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>_U), 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

Expansion and Internal Friction in Unfolded Protein Chain

Similarities in global properties of homopolymers and unfolded proteins provide approaches to mechanistic description of protein folding. Here, hydrodynamic properties and relaxation rates of the unfolded state of carbonmonoxide-liganded cytochrome <i>c</i> (cyt-CO) have been measured using nuclear magnetic resonance and laser photolysis methods. Hydrodynamic radius of the unfolded chain gradually increases as the solvent turns increasingly better, consistent with theory. Curiously, however, the rate of intrachain contact formation also increases with an increasing denaturant concentration, which, by Szabo, Schulten, and Schulten theory for the rate of intramolecular contact formation in a Gaussian polymer, indicates growing intramolecular diffusion. It is argued that diminishing nonbonded atom interactions with increasing denaturant reduces internal friction and, thus, increases the rate of polypeptide relaxation. Qualitative scaling of the extent of unfolding with nonbonded repulsions allows for description of internal friction by a phenomenological model. The degree of nonbonded atom interactions largely determines the extent of internal friction

Unfolding Action of Alcohols on a Highly Negatively Charged State of Cytochrome <i>c</i>

It is well-known that hydrophobic effect play a major role in alcohol–protein interactions leading to structure unfolding. Studies with extremely alkaline cytochrome <i>c</i> (U_B state, pH 13) in the presence of the first four alkyl alcohols suggests that the hydrophobic effect persistently overrides even though the protein carries a net charge of −17 under these conditions. Equilibrium unfolding of the U_B state is accompanied by an unusual expansion of the chain involving an intermediate, I_alc, from which water is preferentially excluded, the extent of water exclusion being greater with the hydrocarbon content of the alcohol. The mobility and environmental averaging of side chains in the I_alc state are generally constrained relative to those in the U_B state. A few nuclear magnetic resonance-detected tertiary interactions are also found in the I_alc state. The fact that the I_alc state populates at low concentrations of methanol and ethanol and the fact that the extent of chain expansion in this state approaches that of the U_B state indicate a definite influence of electrostatic repulsion severed by the low dielectric of the water/alcohol mixture. Interestingly, the U_B ⇌ I_alc segment of the U_B ⇌ I_alc ⇌ U equilibrium, where U is the unfolded state, accounts for roughly 85% of the total number of water molecules preferentially excluded in unfolding. Stopped-flow refolding results report on a submillisecond hydrophobic collapse during which almost the entire buried surface area associated with the U_B state is recovered, suggesting the overwhelming influence of hydrophobic interaction over electrostatic repulsions