Optimization Of Charge-charge Interactions Research Articles

TKSA-MC: A web server for rational mutation through the optimization of protein charge interactions.

The TKSAMC is a web server which calculates protein charge-charge interactions via the Tanford-Kirkwood Surface Accessibility model with the Monte Carlo method for sampling different protein protonation states. The optimization of charge-charge interactions via directed mutations has successfully enhanced the thermal stability of different proteins and could be a key to protein engineering improvement. The server presents the electrostatic free energy contribution of each polar-charged residue to the protein native state stability. Specific residues are suggested to be mutated for improving thermal stability. The choice of a residue is based on its fraction of side chain exposed to solvent and its positive free energy contribution, which tends to destabilize the protein native state. Any residue energy contribution can be shown as a function of pH condition. The web server is freely available at UNESP (São Paulo State University - DF/IBILCE): http://tksamc.df.ibilce.unesp.br and also on GitHub https://github.com/contessoto/tksamc.

Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions

Improving enzyme thermostability is of importance for widening the spectrum of application of enzymes. In this study, a structure-based rational design approach was used to improve the thermostability of a highly active, wide-pH-range-adaptable, and stable endopolygalacturonase (PG8fn) from Achaetomium sp. strain Xz8 via the optimization of charge-charge interactions. By using the enzyme thermal stability system (ETSS), two residues--D244 and D299--were inferred to be crucial contributors to thermostability. Single (D244A and D299R) and double (D244A/D299R) mutants were then generated and compared with the wild type. All mutants showed improved thermal properties, in the order D244A < D299R < D244A/D299R. In comparison with PG8fn, D244A/D299R showed the most pronounced shifts in temperature of maximum enzymatic activity (Tmax), temperature at which 50% of the maximal activity of an enzyme is retained (T50), and melting temperature (Tm), of about 10, 17, and 10.2°C upward, respectively, with the half-life (t1/2) extended by 8.4 h at 50°C and 45 min at 55°C. Another distinguishing characteristic of the D244A/D299R mutant was its catalytic activity, which was comparable to that of the wild type (23,000 ± 130 U/mg versus 28,000 ± 293 U/mg); on the other hand, it showed more residual activity (8,400 ± 83 U/mg versus 1,400 ± 57 U/mg) after the feed pelleting process (80°C and 30 min). Molecular dynamics (MD) simulation studies indicated that mutations at sites D244 and D299 lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity. This study reveals the importance of charge-charge interactions in protein conformation and provides a viable strategy for enhancing protein stability.

How Stable is an Enzyme from a Thermophilic Organism? Denaturation Studies with the Esterase from Pyrococcus Furiosus - The Role of Charge-Charge Interactions

We have expressed the gene of the hyperthermophilic esterase (Pf2001Δ60) from Pyrococcus furiosus. This esterase showed to be active after boiling and has a half-life of 120 min at 75 °C. We decided to study the unfolding of this enzyme by fluorescence spectroscopy induced by urea, guanidinium hydrochloride (GndHCl) and high hydrostatic pressure (HHP). Ours results pointed out that urea is about three times more efficient than GndHCl in promoting structural perturbations in PF2001Δ60 ([D1/2]=7.1 and 2.3 M, respectively), suggesting that ion pairs are important stabilizing factors on its structure. There was almost no change in the tryptophan center of mass of PF2001Δ60 (342 nm) under HHP up to 3.1 kbar, even by staying 120 min under 2,500 bar. Interestingly, the combination of HHP with a subdenaturating concentration of urea (1 M) displaced the center of mass by ≈ 7 nm after 40 min at 2,500 bar. Since HHP enhances the electrostriction, this result reinforces the crucial contribution of salt bridges in esterase's stability. The binding of the bis(8-anilinonaphthalene-1-sulfonate) to PF2001Δ60 was increased by 2.5- and 3-fold after treatment with 2 M GndHCl or under HHP combined with urea, suggesting that these treatments convert the enzyme into a partially folded intermediate with exposed hydrophobic regions. Altogether, ours results may be an indication that the optimization of charge-charge interactions on the protein surface is a key factor for its stability. To our knowledge, this is the first time that HHP is used to access the ion pair contributions to the stability of a hyperthermophilic esterase.

Conformational Stability of Esterase Enzymes from Different Sources

In the last years we have performed a series of studies to characterize the conformational stability of three esterases from thermophilic and mesophilic sources: Aes esterase from Escherichia coli, EST2 from Alicyclobacillus acidocaldarius and AFEST from Archeoglobus fulgidus. These three esterases belong to the Hormone-sensitive lipase group of the superfamily of carboxylester hydrolases. The conformational stability of the three enzymes against temperature, urea and GuHCl has been determined by means of circular dichroism, fluorescence and differential scanning calorimetry measurements. Analysis of experimental data coupled with available structural information allowed us to suggest that the optimization of charge-charge interactions on the protein surface could one of the mechanisms to increase the thermal stability for the three esterases. This idea has been tested in the case of EST2, which shows a fully reversible thermal unfolding, by producing and studying variant forms of wild type enzyme in which a charged residue has been mutated. In the present article the obtained results are critically recollected in order to provide a clear and unified scenario.

Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface.

A simple theoretical model for increasing the protein stability by adequately redesigning the distribution of charged residues on the surface of the native protein was tested experimentally. Using the molecule of ubiquitin as a model system, we predicted possible amino acid substitutions on the surface of this protein which would lead to an increase in its stability. Experimental validation for this prediction was achieved by measuring the stabilities of single-site-substituted ubiquitin variants using urea-induced unfolding monitored by far-UV CD spectroscopy. We show that the generated variants of ubiquitin are indeed more stable than the wild-type protein, in qualitative agreement with the theoretical prediction. As a positive control, theoretical predictions for destabilizing amino acid substitutions on the surface of the ubiquitin molecule were considered as well. These predictions were also tested experimentally using correspondingly designed variants of ubiquitin. We found that these variants are less stable than the wild-type protein, again in agreement with the theoretical prediction. These observations provide guidelines for rational design of more stable proteins and suggest a possible mechanism of structural stability of proteins from thermophilic organisms.