Structural Dynamics of c-Myb DNA-Binding Domain Revealed by DXT and Thermal Analysis

Abstract

The transcriptional factor, c-Myb R2R3, is minimum unit for DNA-binding and shows largely flexible conformation in solution, which is important for specific DNA-binding function. Here we investigated the structural dynamics of c-Myb R2R3 induced by the DNA-binding, using circular dichroism (CD), diffracted X-ray tracking (DXT), and isothermal titration calorimetry (ITC). DXT is recently developed methods and can evaluate the protein structural fluctuations by detecting the movement of a gold-nanocrystal attached to the target protein. Thermal stability of R2R3 was increased in the presence of cognate DNA, suggesting that the structure was changed in more rigid upon the DNA-binding. The resultant curve of the mean square angular displacements (MSD) obtained from DXT clearly showed that the flexibility of R2R3 was decreased upon DNA binding, and the DNA-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using ITC. The results of the MSD curves also indicate that the translational length reduces by approximately half upon DNA binding.