| Abstract
| - The site-specific interaction of the basic leucinezipper protein C62GCN4, which correspondsto the C-terminal sequence 220−281 of the yeast transcription factorGCN4, with the AP-1 and ATF/CREB DNA recognition sites was analyzed by isothermal titrationmicrocalorimetry. Free C62GCN4 isa dimer composed of a C-terminal leucine zipper and a basic, mainlyunstructured DNA binding domain.Upon association with the target DNA, C62GCN4 folds to a fullyα-helical dimer [Ellenberger et al.(1992) Cell 71, 1223−1237; König and Richmond (1993)J. Mol. Biol. 233, 139−154]. Theprotein-bound AP-1 site is straight, and the protein-bound ATF/CREB site is bentby 20° toward the leucinezipper domain. The coupling between protein folding and DNAassociation resulting in two conformationally different complexes with C62GCN4 poses interestingthermodynamic problems. The associationwas strongly exothermic for both DNA target sites. The freeenergies of binding were indistinguishablein buffers of low salt concentration, and no change of the protonationstate of C62GCN4 and/or the DNAtarget site occurred on formation of the complexes. Both complexesexhibited large and negative heatcapacity changes. The empirical correlation between buriednonpolar and polar surfaces and the reductionin heat capacity concomitant to complexation did hold for the reactionwith the AP-1 site at low saltconcentration. However, in the case of the ATF/CREB site, thechange in heat capacity was larger thancould be accounted for by the burial of solvent-accessible surface.Potential sources of the extra decrementin the heat capacity could be restrictions in the vibrational modes ofpolar groups and of bound watermolecules at the protein−DNA interface, thought to result from thebending of the ATF/CREB site. Inthe presence of high concentrations of glutamate and NaCl, the complexwith the ATF/CREB site wassignificantly weaker than the complex with the AP-1 site.
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