Protein folding in the absence of chemical denaturants - Reversible pressure denaturation of the noncovalent complex formed by the association of twoprotein fragments

Small monomeric proteins are the best models for studying protein folding, but they are often too stable for denaturation using pressure as the sole p erturbant. In the present work we subject [CI-2(1-40).(41-64)], a noncovale nt complex formed by the association of two complementary fragments of the chymotrypsin inhibitor-2, to high pressure to investigate the folding mecha nism of a model protein. Pressures up to 3.5 kilobar do not affect the inta ct protein, but it can be unfolded reversibly by pressure in the presence o f subdenaturing concentrations of guanidine chloride, with free energy and molar volume changes of 2.5 kcal mol(-1) and 42.5 ml mol(-1), respectively. In contrast, the complex can be reversibly denatured by high pressure with out the addition of chemical denaturants. However, the process is clearly i ndependent of the protein concentration, indicating lack of dissociation. W e determined a change in the free energy of 1.4 kcal mol(-1) and a molar vo lume change of 35 ml mol(-1) for the pressure denaturation of the complex. A persistent quenching of the tryptophan adds further evidence for the pres ence of residual structure in the high pressure-denatured state. This state also appears to be compact as the small volume change indicates, compared with pressure denaturation of naturally occurring dimers, Based on observat ions of a number of pressure-denatured states and on characteristics of lar ge CI-2 fragments with a solvent accessible core but maintaining tertiary i nteractions, the structure of the pressure-denatured state of the CI-2 comp lex could be explained by an ordered molten globule-like conformation.