Background: Occ channels mediate small molecule uptake in Pseudomonads. Results: We have analyzed a number of site-directed mutants for two Occ channels. Conclusion: Pores of OccD subfamily members are highly flexible. The central basic ladder residues interact with the substrate carboxyl group and are essential for transport. Significance: The data provide the first atomistic insights into transport by an important class of OM channels.

Permeation of small molecules across cell membranes is a ubiquitous process in biology and is dependent on the principles of physical chemistry at the molecular level. Here we use atomistic molecular dynamics simulations to calculate the free energy of permeation of a range of small molecules through a model of the outer membrane of Escherichia coli, an archetypical Gram-negative bacterium. The model membrane contains lipopolysaccharide (LPS) molecules in the outer leaflet and phospholipids in the inner leaflet. Our results show that the energetic barriers to permeation through the two leaflets of the membrane are distinctly asymmetric; the LPS headgroups provide a less energetically favorable environment for organic compounds than do phospholipids. In summary, we provide the first reported estimates of the relative free energies associated with the different chemical environments experienced by solutes as they attempt to cross the outer membrane of a Gram-negative bacterium. These results provide key insights for the development of novel antibiotics that target these bacteria.

In the following review we use recent examples from the literature to discuss progress in the area of atomistic and coarse-grained molecular dynamics simulations of selected bacterial membranes and proteins, with a particular focus on Gram-negative bacteria. As structural biology continues to provide increasingly high-resolution data on the proteins that reside within these membranes, simulations have an important role to play in linking these data with the dynamical behavior and function of these proteins. In particular, in the last few years there has been significant progress in addressing the issue of biochemical complexity of bacterial membranes such that the heterogeneity of the lipid and protein components of these membranes are now being incorporated into molecular-level models. Thus, in future we can look forward to complementary data from structural biology and molecular simulations combining to provide key details of structure-dynamics-function relationships in bacterial membranes.

Molecular modelling and simulations have been employed to study the membranes of Gram-negative bacteria for over 20 years. Proteins native to these membranes, as well as antimicrobial peptides and drug molecules have been studied using molecular dynamics simulations in simple models of membranes, usually only comprising one lipid species. Thus, traditionally, the simulations have reflected the majority of in vitro membrane experimental setups, enabling observations from the latter to be rationalized at the molecular level. In the last few years, the sophistication and complexity of membrane models have improved considerably, such that the heterogeneity of the lipid and protein composition of the membranes can now be considered both at the atomistic and coarse-grain levels of granularity. Importantly this means relevant biology is now being retained in the models, thereby linking the in silico and in vivo scenarios. We discuss recent progress in simulations of proteins in simple lipid bilayers, more complex membrane models and finally describe some efforts to overcome timescale limitations of atomistic molecular dynamics simulations of bacterial membranes.

Pseudomonas aeruginosa is a Gram-negative bacterium that does not contain large, nonspecific porins in its outer membrane. Consequently, the outer membrane is highly impermeable to polar solutes and serves as a barrier against the penetration of antimicrobial agents. This is one of the reasons why such bacteria are intrinsically resistant to antibiotics. Polar molecules that permeate across the outer membrane do so through substrate-specific channels proteins. To design antibiotics that target substrate-channel proteins, it is essential to first identify the permeation pathways of their natural substrates. In P. aeruginosa, the largest family of substrate-specific proteins is the OccD (previously reported under the name OprD) family. Here, we employ equilibrium and steered molecular-dynamics simulations to study OccD1/OprD, the archetypical member of the OccD family. We study the permeation of arginine, one of the natural substrates of OccD1, through the protein. The combination of simulation methods allows us to predict the pathway taken by the amino acid, which is enabled by conformational rearrangements of the extracellular loops of the protein. Furthermore, we show that arginine adopts a specific orientation to form the molecular interactions that facilitate its passage through part of the protein. We predict a three-stage permeation process for arginine.

Responsible research and innovation (RRI) has been the preferred idiom for interrogating the social, ethical and political dimensions of science, technology and innovation for roughly a decade. The uptake of RRI into prominent policy organisations has resulted in a proliferation of policy frameworks as policy makers have attempted to articulate what it means for them to enact RRI. Here, we draw on our experience developing an RRI framework in the ERA Cofund on Biotechnology. We discuss three ways that treating RRI as a form of knowledge production has allowed us to engage with the institutional dimensions of science: as research within scientific projects; as administrative knowledge; and as methodological knowledge. We argue that Science and Technology Studies' concern with knowledge making offers a valuable route to approach RRI within research funding organisations, and reflect on how this approach might be developed in the next European Commission Framework Programme.

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