DNA-binding function of c-Myb R2R3 around thermal denaturation temperature

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The DNA-binding domain of transcriptional factor c-Myb R2R3 exhibits significant solution fluctuations.
• Understanding protein dynamics is crucial for elucidating DNA-binding mechanisms.

Purpose of the Study:

• To investigate the impact of protein fluctuation on DNA-binding and folding thermodynamics of the c-Myb R2R3* mutant.
• To explore the relationship between temperature-induced conformational changes and DNA binding affinity.

Main Methods:

• Isothermal titration calorimetry (ITC) to assess DNA-binding thermodynamics.
• Differential scanning calorimetry (DSC) to analyze protein folding and thermal denaturation.
• Characterization of protein conformational states in both DNA-bound and unbound forms.

Main Results:

• A highly negative heat capacity change above 35°C indicates increased protein fluctuation with temperature, enhancing conformational changes upon DNA binding.
• Differential scanning calorimetry revealed a broad thermal denaturation transition for R2R3* that sharpens and increases in enthalpy upon DNA binding.
• The R2R3* protein can specifically bind DNA near its thermal denaturation temperature, suggesting a role for flexibility in binding.

Conclusions:

• DNA binding induces a transition from a more fluctuating to a less fluctuating state for R2R3*, significantly impacting binding enthalpy.
• Protein flexibility plays a critical role in the specific binding of c-Myb R2R3* to DNA, particularly around its denaturation temperature.
• Conformational flexibility allows for specific and non-specific DNA interactions through transitions between ordered and disordered protein states.