Structural dynamics of a DNA-binding protein analyzed using diffracted X-ray tracking

Abstract

Diffracted X-ray tracking (DXT) is one of methods for the real-time evaluation of protein structural dynamics by detecting the movement of a gold-nanocrystal attached to a target protein. However, one of the technical concerns is the size of the gold-nanocrystals, which are larger than the protein. In our previous results of mean square angular displacement curves in DXT analysis, dynamical movements of the DNA-binding protein, c-Myb R2R3, were observed in only one population in either DNA-unbound or -bound state, and was found to decrease upon DNA binding. In this study, c-Myb R2R3 dynamical movements were re-evaluated with a low density of the protein immobilized on the DXT substrate, to decrease the possibility that the gold-nanocrystals attached to more than one R2R3 molecule. We observed two dynamical moving populations in the DNA-bound state, which could be classified due to electrostatic attraction and repulsion between the DNA-protein complexes, and determined the apparent angular diffusion constant, which was similar to the value calculated in our previous study. We showed more real movement of the protein could be observed by lowering the immobilization density of the protein.

Keywords: DNA-binding protein, immobilization density; Diffracted X-ray tracking; Protein structural dynamics.