Thermally versus Chemically Denatured Protein States

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.

Chain Compaction and Synergistic Destabilization of Globular Proteins by Mixture of Denaturants

“…Structural analysis of thermal‐ and chemical‐induced denatured states of proteins suggest that the denatured states attained by different perturbation methods differ in their conformation. Also, the chemically denatured states show larger expansion than thermally denatured conformations . In the present study as well, the chemical denaturation (ΔΔ G unf ) showed slightly higher energy changes than the thermally denatured states (ΔΔ G T ).…”

“…Also, the chemically denatured states show larger expansion than thermally denatured conformations. [15] In the present study as well, the chemical denaturation (ΔΔG unf ) showed slightly higher energy changes than the thermally denatured states (ΔΔG T ).…”

Stabilization Effect of Intrinsically Disordered Regions on Multidomain Proteins: The Case of the Methyl-CpG Protein 2, MeCP2

“…Moreover, NTD-MBD-ID showed a stabilization energy of 2.5 kcal/mol determined from fluorescence thermal denaturations, while it showed a stabilization energy of 2.8 determined by chemical denaturation. Of course, those quantities do not necessarily must be in agreement, because, although the initial state in both unfolding processes is the same (the native state), the final state may be different: the unfolded state after thermal denaturation may be structurally different from the unfolded state after chemical denaturation [49,50]. The same occurs with the unfolding process itself, which may proceed through different routes and involving different intermediate states depending on whether temperature or denaturant concentration drives the unfolding process.…”

Controlling Structure and Dimensions of a Disordered Protein via Mutations

“…We also observe a similar correlation between dimensions and secondary-structure content at different salt concentrations for the CytR WT combining SEC and analytical ultracentrifugation data ( Figure S4 ). These observations are strong evidence that the magnitude of the far-UV CD signal at 222 nm (in absolute units) for a given protein can be an intimate indicator of the molecular dimension even in the disordered regime, 24 particularly for helical protein domains, and if appropriately calibrated.…”