Exploration of Free Energy Surfaces Across a Membrane Channel Using Metadynamics and Umbrella Sampling.

To reach their site of action, it is essential for antibiotic molecules to cross the bacterial outer membrane. The progress of enhanced sampling techniques in molecular dynamics simulations enables us to understand these translocations at an atomic level. To this end, calculations of free energy surfaces for these permeation processes are of key importance. Herein, we investigate the translocation of a variety of anionic solutes through the outer membrane pore OprO of the Gram-negative bacterium using the metadynamics and umbrella sampling techniques at the all-atom level. Free energy calculations have been performed employing these two distinct methods in order to illustrate the difference in computed free energies, if any. The investigated solutes range from a single atomic chloride ion over a multiatomic monophosphate ion to a more bulky fosmidomycin antibiotic. The role of complexity of the permeating solutes in estimating accurate free energy profiles is demonstrated by performing extensive convergence analysis. For simple monatomic ions, good agreement between the well-tempered metadynamics and the umbrella sampling approaches is achieved, while for the permeation of the monophosphate ion differences start to appear. In the case of larger molecules such as fosmidomycin it is a tough challenge to achieve converged free energy profiles. This issue is mainly due to neglecting orthogonal degrees of freedom during the free energy calculations. Nevertheless, the freely driven metadynamics approach leads to clearly advantageous results. Additionally, atomistic insights of the translocation mechanisms of all three solutes are discussed.