@article {43845, title = {The Osmolyte TMAO Modulates Protein Folding Cooperativity by Altering Global Protein Stability.}, journal = {Biochemistry}, year = {2018}, month = {2018 Sep 24}, abstract = {

The folding of many globular proteins from the unfolded (U) to the native (N) state appears to be describable by a two-state N <-> U model, which has led to the general belief that protein folding occurs in a highly cooperative manner. One reason for the widespread belief in "two-state folding" is that protein folding reactions are invariably studied by ensemble averaging probes and not by probes that can distinguish as well as quantify the multiple conformations that may be present. Consequently, how cooperativity is affected by protein stability, protein sequence, and solvent conditions is poorly understood. In this study, hydrogen exchange coupled to mass spectrometry (HX-MS) of the PI3K SH3 domain was carried out in the presence of a stabilizing osmolyte, trimethylamine N-oxide (TMAO). By showing that HX occurs under the EX1 regime even in the presence of 2 M TMAO, we were able to examine the temporal evolution of the populations of the different conformations present together. A strong link between protein folding cooperativity and protein stability is revealed. Increasing stability is accompanied by an increase in the ruggedness of the free energy landscape as well as diminished cooperativity; the number of amide sites simultaneously opening up their structure decreases with an increase in TMAO concentration. A comparison of the effect of TMAO to that of urea on the intrinsic dynamics of the PI3K SH3 domain indicates that TMAO counteracts the effect of urea not only on protein stability but also on protein folding cooperativity.

}, issn = {1520-4995}, doi = {10.1021/acs.biochem.8b00698}, author = {Jethva, Prashant N and Udgaonkar, Jayant B} } @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 = {

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.

}, 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 {25254, title = {Modulation of the Extent of Cooperative Structural Change During Protein Folding by Chemical Denaturant.}, journal = {J Phys Chem B}, volume = {121}, year = {2017}, month = {2017 Sep 07}, pages = {8263-8275}, abstract = {

Protein folding and unfolding reactions invariably appear to be highly cooperative reactions, but the structural and sequence determinants of cooperativity are poorly understood. Importantly, it is not known whether cooperative structural change occurs throughout the protein, or whether some parts change cooperatively and other parts change noncooperatively. In the current study, hydrogen exchange mass spectrometry has been used to show that the mechanism of unfolding of the PI3K SH3 domain is similar in the absence and presence of 5 M urea. The data are well described by a four state N <-> IN <-> I2 <-> U model, in which structural changes occur noncooperatively during the N <-> IN and IN <-> I2 transitions, and occur cooperatively during the I2 <-> U transition. The nSrc-loop and RT-loop, as well as β strands 4 and 5 undergo noncooperative unfolding, while β strands 1, 2, and 3 unfold cooperatively in the absence of urea. However, in the presence of 5 M urea, the unfolding of β strand 4 switches to become cooperative, leading to an increase in the extent of cooperative structural change. The current study highlights the relationship between protein stability and cooperativity, by showing how the extent of cooperativity can be varied, using chemical denaturant to alter protein stability.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.7b04473}, author = {Jethva, Prashant N and Udgaonkar, Jayant B} }