The Effects of Hofmeister Salts on the Cytochrome c Folding Pathway in Solution and within Sol-Gel Glasses

The ferric cytochrome c (Cyt c) (un)folding mechanism in the presence of steric constraints and altered solvating water structure is examined. Sol-gel encapsulation was used to constrain the protein to a volume slightly larger than the native state. Hofmeister salts were added to alter water order. UV/VIS absorption spectroscopy and a basis spectra fitting analysis were used to determine the populations of species present 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. An additional unfolded state found only within the sol-gel contains an unligated four-coordinate heme sequestered from aqueous solution. In solution, the native HM state unfolds primarily into the HH state, while unfolding within the gel produces comparable amounts of the HH, HW, and four-coordinate states. This indicates that the steric constraints within the gel pores hinder some backbone motions. Four anions (HPO4−2, H2PO4−1, SO4−2 and Cl−1) and three cations (Gdn+1, K+1, and NH4+1) that lie within the Hofmeister series were utilized. Gdn+1 lies at one end of the series and promotes unfolding of the protein. Presence of an additional ion can counter the denaturing effects of the Gdn+1 to an extent dependant on its proximity to Gdn+1 in the series. Both the (un)folding kinetics and the accessible conformations were found to depend on the identity of ions present. A model discussing changes in protein stability as a function of water order and the hydrophobic effect is used to interpret these results. Water order depends on the degree of confinement in the gel pores and the properties of the ions present.