Volume changes of the molten globule transitions of horse heart ferricytochrome c: A thermodynamic cycle

Volume changes among the unfolded (U), native (N), and molten globule (MG) conformations of horse heart ferricytochrome c have been measured. U to N (pH 2 to pH 7) was determined in the absence of added salt to be —136 ± 5 mL/mol protein. U to MG (pH 2, no added salt to pH 2, 0.5 M KCl) yielded +100 ± 6 mL/mol. MG to N was broken into two steps, N to NClX at pH 7 by addition of buffered KCl to buffered protein lacking added salt (NClX = N interacting with an unknown number, X, of chloride ions), and MG to NClX by jumping MG at pH 2 in 0.5 M KCl to pH 7 at the same salt concentration. The ΔV of N to NClX was —30.9 ± 1.4 mL/mol protein, whereas MG to NClX entailed a ΔV of —235 ± 6 mL/mol. Within experimental error, the results add up to zero for a complete thermodynamic cycle. We believe this to be the first volumetric cycle to have been measured for the conformational transitions of a protein. The results are discussed in terms of hydration contributions from deprotonation of the protein, other hydration effects, and the formation and/or enlargement of packing defects in the protein's tertiary structure during the steps of folding.