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

• Published in: Journal of molecular biology 2003-07, Vol.330 (3), p.621-637
• Main Authors: Marti, Daniel N., Rudolf Bosshard, Hans
• Format: Article
• Language: English
• Subjects: 1H NMR; Amino Acid Sequence; Arginine - genetics; DNA-Binding Proteins - chemistry; Glutamic Acid - chemistry; Histidine - chemistry; Hydrogen-Ion Concentration; Leucine Zippers - genetics; Leucine Zippers - physiology; Lysine - genetics; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; mutation; Norleucine - genetics; p Ka values; Peptides - chemistry; Protein Folding; Protein Kinases - chemistry; protein thermodynamics; Saccharomyces cerevisiae Proteins - chemistry; salt bridges; Salts - chemistry; Static Electricity; Thermodynamics
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/S0022-2836(03)00623-5

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 AB SS. The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the p K a induced by folding of the coiled coil structure. p K a F values of ten Glu and two His side-chains in folded AB SS and the corresponding p K a U values in unfolded peptides with partial sequences of AB SS 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 AB SS 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 AB SS 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 p K a 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.