(156f) Computer Simulations and Free Energy Calculations of Protein-DNA Binding. Application to the TetR:TetO System

The binding of proteins to DNA is a crucial step in countless cellular processes and gene regulatory networks. In particular, the binding of Tet repressor protein (TetR) to its 19-base pair operator site (TetO) on DNA is employed as part of many synthetic biology applications. In order to fine-tune the dynamics and phenotypic behaviour of such gene networks, mutations in the TetO nucleic acid sequence can be employed; however, the effect of such mutations is often difficult to predict and quantify. In the present work, we have carried out fully atomistic molecular dynamics simulations along with free energy perturbation calculations to compute the binding free energy differences between several TetO mutants and the wild-type TetR protein. The computed free energy differences were compared to experimentally observed gene expression levels, and a reasonable correlation in the mutant ranking was found. We have additionally analyzed the atomistic-level interactions that are responsible for TetR binding to TetO, and have been able to identify some of the key protein residues that are responsible for binding as well as differentiation between different base pair sequences. The formation of hydrogen bonds as well as salt bridges between TetR and TetO is investigated in great detail. The methods presented herein demonstrate the applicability of molecular simulations to ?in silico? design of gene network components at an atomistic level.