ENERGETICS OF FOLDING AND DNA-BINDING OF THE MAT-ALPHA-2 HOMEODOMAIN

Homeodomains are a class of DNA-binding protein domains which play an important role in genetic regulation in eukaryotes. We have characteri zed the thermodynamics of folding and sequence-specific association wi th DNA of the MAT alpha 2 homeodomain of yeast. Using differential sca nning and isothermal titration calorimetry, we measured the enthalpy, heat capacity, and Gibbs free energy changes of these processes. The p rotein-DNA interaction is enthalpically driven at physiological temper atures, DSC data on the process of melting the protein-DNA complex at different salt concentrations were dissected into its endothermic comp onents, yielding the enthalpy change and dissociation constant of bind ing. A comparison of the circular dichroism spectra of the free and DN A-bound protein species revealed the formation of additional alpha-hel ical structure upon binding to DNA. We propose that the latter half of helix 3, the recognition helix, is substantially unfolded in the free protein under the conditions used, as has been observed with other ho meodomains [Tsao, D. H. H., et al. (1994) Biochemistry 33, 15053-15060 ; Cox, M., et al. (1995) J. Biomol. NMR 5, 23-32]. Formation of protei n structure is induced by DNA binding, and the energies measured for a ssociation therefore include a component due to folding.