Electrostatic Contributions to T4 Lysozyme Stability: Solvent-Exposed Charges versus Semi-Buried Salt Bridges

Abstract

We carried our Poisson-Boltzmann (PB) calculations for the effects of charge reversal at five exposed sites (K16E, R119E, K135E, K147E, and R154E) and charge neutralization and proton titration of the H31-D70 semi-buried salt bridge on the stability of T4 lysozyme. Instead of the widely used solvent-exclusion (SE) surface, we used the van der Waals (vdW) surface as the boundary between the protein and solvent dielectrics (a protocol established in our earlier study on charge mutations in barnase). By including residual charge-charge interactions in the unfolded state, the five charge reversal mutations were found to have DeltaDeltaG(unfold) from -1.6 to 1.3 kcal/mol. This indicates that the variable effects of charge reversal observed by Matthews and co-workers are not unexpected. The H31N, D70N, and H31N/D70N mutations were found to destabilize the protein by 2.9, 1.3, and 1.6 kcal/mol, and the pK(a) values of H31 and D70 were shifted to 9.4 and 0.6, respectively. These results are in good accord with experimental data of Dahlquist and co-workers. In contrast, if the SE surface were used, the H31N/D70N mutant would be more stable than the wild-type protein by 1.3 kcal/mol. From these and additional results for 27 charge mutations on five other proteins, we conclude that 1) the popular view that electrostatic interactions are generally destabilizing may have been based on overestimated desolvation cost as a result of using the SE surface as the dielectric boundary; and 2) while solvent-exposed charges may not reliably contribute to protein stability, semi-buried salt bridges can provide significant stabilization.

Computational Methods for Biomolecular Electrostatics

“…In general, proteins have multiple ionizable sites and the protonation energetics of these different sites are coupled, as discussed below. Single site pK a predictions have successfully reproduced measured pK a s for different residues in several different proteins (Dong and Zhou, 2002;Dong et al, 2003) and therefore have some predictive power. However, a more complete treatment of ionizable residues in proteins considers the coupling between all the ionizable sites.…”

Site-specific contributions to the pH dependence of protein stability

“…The pH-independent stability differences of ⌬G FU , such as that between the Arg-20 3 Ala mutant and wild-type protein, reflect changes of enthalpic or entropic contributions to the thermodynamic stability of either the folded and͞or the unfolded state on modification of the side-chain. The contribution of solvent-exposed charges (Arg-20, Lys-21) to the thermodynamic stability of a protein can exhibit considerable variability (35). The experimental pH stability profile for His-7 3 Ala, however, is notably different from that for the wild-type protein.…”

Do electrostatic interactions destabilize protein–nucleic acid binding?

“…[14][15][16][17] Briefly, the calculation began with a coarse grid with a 1.5 Å spacing, and then a finer grid with a 0.5 Å spacing, both centered at the geometric center of the solute molecule. A final grid with a 0.25 Å spacing was centered at the site of mutation.…”

“…This adds to a large body of data for the effects of charge mutations on protein folding and binding stability that points to the same conclusion. [14][15][16][17]33,34 A caveat on mutational data is that electrostatic interactions are assumed to make dominant contributions to the effects of mutation. Experiments measure the total effects of mutations, which may have both electrostatic and nonelectrostatic contributions, but PB calculations only give the electrostatic contributions.…”

“…We have explored an alternative choice, i.e., the van der Waals (vdW) surface, and found that the electrostatic interaction energy is quite sensitive to the choice of the dielectric boundary. [14][15][16][17] In the case of protein-protein binding, the sign of the electrostatic interaction energies can be changed from positive to negative when the choice of dielectric boundary is changed from the molecular surface to the vdW surface. 16,17 In this article, we study the effect of the dielectric boundary on the electrostatic interaction energies of two protein-RNA complexes, and re-examine the previous conclusion that electrostatic interactions destabilize proteinnucleic acid binding.…”