Surface salt bridges stabilize the GCN4 leucine zipper.
We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcal/ mol, an apparent elevation of the melting temperature relative to wild type of about 22 degrees C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences.
Spek
E J
EJ
Department of Chemistry, New York University, New York 10003, USA.
Bui
A H
AH
Lu
M
M
Kallenbach
N R
NR
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004268:DNA-Binding Proteins; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts / Q000737:chemistry*; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts; D011494:Protein Kinases; D000409:Alanine