Free energy landscape of lysozyme: Multiple near-native conformational states and rollover in the urea dependence of folding energy

Abstract

Deviation from linearity of the equilibrium folding free energy (ΔG) 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 ΔG rolls over from linearity under mild to strongly native-like conditions. The urea dependence of ΔG 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 ΔG 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 ΔG 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 ΔG occurs due to these near-native alternative states in the native ensemble used for defining the unfolding equilibrium constant (KU), 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. © 2014 American Chemical Society.