Unfolding of Cytochrome C in the Presence of Hofmeister Ions

Abstract

The ferric cytochrome c (Cyt c) (un)folding mechanism in the presence of ions from the Hofmeister series is examined. Unfolding was initiated both thermally and with chemical denaturants. Hofmeister ions were added singly and in pairs to alter the stability of the native folded state, the unfolded state, and two partially folded intermediates. Protein stability was characterized by either the midpoint of the chemical denaturization curve or by the melting temperature in the thermal studies. UV/VIS absorption spectroscopy and a basis spectra fitting analysis were used to determine the populations of each protein conformation along the folding pathway. These species can be differentiated by their axial heme ligands. Four species exist in solution: the native HM state (His18/Met80), the partially folded HW (His18/water) and HH (His18/His33) intermediates, and the 5C (water) unfolded state. The results indicate that the thermal and chemical denaturization pathways are not the same and that both involve significant backbone rearrangement. The relative populations of the conformational states depend on how the protein is denatured. For the same concentration, guanidinium causes more unfolding than does urea. The thermal unfolding pathway appears to involve a more gradual unfolding of the protein and a different sequence of change in the heme iron axial ligands. Additionally, it was found that addition of multiple ions changed the protein's stability in an additive manner. These results are discussed in terms of the hydrophobic effect, partitioning of the ions to the protein surface, and an altered water structure around the protein.