Using Reweighted Pulling Simulations to Characterize Conformational Changes in Riboswitches

Riboswitches are RNA sequences located in noncoding portions of mRNA that can sense specific ligands and subsequently control gene expression. The ligand-binding event induces conformational changes in the riboswitch that are then transmitted to the gene expression apparatus. Probing the mechanisms of such a fine regulation at atomic resolution is very difficult experimentally and molecular dynamics (MD) could be used to quantify the ligand-dependent behavior of a riboswitch. However, since the accessible time scale of fully atomistic simulations is limited, this can only be done using enhanced sampling techniques. Here, we discuss the application of steered MD to the characterization of the ligand-dependent stability of the aptamer terminal helix in the add adenine-sensing riboswitch. The employed techniques are discussed in detail and sample input files are provided. We show that with a limited computational effort it is possible to quantify, in terms of free energy, the stacking interaction between the ligand and the terminal helix, obtaining results in agreement with thermodynamic experiments.