Translocation of small molecules through engineered outer-membrane channels from Gram-negative bacteria

A hybrid computational approach based on molecular dynamics simulations and deep learning algorithms facilitate fast and accurate estimation of the free energy of permeation of small drug-like molecules across lipid bilayers.

Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. In Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, and S. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed in Streptococcus pneumoniae (IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling of S. aureus PBP4 by Bocillin FL. In PBP competition assays with S. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50 of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets.

Decision letter: Improved ANAP incorporation and VCF analysis reveal details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain

The translocation of small molecules and polymers is an integral process for the functioning of living cells. Many of the basic physical, chemical, and biological interactions have not yet been studied because they are not directly experimentally accessible. We have shown that a combination of optical tweezers, single solid-state nanopores, and electrophysiological ionic current detection enable deeper insight into the behavior of polymers in confinement. Here we describe the experimental procedures that are necessary to manipulate single biopolymers in a single nanopore, not only by electrical fields, but also through mechanical forces using optical tweezers.

Barrier polymers are used for many packaging and protective applications. As barriers they separate a system, such as an article of food or an electronic component, from an environment. Barrier polymers limit movement of substances, called permeants. The movement can be through the polymer or, in some cases, merely into the polymer. After crossing the barrier polymer, the permeant moves to the polymer surface, desorbs, and moves away. Permeant movement is a physical process that has both a thermodynamic and a kinetic component. For polymers without special surface treatments, the thermodynamic contribution is in the solution step. The permeant partitions between the environment and the polymer according to thermodynamic rules of solution. The kinetic contribution is in the diffusion. The net rate of movement is dependent on the speed of permeant movement and the availability of new vacancies in the polymer. The traditional definition of a barrier polymer required an oxygen permeability less than \documentclass{article}\usepackage{amssymb}\pagestyle{empty}\begin{document}${2{\hskip0.167em}{\hskip0.167em}{\rm{nmol}}/{(}{\rm{m}} {\hskip-0.167em}{\hskip-0.167em}{\cdot{}}{\hskip-0.167em}{\hskip-0.167em} {\rm{s}}{\hskip-0.167em}{\hskip-0.167em}{\cdot{}}{\hskip-0.167em} {\hskip-0.167em}{\rm{GPa}}{)}}$\end{document} (originally, less than \documentclass{article}\usepackage{amssymb}\pagestyle{empty}\begin{document}${{(}1{\hskip0.167em}{\hskip0.167em}{\rm{cc}}{\hskip-0.167em} {\hskip-0.167em}{\cdot{}}{\hskip-0.167em}{\hskip-0.167em}{\rm{mil}}{)}/{(}100{\hskip0.167em}{\hskip0.167em}{\rm{in}} ^{2}{\hskip-0.167em} {\hskip-0.167em}{\cdot{}}{\hskip-0.167em}{\hskip-0.167em}{\rm{d}} {\hskip-0.167em}{\hskip-0.167em}{\cdot{}}{\hskip-0.167em}{\hskip-0.167em} {\rm{atm}}{)}}$\end{document} ) at room temperature. Poly(ethylene terephthalate) (PET), with an oxygen permeability of \documentclass{article}\usepackage{amssymb}\pagestyle{empty}\begin{document}${8{\hskip0.167em}{\hskip0.167em}{\rm{nmol}}/{(}{\rm{m}} {\hskip-0.167em}{\hskip-0.167em}{\cdot{}}{\hskip-0.167em}{\hskip-0.167em} {\rm{s}}{\hskip-0.167em}{\hskip-0.167em}{\cdot{}}{\hskip-0.167em} {\hskip-0.167em}{\rm{GPa}}{)}}$\end{document} , is not considered a barrier polymer by the old definition; however, it is an adequate barrier polymer for holding carbon dioxide in a 2‐L bottle for carbonated soft drinks. Many months are required to lose enough carbon dioxide (15% of initial) to be objectionable. The polymers that are good barriers to permanent gases, especially oxygen, have important commercial significance. Vinylidene chloride copolymers are available as resins for extrusion, latices for coating, and resins for solvent coating. Vinylidene chloride copolymers are marketed under a variety of trade names. Saran is a trademark of The Dow Chemical Company for vinylidene chloride copolymers. Other trade names include Daran (W.R. Grace), Amsco Res (Union Oil), and Serfene (Morton Chemical) in the United States; and Haloflex (Imperial Chemical Industries, Ltd.), Diofan (BASF), Ixan (Solvay and Cie SA), and Polyidene (Scott‐Bader) in Europe. Hydrolyzed ethylene–vinyl acetate copolymers, commonly known as ethylene–vinyl alcohol (EVOH) copolymers, are usually used as extrusion resins, although some may be used in solvent‐coating applications. Copolymers of acrylonitrile are used in extrusion and molding applications. Commercially important comonomers for barrier applications include styrene and methyl acrylate. Polyamide polymers can provide a good‐to‐moderate barrier to permeation by permanent gases. Two often‐used polymers have adequate properties for some applications. Poly(ethylene terephthalate) (PET) is used to make films and bottles. Poly(vinyl chloride) (PVC) is a moderate barrier to permanent gases. Plasticized poly(vinyl chloride) is used as a household wrapping film. In regard to water vapor transmission (WVTR) values, those polymers that are good oxygen barriers are often poor water‐vapor barriers and vice versa. Polymer molecules without dipole–dipole interactions, such as polyolefins, dissolve very little water and have low WVTR and permeability values. The permeation of flavor, aroma, and solvent molecules in polymers follows the same physics as the permeation of small molecules, but with two significant differences. For these larger molecules, the diffusion coefficients are much lower and the solubility coefficients are much higher. Furthermore, the large solubility coefficient can lead to enough sorption of the large molecule that plasticization occurs in the polymer, which can increase the diffusion coefficient. Generally, vinylidene chloride copolymers and glassy polymers such as polyamides and EVOH are good barriers to flavor and aroma permeation, whereas the polyolefins are poor barriers. Several physical factors can affect the barrier properties of a polymer. These include temperature, humidity, orientation, and cross‐linking. Typically, the permeability increases 5 to 10% for every increase of 1°C. When a polymer equilibrates with a humid environment, it absorbs water. This can plasticize the polymer and increase the permeability. The effect of orientation on the permeability of polymers is difficult to assess; diffusion in some polymers is unaffected by orientation; in others, increases or decreases are observed. Cross‐linking has been shown in a few cases to decrease the diffusion coefficient. Reasonable prediction can be made of the permeabilities of low molecular weight gases such as oxygen, nitrogen, and carbon dioxide in many polymers. The diffusion coefficients are not complicated by the shape of the permeant, and the solubility coefficients of each of these molecules do not vary much from polymer to polymer. Reasonable predictions of the permeabilities of larger molecules such as flavors, aromas, and solvents are not easily made. The diffusion coefficients are complicated by the shape of the permeant, and the solubility coefficients for a specific permeant can vary widely from polymer to polymer. The permachor method is an empirical method for predicting the permeabilities of oxygen, nitrogen, and carbon dioxide in polymers. In this method a numerical value is assigned to each constituent part of the polymer. An average number is derived for the polymer, and a simple equation converts the value into a permeability. The model has been modified to liquid permeation with some success. For larger molecules, independent predictions of the diffusion coefficients and the solubility coefficients are required. Predicting the diffusion coefficient for a permeant in a polymer requires knowing one other diffusion coefficient in the polymer. The solubility coefficients are more difficult to predict. Although advances are being made, the best method is probably to use a few known solubility coefficients in the polymer to predict others. Measuring the barrier properties of polymers is important for several reasons. The effects of formulation or process changes need to be known, new polymers need to be evaluated, data are needed for a new application before a large investment has been made, and fabricated products need to have performance verified. Two methods of measuring water‐vapor transmission rates (WVTR) are commonly used. The newer method uses a Permatran‐W (Modern Controls, Inc.). The other method is the ASTM cup method. Measuring the permeation of carbon dioxide occurs far less often than measuring the permeation of oxygen or water. The simplest method uses the Permatran‐C instrument (Modern Controls, Inc.). Many methods are used to characterize the transport of flavor, aroma, and solvent molecules in polymers. Each has some value, and no one method is suitable for all situations. Any experiment should obtain the permeability, the diffusion coefficient, and the solubility coefficient. The primary application for barrier polymers is food and beverage packaging. Barrier polymers are also used for packaging medical products, agricultural products, cosmetics, and electronic components and in moldings, pipe, and tubing. The use of safe materials is vital for barrier applications, particularly for food, medical, and cosmetics packaging. Suppliers of specific barrier polymers can provide the necessary details to ensure safe processing and use of barrier polymers.

Our earlier studies with outer membrane permeability in E. coli showed that an insertion mutation in lpp gene (encoding Braun's lipoprotein) drastically changed the outer membrane permeability, resulting in significant acceleration of whole-cell catalyzed reactions. In order to gain a mechanistic understanding of the nature of permeability change, the lpp region was sequenced. The results revealed that Lpp was not expressed in the insertion mutant, suggesting that the absence, rather than the alteration, of Lpp is responsible for the observed permeability change. This surprising result prompts us to investigate the possibility of establishing lpp deletion as a general permeabilization method. Two lpp deletion mutants were generated from strains with different genetic background and the effect of lpp deletion on cell physiology was investigated. While lpp deletion had no significant effect on cell growth, carbon metabolism, and fatty acid compositions, it enhanced permeability of various small molecules, consistent with the results with the insertion mutant. This phenotype is useful in a wide range of biotechnological applications. We illustrate here the use of the mutant with organophosphate hydrolysis and L-carnitine synthesis, where permeability is known to be a limiting factor. Both processes were significantly improved with the mutant because of enhanced permeability through the outer membrane. Therefore, this study has established an easy yet generally applicable method for permeabilizing E. coli cells without significant adverse effects. Further, as lpp homolog is known to exist in gram-negative bacteria, we expect that this method will be applicable to other gram-negative bacteria.

Permeation of small molecules through polymers confined in mesoporous media

Nonjunctional membrane in many cells contains connexin gap junction hemichannels (or connexons) that can open to allow permeation of small molecules. Opening of Cx43 hemichannels is infrequent in normal extracellular Ca(2+) and enhanced by low Ca(2+), positive membrane potentials, and dephosphorylation of critical residues. Here we report that lowering intracellular redox potential increases Cx43 hemichannel open probability under otherwise normal conditions. We studied dye uptake and single-channel activity in HeLa cells transfected with wild-type Cx43, Cx43 with enhanced GFP attached to its C terminus (Cx43-EGFP), and Cx43 with enhanced GFP attached to its N terminus (EGFP-Cx43). Dithiothreitol [(DTT) 10 mM], a membrane permeant-reducing agent, increased the rate of dye uptake by cells expressing Cx43 and Cx43-EGFP, but not by parental cells or cells expressing EGFP-Cx43. Induced dye uptake was blocked by La(3+), by a peptide gap junction and hemichannel blocker (gap 26), and by flufenamic acid. DTT increased Cx43-EGFP hemichannel opening at positive voltages. Bath application of reduced glutathione, a membrane impermeant-reducing agent, did not increase dye uptake, but glutathione in the recording pipette increased hemichannel opening at positive voltages, suggesting that it acted intracellularly. DTT caused little change in levels of surface Cx43 or Cx43-EGFP, or in intracellular pH. These findings suggest that lowering intracellular redox potential increases the opening of Cx43 and Cx43-EGFP hemichannels, possibly by action on cytoplasmic cysteine residues in the connexin C terminus.

Effect of physical aging on sorption and permeation of small molecules in polyimide membranes

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Nitrogen-doped carbon dots (CDs) were prepared from essential fluorescent amino acids, such as lysine (Lys), phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) by two routes, microwave and teflon-lined autoclave. The fluorescent properties of these amino acids were significantly improved in their corresponding CD forms, especially for those prepared via the hydrothermal process. A significantly high fluorescent intensity was measured in the emission range of 420-470nm at the excitation wavelengths of 300-350nm. While a negatively zeta potential value of - 9.8 ± 2.6mV was obtained for Lys CDs suspension, the CDs of Phe, Tyr, and Trp afforded strong positive zeta potentials, + 24.0 ± 2.4, + 12.5 ± 3.1, and + 31.9 ± 3.8 mV, respectively. The Lys CDs were found not to be antimicrobial even at a 10 mg/mL concentration, which is likely due to their negative surface charge that weakens electrostatic interactions with negatively charged microbial membranes. Whereas the Phe, Tyr, and Trp CDs had a 2.5mg/mL minimum inhibition concentration (MIC) against a Gram-negative bacterium, Klebsiella pneumoniae. The Phe CDs commenced the highest antibacterial effect against Bacillus subtilis (ATCC 6633) Gram-positive bacteria, but the lowest MIC value of 1.25mg/mL was determined for Tyr CDs against Candida albicans (ATCC 10231) fungus. Furthermore, a UV light exposure, 30min treatment of UV-A light with a 6.88mW/cm2 irradiance value on amino acids CD exhibited improved photodynamic activity. The natural amino acid-derived CDs show great biocompatibility on L929 fibroblast cells with > 86% cell viability, for all formulations except Tyr CDs, retaining 78 ± 3% viability even at 1000μg/mL concentration, and blood compatibility at 500μg/mL concentration. Therefore, these CDs derived from fluorescent amino acids are photoactivated and are of excellent nanosized materials in a variety of biomedical in vitro and in vivo uses, including diagnostic, sensor, and therapeutic applications.

In the quest to elucidate the solid‐state structures of polymers, insight into the amorphous phase is particularly elusive. Although the permeability of small molecules is often measured as an important performance property, numerous researchers have found that a deeper analysis of the transport characteristics provides insight into polymer morphology, especially if used in combination with more usual characterization techniques. The transport of small gas molecules senses the permeable amorphous structure and probes the nature of the free volume. In recent years, our interest in the gas barrier of polyesters has resulted in an unusual opportunity to investigate the nature of the free volume in the polymer glassy state. This effort has been aided by access to aromatic polyesters with designed variations in their chemical structure. This review focuses on oxygen transport, supplemented with other methods of physical analysis, as a probe of the excess‐hole free volume. The review addresses the profound effects of orientation and crystallization on the free volume of the glassy state. The discussion also presents a simple odel for the gas permeability of the isotropic glass based on lattice concepts and tests more sophisticated models for the gas permeability of semicrystalline polymers. The final section addresses other opportunities for fruitful applications of oxygen transport as a solid‐state structure probe. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 1047–1063, 2005

Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 +/- 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes.

Effects of Channel Forming Peptides on Lipid Bilayer Dynamics and Leaflet Coupling

Modification of a tunable UV-visible capillary electrophoresis detector for simultaneous absorbance and fluorescence detection: profiling of body fluids for drugs and endogenous compounds