Yasin, U. Mahammad Sashi, Pulikallu Bhuyan, Abani K. Free Energy Landscape of Lysozyme: Multiple Near-Native Conformational States and Rollover in the Urea Dependence of Folding Energy Deviation from linearity of the equilibrium folding free energy (Δ<i>G</i>) of proteins along the reaction coordinate is scarcely known. Optical spectroscopic observables and NMR-measured average molecular dimensional property of lysozyme with urea at pH 5 reveal that Δ<i>G</i> rolls over from linearity under mild to strongly native-like conditions. The urea dependence of Δ<i>G</i> is graphed in the 0–7 M range of the denaturant by employing a series of guanidine hydrochloride (GdnHCl)-induced equilibrium unfolding transitions, each in the presence of a fixed level of urea. The observed linear dependence of Δ<i>G</i> on urea under denaturing conditions begins to deviate as moderately native-like conditions are approached and eventually rolls over under strongly native-like conditions. This is atypical of the upward curvature in the Δ<i>G</i> vs denaturant plot predicted by the denaturant binding model. On increasing the denaturant concentration from 0 to 5 M, the hydrodynamic radius of lysozyme shrinks by ∼2 Å. We suggest subdenaturing levels of urea affect the population distribution among multiple near-native isoenergetic conformational states so as to promote them sequentially with increments of the denaturant. We use a multiple-state sequential model to show that the keel over of Δ<i>G</i> occurs due to these near-native alternative states in the native ensemble used for defining the unfolding equilibrium constant (<i>K</i><sub>U</sub>), which we assume to vary linearly with urea. The results and the model appear to indicate a rugged flat bottom in the free energy landscape wherein population distribution of native-like states is modulated by urea-affected interstate motions. equilibrium;urea;denaturant binding model;Free Energy Landscape;Δ G vs denaturant plot;Optical spectroscopic observables;Δ G rolls;Å.;population distribution;KU;Δ G 2014-06-19
    https://acs.figshare.com/articles/journal_contribution/Free_Energy_Landscape_of_Lysozyme_Multiple_Near_Native_Conformational_States_and_Rollover_in_the_Urea_Dependence_of_Folding_Energy/2281402
10.1021/jp501879s.s001