TitleChemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.
Publication TypeJournal Article
Year of Publication2017
AuthorsMalhotra P, Jethva PN, Udgaonkar JB
Date Published2017 Aug 08
KeywordsAmino Acid Substitution, Deuterium Exchange Measurement, Energy Transfer, Guanidine, Indicators and Reagents, Kinetics, Menispermaceae, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Plant Proteins, Protein Conformation, Protein Denaturation, Protein Folding, Protein Stability, Thermodynamics

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

Alternate JournalBiochemistry
PubMed ID28714672