Effects of pH and Salt Concentration on Stability of a Protein G Variant Using Coarse-Grained Models

Abstract

The importance of charge-charge interactions in the thermal stability of proteins is widely known. pH and ionic strength play a crucial role in these electrostatic interactions, as well as in the arrangement of ionizable residues in each protein-folding stage. In this study, two coarse-grained models were used to evaluate the effect of pH and salt concentration on the thermal stability of a protein G variant (1PGB-QDD), which was chosen due to the quantity of experimental data exploring these effects on its stability. One of these coarse-grained models, the TKSA, calculates the electrostatic free energy of the protein in the native state via the Tanford-Kirkwood approach for each residue. The other one, CpHMD-SBM, uses a Coulomb screening potential in addition to the structure-based model C. Both models simulate the system in constant pH. The comparison between the experimental stability analysis and the computational results obtained by these simple models showed a good agreement. Through the TKSA method, the role of each charged residue in the protein's thermal stability was inferred. Using CpHMD-SBM, it was possible to evaluate salt and pH effects throughout the folding process. Finally, the computational pK values were calculated by both methods and presented a good level of agreement with the experiments. This study provides, to our knowledge, new information and a comprehensive description of the electrostatic contribution to protein G stability.

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

“…The charge-charge interaction optimization by mutation is applied to protein engineering. 9,16,20,29,30,34 The indicated residue is a candidate for mutation for increasing the protein thermal stability in the chosen pH and temperature conditions. It is recommended that the TKSA-MC also be run for the mutated protein, and the pH range option be run to check the pH-dependence of the protein stability due to electrostatic contribution ΔG elec .…”

“…where R is the ideal gas constant; T is the temperature; q i is the charge of the ionizable residue i in its deprotonated state; x i is 0 or 1, according to the protonation state of the residue i; and pK a, ref, i is the reference pK a adopted of the compound model (3.6 for C-terminal, 4.0 for Aspartic acid, 4.5 for Glutamic acid, 6.3 for Histidine, 7.5 for N-terminal, 10.6 for Lysine, and 12.0 for Arginine). 9 The probability that the protein will be in its native state with a particular state of protonation, ρ N (χ), is given by,…”

“…[1][2][3] Among these interactions, electrostatic effects are widely known to be crucial for thermal stability. [4][5][6][7][8][9] Solution conditions, such as salt concentration and pH, tend to have a great influence on protein stability. [10][11][12][13] Makhatadze and colleagues have explored the optimization of charge-charge interactions via directed mutations and have successfully enhanced the thermal stability of different proteins.…”

TKSA-MC: A Web Server for rational mutation through the optimization of protein charge interactions

“…The charge-charge interaction optimization by mutation is applied to protein engineering. 9,16,[26][27][28][29] The indicated residue is a candidate for mutation for increasing the protein thermostability in the chosen pH and temperature conditions. It is recommended that the TKSA-MC also be run for the mutated protein, and the pH range option be run to check the pH-dependence of the protein stability due to electrostatic contribution ∆G elec .…”

“…[1][2][3] Among these interactions, electrostatic effects are widely known to be crucial for thermal stability. [4][5][6][7][8][9] Solution conditions, such as salt concentration and pH tend to have a great influence on protein stability. [10][11][12][13] Makhatadze and co-workers have explored the optimization of charge-charge interactions via directed mutations and have successfully enhanced the thermal stability of different proteins.…”