Non-specific vs specific DNA binding free energetics of a transcription factor domain protein for target search and recognition

Transcription factor (TF) proteins regulate gene expression by binding to specific sites on the genome. In the facilitated diffusion model, an optimized search process is achieved by the TF protein alternating between 3D diffusion in the bulk and 1D diffusion along DNA. While undergoing 1D diffusion, the protein can switch from a search mode for fast diffusion along non-specific DNA to a recognition mode for stable binding to specific DNA. It was recently noticed that for a small TF domain protein, re-orientations on DNA other than conformational changes happen between the non-specific and specific DNA binding. We here conducted all-atom molecular dynamics simulations with steering forces to reveal the protein-DNA binding free energetics, with a difference between the non-specific and specific binding about 10 kBT, confirming that the search and recognition modes are distinguished only by protein orientations on the DNA. As the binding free energy difference differs from that being estimated from experimental measurements about 4-5 kBT on 15-bp DNA constructs, we hypothesize that the discrepancy comes from DNA sequences flanking the 6-bp central binding sites impacting on the dissociation kinetics measurements. The hypothesis is supported by a simplified spherical protein-DNA model along with stochastic simulations and kinetic modeling.