Alteration of the Cytochrome C Folding Pathway in Solution and Within Sol-Gel Glasses by Addition of Hofmeister Salts

Abstract

The ferric cytochrome c (un)folding mechanism in the presence of steric constraints and salts is examined. Unfolding was initiated both thermally and with chemical denaturants. Sol-gel encapsulation was used to constrain the protein to a volume close to the “molten globule state.” Hofmeister salts were added singly and in pairs to alter protein stability. 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. An additional unfolded state found only within the sol-gel contains an unligated four-coordinate heme sequestered from aqueous solution. Solution results indicate that the thermal and chemical denaturization pathways are not the same, but that both involve significant backbone rearrangement. Further, while every species observed in solution is also observed in the sol-gel, their relative populations are different, indicating that backbone motions are hindered by encapsulation within the sol-gel matrix. The ranking of salts in the Hofmeister series (from stabilizing to destabilizing protein structure) was found to persist in the sol-gel samples, and in some cases a larger effect on protein stability was found in the sol-gels. Additionally, it was found that addition of multiple salts changed the protein's stability in an additive manner. Both the (un)folding kinetics and the accessible conformations were found to depend on the identity of ions present. These results are discussed in terms of the hydrophobic effect and an altered water structure within the confines of the gel pores.