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 AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(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 alpha-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 alpha-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 pK(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.
Marti
Daniel N
DN
Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. dmarti@access.unizh.ch
Bosshard
Hans Rudolf
HR
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D016350:Leucine Zippers / Q000235:genetics / Q000502:physiology*; D008239:Lysine / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009646:Norleucine / Q000235:genetics; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D012492:Salts / Q000737:chemistry; D055672:Static Electricity; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts; D018698:Glutamic Acid; D006639:Histidine; D009646:Norleucine; D001120:Arginine; D011494:Protein Kinases; D008239:Lysine