Electrostatic Interactions in Leucine Zippers: Thermodynamic Analysis of the Contributions of Glu and His Residues and the Effect of Mutating Salt Bridges

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper ABSS. The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pKa induced by folding of the coiled coil structure. pKaF values of ten Glu and two His side-chains in folded ABSS and the corresponding pKaU values in unfolded peptides with partial sequences of ABSS were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (−5.6 kJ mol−1) and two are interacting with the positive α-helix dipoles and are thus stabilizing (+3.8 kJ mol−1) in charged form. The two His residues positioned in the C-terminal moiety of ABSS interact with the negative α-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (−2.6 kJ mol−1). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (−1.1 kJ mol−1). The sum of the individual contributions of protonated Glu and His to the higher stability of ABSS at acidic pH (−5.4 kJ mol−1) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (−5.3 kJ mol−1). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys→norleucine, Arg→norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pKa of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge–charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.