MULTISTATE THERMAL TRANSITIONS OF PROTEINS - DNA-BINDING DOMAIN OF THE C-MYB ONCOPROTEIN

The DNA binding domain of c-Myb, protein, a regulator of the transcrip tion, consists of three homologous tandem repeats (R1, R2 and R3) with 51-52 amino acid residues. Previously, we found that the thermal tran sition of the binding domain (R1R2R3) is three-state transition and th at the intermediate state is related to the lower stability of R2 than the other two. In order to clarify the thermodynamic characteristics of the most unstable R2, we synthesized two mutants of R2, R2(V103I) a nd R2(V103L), and examined their thermodynamic properties by circular dichroism and differential scanning calorimetry. The thermal transitio n temperature (T-d) and enthalpy change (Delta H) obtained by CD for R 2(V103I) and R2(V103L) were 47.1 and 62.6 degrees C, and 144 and 166 k J mol(-1), respectively. The corresponding values by DSC were 47.5 and 62.8 degrees C, and 140.1 and 173.4 kJ mol(-1), respectively. Especia lly, the values of T-d and Delta H for R2(V103L) by CD (DSC) are signi ficantly higher than those for R2, which are 43.7 (43.8) degrees C and 125.4 (133.6) kJ mol(-1). The larger stabilizing effect by the substi tution was found for Leu mutant. The results suggest that not only the cavity effect but also the contributions of conformational change in native and denatured states, and of the interactions of the side chain s with water should be taken into account.