Thermodynamic characterization of the structural stability of the coiled-coil region of the bZIP transcription factor GCN4.
The thermal stability of a 56 amino acid fragment of GCN4 has been studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. This fragment contains the leucine zipper and part of the basic region. The thermal unfolding of GCN4-56 is a reversible process and can be well represented by a reaction of the form N2<-->2U, indicating that the unfolding of the leucine zipper is a two-state process in which the helices are only stable when they are in the coiled-coil conformation. As expected, the transition temperature is concentration dependent. At pH 7.06 and a protein concentration of 5 x 10(-4) M the transition temperature is close to 70 degrees C while at 5 x 10(-6) M it is close to 50 degrees C. The enthalpy change for unfolding is 31.5 kcal mol-1 at 70 degrees C. Since the isolated helices are unstable, interactions at the interface between the two helices play a key role in the stabilization of the native dimer. These interactions primarily involve the burial of apolar surface from the solvent (hydrophobic effect) and electrostatic interactions. Structural thermodynamic calculations have permitted a dissection of the magnitude of the various contributions to the total Gibbs free energy of stabilization.
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
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Thompson
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Vinson
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Freire
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry*; D016350:Leucine Zippers; D008433:Mathematics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins; D013056:Spectrophotometry, Ultraviolet
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases