The rapid spreading of antimicrobial resistance in Gram-negative bacteria has become a major threat for humans as well as animals. As one of the main factors involved, the permeability of the outer membrane has attracted a great deal of attention recently. However, the knowledge regarding the translocation mechanisms for most available antibiotics is so far rather limited. Here, a theoretical study concerning the diffusion route of ciprofloxacin across the outer membrane porin OmpC from E. coli is presented. To this end, we establish a protocol to characterize meaningful permeation pathways by combining metadynamics with the zero-temperature string method. It was found that the lowest-energy pathway requires a reorientation of ciprofloxacin in the extracellular side of the porin before reaching the constriction region with its carboxyl group ahead. Several affinity sites have been identified, and their metastability has been evaluated using unbiased simulations. Such a detailed understanding is potentially very helpful in guiding the development of next generation antibiotics.

Despite tremendous successes in the field of antibiotic discovery seen in the previous century, infectious diseases have remained a leading cause of death. More specifically, pathogenic Gram-negative bacteria have become a global threat due to their extraordinary ability to acquire resistance against any clinically available antibiotic, thus urging for the discovery of novel antibacterial agents. One major challenge is to design new antibiotics molecules able to rapidly penetrate Gram-negative bacteria in order to achieve a lethal intracellular drug accumulation. Protein channels in the outer membrane are known to form an entry route for many antibiotics into bacterial cells. Up until today, there has been a lack of simple experimental techniques to measure the antibiotic uptake and the local concentration in subcellular compartments. Hence, rules for translocation directly into the various Gram-negative bacteria via the outer membrane or via channels have remained elusive, hindering the design of new or the improvement of existing antibiotics. In this review, we will discuss the recent progress, both experimentally as well as computationally, in understanding the structure−function relationship of outer-membrane channels of Gram-negative pathogens, mainly focusing on the transport of antibiotics.

To quantify the flow of small uncharged molecules into and across nanopores, one often uses ion currents. The respective ion-current fluctuations caused by the presence of the analyte make it possible to draw some conclusions about the direction and magnitude of the analyte flow. However, often this flow appears to be asymmetric with respect to the applied voltage. As a possible reason for this asymmetry, we identified the electroosmotic flow (EOF), which is the water transport associated with ions driven by the external transmembrane voltage. As an example, we quantify the contribution of the EOF through a nanopore by investigating the permeation of α-cyclodextrin through CymA, a cyclodextrin-specific channel from Klebsiella oxytoca. To understand the results from electrophysiology on a molecular level, all-atom molecular dynamics simulations are used to detail the effect of the EOF on substrate entry to and exit from a CymA channel in which the N-terminus has been deleted. The combined experimental and computational results strongly suggest that one needs to account for the significant contribution of the EOF when analyzing the penetration of cyclodextrins through the CymA pore. This example study at the same time points to the more general finding that the EOF needs to be considered in translocation studies of neutral molecules and, at least in many cases, should be able to help in discriminating between translocation and binding events.

Because of the size of light-harvesting complexes and the involvement of electronic degrees of freedom, computationally these systems need to be treated with a combined quantum–classical description. To this end, Born−Oppenheimer molecular dynamics simulations have been employed in a quantum mechanics/molecular mechanics (QM/MM) fashion for the ground state followed by excitation energy calculations again in a QM/MM scheme for the Fenna−Matthews−Olson (FMO) complex. The self-consistent-charge density functional tight-binding (DFTB) method electrostatically coupled to a classical description of the environment was applied to perform the ground-state dynamics. Subsequently, long-range-corrected time-dependent DFTB calculations were performed to determine the excitation energy fluctuations of the individual bacteriochlorophyll a molecules. The spectral densities obtained using this approach show an excellent agreement with experimental findings. In addition, the fluctuating site energies and couplings were used to estimate the exciton transfer dynamics.

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 Pseudomonas aeruginosa 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.