THE DNA-BINDING DOMAIN OF DROSOPHILA-MELANOGASTER C-MYB UNDERGOES A MULTISTATE DENATURATION

The DNA-binding domain of Drosophila c-Myb protein has been studied us ing different spectroscopic probes, namely CD, fluorescence, acrylamid e quenching and NMR, to determione the structure of some of its sub-do mains and their relative stabilities in aqueous solutions. While CD an d fluorescence spectroscopy showed that the protein had completely los t its tertiary and secondary structures in approximately 3 M urea, sol vent accessibility of the tryptophan residues was still partial, as de termined by acrylamide quenching. This suggested the presence of signi ficant amounts of residual structure which persisted until the urea co ncentration was raised to approximately 6.0 M. Thermal-denaturation ex periments also indicated the presence of an intermediate in the unfold ing pathway. The experimental data could be fitted assuming a minimum of three states in both modes of denaturation. The thermodynamic param eters for the apparent three-state transition have been determined. Fr om the protein stability curve, we have determined that Drosophila mel anogaster Myb R123 has maximal stability at 16 degrees C and pH 7.0.