Contribution of buried aspartic acid to the stability of the PDZ2 protein

Statistical analysis of protein structures shows that buried aspartic acid residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 potential ionic interactions with other protein groups. To estimate the energetic contribution of such buried groups to the Gibbs free energy of proteins, we measured the effects of amino acid substitutions of D56 in a model protein PDZ2 on its stability. We used temperature-induced unfolding monitored by DSC and denaturant-induced unfolding monitored by the changes in fluorescence intensity. We find that all substitutions of D56 lead to protein unfolding, thus suggesting that this buried hydrogen bonded aspartic acid has a significant contribution to the stability. To quantify the changes in the Gibbs free energy, one of the variants, D56N was stabilized by addition of the protective osmolyte TMAO. Comparison of the stability of the D56N variant with the wild-type PDZ2 in the presence and absence of TMAO allowed us to estimate the contribution of D56 to the protein stability to be 18 kJ · mol−1. These findings are discussed in terms of contribution of buried ionizable groups to protein stability.

► Buried Asp residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 salt bridges.
► Contribution of buried Asp to stability was estimated using model protein PDZ2.
► The energetic contribution of Asp56 to PDZ2 stability estimated to be 18 kJ · mol−1.
► Findings are discussed in terms of contribution of Asp residues to protein stability.