Quantitative Analysis of Protein Unfolded State Energetics: Experimental and Computational Studies Demonstrate That Non-Native Side-Chain Interactions Stabilize Local Native Backbone Structure

Abstract

Proteins fold on relatively smooth free energy landscapes which are biased toward the native state, but even simple topologies which fold rapidly can experience roughness on their free energy landscape. The details of these interactions are difficult to elucidate experimentally. Closely related to the problem of deciphering the details of the free energy landscape is the problem of defining the interactions in the denatured state ensemble (DSE) which is populated under native conditions, that is, under conditions where the native state is stable. The DSE of many proteins deviates from random coil models, but quantifying and defining the energetics of the transiently populated interactions in this ensemble is extremely challenging. Characterization of the DSE of proteins which fold to compact structures is also relevant to studies of intrinsically disordered proteins (IDPs) since interactions in the dynamic ensemble populated by IDPs can modulate their behavior. Here we show how experimental thermodynamic and pK a measurements can be combined with computational thermodynamic integration to quantify interactions in the DSE. We show that non-native side chain interactions can stabilize native backbone structure in the DSE and demonstrate that that even rapidly folding proteins can form energetically significant non-native interactions in their DSE. As an example, we characterize a non-native salt bridge that stabilizes local native backbone structure in the DSE of a widely studied fast-folding protein, the villin headpiece helical domain. The combined computational experimental approach is applicable to other protein unfolded states and provides insight that is impossible to obtain with either method alone.

Conformational Stability and Denaturation Processes of Proteins Investigated by Electrophoresis under Extreme Conditions

“…In addition to an exploration of the effect of point mutations on already folded—experimental and predicted—3D structures, MD simulations are used to address the folding process, in spite of Levinthal’s paradox [ 114 , 120 , 121 , 122 ]; unfolding [ 96 , 97 , 123 ]; and dynamics of intrinsically disordered proteins (IDPs) [ 82 , 124 ]. Such tasks particularly highlight the problem of computational cost and conformational sampling in MD simulations [ 125 ].…”

Conformational Characteristics and Phase Behavior of Intrinsically Disordered Proteins─Where Physical Chemistry Meets Biology

“…Zou et al. quantified interactions involved in stabilizing the local backbone protein structure in denatured state ensembles by integrating experimental and computational approaches . A study by Ferrie and Petersson utilized the Monte Carlo approach to develop a PyRosetta-based algorithm to capture the conformational disorder in both disordered and partially ordered systems …”

“…25 Zou et al quantified interactions involved in stabilizing the local backbone protein structure in denatured state ensembles by integrating experimental and computational approaches. 26 A study by Ferrie and Petersson utilized the Monte Carlo approach to develop a PyRosetta-based algorithm to capture the conformational disorder in both disordered and partially ordered systems. 27 Amin et al quantified the role of conformational disorder, sequence-specific protein−protein interactions in IDPs and established the sequence-dependent phase transition behavior of proteins by developing an analytical approach taking into account a novel two-chain charge pattern parameter for fuzzy binary complexes.…”