The development of new antibiotics against Gramnegative bacteria is hampered by the powerful protective properties of their cell envelope. This envelope consists of two membranes augmented by efflux transporters, which act in synergy to restrict cellular access to a broad range of chemical compounds. Recently, a kinetic model of this system has been constructed. The model revealed a complex, nonlinear behavior of the system, complete with a bifurcation, and matched very well to experimental uptake data.Here, we expand the model to include multiple transporters and apply it to an experimental analysis of antibiotic accumulation in wild-type and efflux-deficient Escherichia coli. We show that transporters acting across the inner and outer membranes have synergistic effects with each other. In contrast, transporters acting across the same membrane are additive as a rule but can be synergistic under special circumstances owing to a bifurcation controlled by the barrier constant. With respect to ethidium bromide, the inner membrane transporter MdfA was synergistic to the TolC-dependent efflux across the outer membrane. The agreement between the model and drug accumulation was very good across a range of tested drug concentrations and strains. However, antibiotic susceptibilities related only qualitatively to the accumulation of the drugs or predictions of the model and could be fit to the model only if additional assumptions were made about the physiological consequences of prolonged cell exposure to the drugs. Thus, the constructed model correctly predicts transmembrane permeation of various compounds and potentially their intracellular activity.

We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for β-1,4-galactosyltransferase (β4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock β4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.

Understanding the viral peptide detection, partitioning and subsequent host membrane composition-based response is required for gaining insights into viral mechanism. Here, we probe the crucial role of presence of membrane...

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