Abstract
Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments. Despite decades of work, the degree of structure and the compactness of denatured states populated in these experiments are still open questions. Here, building on previous works, we show that thermally and chemically denatured protein states are distinct from the viewpoint of far-ultraviolet circular dichroism experiments that report on the local conformational status of peptide bonds. The differences identified are independent of protein length, structural class, or experimental conditions, highlighting the presence of two sub-ensembles within the denatured states. The sub-ensembles, UT and UD for thermally induced and denaturant-induced unfolded states, respectively, can exclusively exchange populations as a function of temperature at high chemical denaturant concentrations. Empirical analysis suggests that chemically denatured states are ∼50% more expanded than the thermally denatured chains of the same protein. Our observations hint that the strength of protein backbone-backbone interactions and identity versus backbone-solvent/co-solvent interactions determine the conformational distributions. We discuss the implications for protein folding mechanisms, the heterogeneity in relaxation rates, and folding diffusion coefficients.
Highlights
Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments
While it is well established that a folded protein can be denatured by different perturbations, there has always been a question about the nature of ensembles that are populated.[1−4] This has been further renewed due to the explosion of research in intrinsically disordered proteins (IDPs), their functional significance, their dimensions, and the advantages and limitations of experimental techniques used to characterize them.[5−7]
Earlier studies have pointed to differences in the magnitude of the CD signal between thermally and chemically denatured states on specific proteins.[9−20] Here, we extend these works by collecting CD data from the literature, show that the differences observed previously are robust to structure−sequence changes, and explain their implications
Summary
Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments.
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