Modulation of folding energy landscape by charge-charge interactions: linking experiments with computational modeling.

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.

Kinetic consequences of native state optimization of surface-exposed electrostatic interactions in the Fyn SH3 domain.

Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture.

Universal convergence of the specific volume changes of globular proteins upon unfolding.

Both pressure and temperature are important environmental variables, and to obtain a complete understanding of the mechanisms of protein folding, it is necessary to determine how protein stability is dependent on these fundamental thermodynamic parameters. Although the temperature dependence of protein stability has been widely explored, the dependence of protein stability on pressure is not as well studied. In this paper, we report the results of the direct thermodynamic determination of the change in specific volume (DeltaV/V) upon protein unfolding, which defines the pressure dependence of protein stability, for five model proteins (ubiquitin, eglin c, ribonuclease A, lysozyme, and cytochrome c). We have shown that the specific volumetric changes upon unfolding for four of the proteins (ubiquitin, eglin c, ribonuclease A, and lysozyme) appear to converge to a common value at high temperatures. Analysis of various contributions to the change in volume upon protein unfolding allowed us to put forth the hypothesis that the change in volume due to hydration is very close to zero at this temperature, such that DeltaV/V is defined largely by the total volume of cavities and voids within a protein, and that this is a universal property of all small globular proteins without prosthetic groups. To test this hypothesis, additional experiments were performed with variants of eglin c that had site-directed substitutions at two buried positions, to create an additional cavity in the protein core. The results of these experiments, coupled with the structural analysis of cytochrome c showing a lower packing density compared to those of the other four proteins, provided further support for the hypothesis. Finally, we have shown that the deviation of the high-temperature DeltaV value of a given protein from the convergence value can be used to determine the size of the excess cavities in globular proteins.

Protein stabilization by the rational design of surface charge-charge interactions.

The design of proteins with increased stability has many important applications in biotechnology. In recent years, strategies involving directed evolution, sequence-based design, or computational design have proven successful for generating stabilized proteins. A brief overview of the various methods that have been used to increase protein stability is presented, followed by a detailed example of how the rational design of surface charge-charge interactions has provided a robust method for protein stabilization.

Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge charge interactions.

Computational design of surface charge-charge interactions has been demonstrated to be an effective way to increase both the thermostability and the stability of proteins. To test the robustness of this approach for proteins with predominantly beta-sheet secondary structure, the chicken isoform of the Fyn SH3 domain was used as a model system. Computational analysis of the optimal distribution of surface charges showed that the increase in favorable energy per substitution begins to level off at five substitutions; hence, the designed Fyn sequence contained four charge reversals at existing charged positions and one introduction of a new charge. Three additional variants were also constructed to explore stepwise contributions of these substitutions to Fyn stability. The thermodynamic stabilities of the variants were experimentally characterized using differential scanning calorimetry and far-UV circular dichroism spectroscopy and are in very good agreement with theoretical predictions from the model. The designed sequence was found to have increased the melting temperature, DeltaT (m) = 12.3 +/- 0.2 degrees C, and stability, DeltaDeltaG(25 degrees C) = 7.1 +/- 2.2 kJ/mol, relative to the wild-type protein. The experimental data suggest that a significant increase in stability can be achieved through a very small number of amino acid substitutions. Consistent with a number of recent studies, the presented results clearly argue for a seminal role of surface charge-charge interactions in determining protein stability and suggest that the optimization of surface interactions can be an attractive strategy to complement algorithms optimizing interactions in the protein core to further enhance protein stability.

Use of pressure perturbation calorimetry to characterize the volumetric properties of proteins.

Pressure perturbation calorimetry (PPC) is a new technique that makes possible to study the volumetric changes that occur upon protein unfolding. Here, we summarize the thermodynamic foundation of the method and introduce a two-state model for the analysis of the unfolding data monitored by PPC. Several examples of data analysis illustrating potential pitfalls and solutions are discussed.

A computational approach for the rational design of stable proteins and enzymes: optimization of surface charge-charge interactions.

The design of stable proteins and enzymes is not only of particular biotechnological importance, but also addresses some important fundamental questions. While there are a number of different options available for designing or engineering stable proteins, the field of computational design provides fast and universal methods for stabilizing proteins of interest. One of the successful computational design strategies focuses on stabilizing proteins through the optimization of charge-charge interactions on the protein surface. By optimizing surface interactions, it is possible to alleviate some of the challenges that accompany efforts to redesign the protein core. The rational design of surface charge-charge interactions also allows one to optimize only the interactions that are distant from binding sites or active sites, making it possible to increase stability without adversely affecting activity. The optimization of surface charge-charge interactions is discussed in detail along with the experimental evidence to demonstrate that this is a robust and universal approach to designing proteins with enhanced stability.