Experimental study of the protein folding landscape: Unfolding reactions in cytochrome c

Abstract

Hydrogen exchange results for cytochrome c have been interpreted in terms of transient hydrogen bond-breaking reactions that include large unfolding reactions and small fluctuational distortions. The differential sensitivity of these opening reactions to denaturant, temperature, and protein stability makes it possible to distinguish the different opening reactions and to characterize their structural and thermodynamic parameters. The partially unfolded forms (PUFs) observed are few and discrete, evidently because they are produced by the reversible unfolding of the protein's several intrinsically cooperative secondary structural elements. The PUFs are robust, evidently because the structural elements do not change over a wide range of conditions. The discrete nature of the PUFs and their small number is as expected for classical folding intermediates but not for theoretically derived folding models apparently because the simplified non-protein models usually analyzed in theoretical studies encompass only a single cooperative unit rather than multiple separable units.