
@article {22020,
	title = {Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.},
	journal = {Biochemistry},
	volume = {56},
	year = {2017},
	month = {2017 Aug 08},
	pages = {4053-4063},
	abstract = {<p>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.</p>
},
	keywords = {Amino 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},
	issn = {1520-4995},
	doi = {10.1021/acs.biochem.7b00367},
	author = {Malhotra, Pooja and Jethva, Prashant N and Udgaonkar, Jayant B}
}
@article {1027,
	title = {Rise of the Helix from a Collapsed Globule during the Folding of Monellin.},
	journal = {Biochemistry},
	volume = {54},
	year = {2015},
	month = {2015 Sep 1},
	pages = {5356-65},
	abstract = {<p>Early kinetic intermediates observed during the folding of many proteins are invariably compact and appear to possess some secondary structure. Consequently, it has been difficult to understand whether compaction drives secondary structure formation or secondary structure formation facilitates compaction during folding. In this study of the folding of single-chain monellin, it is shown that a kinetic molten globule (MG) is populated at 2 ms of folding. Far-UV circular dichroism (CD) measurements show that the kinetic MG is devoid of any helical structure even under the most stabilizing folding conditions. Multisite fluorescence resonance energy transfer (FRET) measurements show that the kinetic MG is compact with different segments having contracted to different extents. It is shown that the sequence segment that goes on to form the sole helix in the native protein is fully collapsed in the kinetic MG. This segment expands to accommodate the helix as the kinetic MG folds further to the native state, while other segments of the protein contract. Helix formation starting from the kinetic MG is shown to occur in multiple kinetic steps, whether measured by far-UV CD or by FRET.</p>
},
	keywords = {Amino Acid Substitution, Circular Dichroism, Cysteine, Fluorescence Resonance Energy Transfer, Hydrophobic and Hydrophilic Interactions, Kinetics, Models, Molecular, Mutagenesis, Site-Directed, Plant Proteins, Protein Conformation, Protein Folding, Protein Stability, Protein Structure, Secondary, Spectrometry, Fluorescence},
	issn = {1520-4995},
	doi = {10.1021/acs.biochem.5b00730},
	author = {Goluguri, Rama Reddy and Udgaonkar, Jayant B}
}
