Abstract

Small monomeric proteins are the best models for studying protein folding, but they are often too stable for denaturation using pressure as the sole perturbant. In the present work we subject [CI-2(1-40).(41-64)], a noncovalent complex formed by the association of two complementary fragments of the chymotrypsin inhibitor-2, to high pressure to investigate the folding mechanism of a model protein. Pressures up to 3.5 kilobar do not affect the intact protein, but it can be unfolded reversibly by pressure in the presence of 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 without the addition of chemical denaturants. However, the process is clearly independent of the protein concentration, indicating lack of dissociation. We determined a change in the free energy of 1.4 kcal mol-1 and a molar volume change of 35 ml mol-1 for the pressure denaturation of the complex. A persistent quenching of the tryptophan adds further evidence for the presence 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 observations of a number of pressure-denatured states and on characteristics of large CI-2 fragments with a solvent accessible core but maintaining tertiary interactions, the structure of the pressure-denatured state of the CI-2 complex could be explained by an ordered molten globule-like conformation. Study holds ProTherm entries: 6415 Extra Details: molar volume; free energy; pressure denaturation;,solvent accessible core; tertiary interaction; molten globule

Submission Details

ID: oK9bZvCK

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:32 p.m.

Version: 1

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