(117c) Investigating Repressor Binding in the Tetracycline Operon by Molecular Dynamics Simulations

The tetracycline operon is an important gene network component commonly used in synthetic biology applications. At the heart of the tet operon system is the highly specific binding of the tet repressor protein (TetR) to its cognate DNA sequence (tetO). We employ molecular dynamics (MD) simulations coupled with the free energy perturbation method to investigate the binding affinity of TetR to different tetO mutants. We also carry out in vivo tests in E. coli for a series of promoters based on these mutants, and obtain reasonable agreement between experimental green fluorescent protein (GFP) repression levels and binding free energy differences computed from molecular simulations. In all cases, the wild type tetO sequence yields the strongest TetR binding, which is observed both experimentally in terms of GFP levels and in simulation in terms of free energy changes. Two of the four tetO mutants we tested yield relatively strong binding, although somewhat weaker than the wild type, whereas the other two mutants tend to be significantly weaker. The clustering and relative ranking of this subset of tetO mutants is generally consistent between our own experimental data, previous experiments with different systems and the free energy changes computed from our simulations. Overall, this work offers detailed quantitative insights into an important synthetic biological system and demonstrates the potential of molecular simulations to quantitatively predict biologically relevant behavior.