Electrostatic contributions to the stability of the GCN4 leucine zipper structure.
Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pK(a) values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pK(a) values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pK(a) values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pK(a) differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pK(a) values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pK(a) predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil.
Matousek
William M
WM
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, USA.
Ciani
Barbara
B
Fitch
Carolyn A
CA
Garcia-Moreno
Bertrand
B
Kammerer
Richard A
RA
Alexandrescu
Andrei T
AT
D050976:Basic-Leucine Zipper Transcription Factors; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D016350:Leucine Zippers; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D055672:Static Electricity; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry*
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors