Modulation of folding energy landscape by charge–charge interactions: Linking experiments with computational modeling

Abstract

The kinetics of folding-unfolding of a structurally diverse set of four proteins optimized for thermodynamic stability by rational redesign of surface charge-charge interactions is characterized experimentally. The folding rates are faster for designed variants compared with their wild-type proteins, whereas the unfolding rates are largely unaffected. A simple structure-based computational model, which incorporates the Debye-Hückel formalism for the electrostatics, was used and found to qualitatively recapitulate the experimental results. Analysis of the energy landscapes of the designed versus wild-type proteins indicates the differences in refolding rates may be correlated with the degree of frustration of their respective energy landscapes. Our simulations indicate that naturally occurring wild-type proteins have frustrated folding landscapes due to the surface electrostatics. Optimization of the surface electrostatics seems to remove some of that frustration, leading to enhanced formation of native-like contacts in the transition-state ensembles (TSE) and providing a less frustrated energy landscape between the unfolded and TS ensembles. Macroscopically, this results in faster folding rates. Furthermore, analyses of pairwise distances and radii of gyration suggest that the less frustrated energy landscapes for optimized variants are a result of more compact unfolded and TS ensembles. These findings from our modeling demonstrates that this simple model may be used to: (i) gain a detailed understanding of charge-charge interactions and their effects on modulating the energy landscape of protein folding and (ii) qualitatively predict the kinetic behavior of protein surface electrostatic interactions.protein folding | protein stability | charge-charge interaction | energy landscape | computational design T he energy landscape theory provides a conceptual framework to describe the ensemble nature of the protein folding process (1-3). However, a more detailed understanding of contributions from specific types of interactions remains an active area of research (4, 5). Particularly, the question of how interactions between charged residues modulate the funneled energy landscape is not well explored. These interactions are long-range and thus can alter the conformational ensemble at every step of the folding process. The interactions between charged residues are also nonspecific and either attractive or repulsive and therefore their potential effects on the folding energy landscape can be highly complex (6, 7). Traditionally, the modulation of electrostatic interactions in proteins was done by changing the pH or to a lesser degree changing the ionic strength of the solution (8, 9). Such approaches are complicated by the difficulties of predicting the titration properties of individual amino acid residues in the context of ensembles of protein conformations that are sampled during the folding reaction (10). A more attractive approach is to modulate electrostatic interactions via substitutions that perturb the thermody...

Alterations of Nonconserved Residues Affect Protein Stability and Folding Dynamics through Charge–Charge Interactions

“…Interestingly, we further observed that optimization of the surface charges of TNfn3 significantly reduces the energetic frustration in the MUT TNfn3 compared to the WT. This reduction of energetic frustration upon optimization of charge− charge interactions was previously demonstrated by us using C α -structure-based model 30 and is also in agreement with recent computer simulations of Naganathan et al 31 We also explored the interrelationship between charge− charge interactions and folding mechanism from the correlated fluctuations of the C α atoms at different states for all the three systems of TNfn3. The correlation map captures both the native and non-native interactions from local fluctuations of C α positions of each residue.…”

Electrostatic control of calcineurin's intrinsically-disordered regulatory domain binding to calmodulin

“…S11). Since lower native contact values will likely encounter greater frustration in folding landscapes compared to near native states, and reduced frustration results in increased rates of folding kinetics (72), we anticipate that induced-fit binding occurs more slowly than the binding of open-state conformations.…”

Protein stability: computation, sequence statistics, and new experimental methods

“…Using Tanford-Kirkwood electrostatics calculations, they showed reasonable agreement to prediction both for stabilizing and destabilizing mutations on the surface of ubiquitin [44]. Makhatadze further demonstrated this for other proteins like acylphosphatase and Cdc42 with little change in activity [45], and has suggested that the stabilization arises mostly from increased folding rates due to elimination of folding frustration in the native sequences [46]. …”