Contribution of cation-π interactions to protein stability

Abstract

Calculations predict that cation-π interactions make an important contribution to protein stability. While there have been some attempts to experimentally measure strengths of cation-p interactions using peptide model systems, much less experimental data are available for globular proteins. We have attempted to determine the magnitude of cation-π interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP, and Trx). In each case, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-p pair was replaced by Leu. Stabilities of wild-type (WT) and mutant proteins were characterized by both thermal and chemical denaturation. Gln and aromatic → Leu mutants were consistently less stable than corresponding Met mutants, reflecting the nonisosteric nature of these substitutions. The strength of the cation-π interaction was assessed by the value of the change in the free energy of unfolding [ΔΔ G° = Δ G°(Met) - Δ G°(WT)]. This ranged from +1.1 to −1.9 kcal/mol (average value −0.4 kcal/mol) at 298 K and +0.7 to −2.6 kcal/mol (average value -1.1 kcal/mol) at the T_m of each WT. It therefore appears that the strength of cation-π interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than calculated values with an average difference |Δ G°_expt - Δ G°_calc|av of 2.9 kcal/mol. At room temperature, the data indicate that cation-π interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However, at elevated temperatures, close to typical T_m's, cation-π interactions are generally stabilizing.