Publications by authors named "Jayesh Arun Bafna"

9 Publications

  • Page 1 of 1

Rapid fabrication of teflon apertures by controlled high voltage pulses for formation of free standing planar lipid bilayer membrane.

Biomed Microdevices 2021 Feb 27;23(1):12. Epub 2021 Feb 27.

Department of Biomedical Engineering, Ankara Yıldırım Beyazıt University, Ankara, Turkey.

Free standing artificial lipid bilayers are widely used in the study of biological pores. In these types of studies, the free standing planar lipid bilayer is formed over a micron-sized aperture consisting of either polymer such as Polytetrafluoroethylene (PTFE, Teflon) or glass. Teflon is chemically inert, has a low dielectric constant, and has a high electrical resistance which combined allow for obtaining low noise recordings. This study investigates the reproducible generation of micropores in the range of 50-100 microns in diameter in a Teflon film using a high energy discharge set-up. The discharger set-up consists of a microprocessor, a transformer, a voltage regulator, and is controlled by a computer. We compared two approaches for pore creation: single and multi-pulse methods. The results showed that the multi-pulse method produced narrower aperture size distributions and is more convenient for lipid bilayer formation, and thus would have a higher success rate than the single-pulse method. The bilayer stability experiments showed that the lipid bilayer lasts for more than 33 h. Finally, as a proof-of-concept, we show that the single and multi-channel electrophysiology experiments were successfully performed with the apertures created by using the mentioned discharger. In conclusion, the described discharger provides reproducible Teflon-pores in a cheap and easy-to-operate manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10544-021-00553-4DOI Listing
February 2021

Kanamycin Uptake into Is Facilitated by OmpF and OmpC Porin Channels Located in the Outer Membrane.

ACS Infect Dis 2020 07 20;6(7):1855-1865. Epub 2020 May 20.

IOM/CNR, Sezione di Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato, Italy.

Despite decades of therapeutic application of aminoglycosides, it is still a matter of debate if porins contribute to the translocation of the antibiotics across the bacterial outer membrane. Here, we quantified the uptake of kanamycin across the major porin channels OmpF and OmpC present in the outer membrane of . Our analysis revealed that, despite its relatively large size, about 10-20 kanamycin molecules per second permeate through OmpF and OmpC under a 10 μM concentration gradient, whereas OmpN does not allow the passage. Molecular simulations elucidate the uptake mechanism of kanamycin through these porins. Whole-cell studies with a defined set of porin mutants provide evidence that translocation of kanamycin via porins is relevant for antibiotic potency. The values are discussed with respect to other antibiotics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.0c00102DOI Listing
July 2020

Rapid lipid bilayer membrane formation on Parylene coated apertures to perform ion channel analyses.

Biomed Microdevices 2020 04 30;22(2):32. Epub 2020 Apr 30.

Institute for Microsensors, -actuators and -systems (IMSAS), University of Bremen, Microsystems Center Bremen (MCB), Bremen, Germany.

We present a chip design allowing rapid and robust lipid bilayer (LBL) membrane formation using a Parylene coated thin silicon nitride aperture. After bilayer formation, single membrane channels can be reconstituted and characterized by electrophysiology. The ability for robust reconstitution will allow parallelization and enhanced screening of small molecule drugs acting on or permeating across the membrane channel. The aperture was realized on a microfabricated silicon nitride membrane by using standard clean-room fabrication processes. To ensure the lipid bilayer formation, the nitride membrane was coated with a hydrophobic and biocompatible Parylene layer. We tested both Parylene-C and Parylene-AF4. The contact angle measurements on both Parylene types showed very good hydrophobic properties and affinity to lipids. No precoating of the Parylene with an organic solvent is needed to make the aperture lipophilic, in contradiction to Teflon membranes. The chips can be easily placed in an array utilizing a 3D printed platform. Experiments show repetitive LBL formation and destruction (more than 6 times) within a very short time (few seconds). Through measurements we have established that the LBL layers are very thin. This allows the investigation of the fusion process of membrane proteins i.e. outer membrane protein (OmpF) in the LBL within a few minutes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10544-020-0473-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192868PMC
April 2020

Structural insights into the main S-layer unit of reveal a massive protein complex with porin-like features.

J Biol Chem 2020 03 18;295(13):4224-4236. Epub 2020 Feb 18.

Department of Plant Physiology, Warsaw University of Life Sciences-SGGW, Nowoursynowska Str. 159, 02776 Warsaw, Poland. Electronic address:

In the extremophile bacterium , the outermost surface layer is tightly connected with the rest of the cell wall. This integrated organization provides a compact structure that shields the bacterium against environmental stresses. The fundamental unit of this surface layer (S-layer) is the S-layer deinoxanthin-binding complex (SDBC), which binds the carotenoid deinoxanthin and provides both, thermostability and UV radiation resistance. However, the structural organization of the SDBC awaits elucidation. Here, we report the isolation of the SDBC with a gentle procedure consisting of lysozyme treatment and solubilization with the nonionic detergent -dodecyl-β-d-maltoside, which preserved both hydrophilic and hydrophobic components of the SDBC and allows the retention of several minor subunits. As observed by low-resolution single-particle analysis, we show that the complex possesses a porin-like structural organization, but is larger than other known porins. We also noted that the main SDBC component, the protein DR_2577, shares regions of similarity with known porins. Moreover, results from electrophysiological assays with membrane-reconstituted SDBC disclosed that it is a nonselective channel that has some peculiar gating properties, but also exhibits behavior typically observed in pore-forming proteins, such as porins and ionic transporters. The functional properties of this system and its porin-like organization provide information critical for understanding ion permeability through the outer cell surface of S-layer-carrying bacterial species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA119.012174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105295PMC
March 2020

Electrophysiological Characterization of Transport Across Outer-Membrane Channels from Gram-Negative Bacteria in Presence of Lipopolysaccharides.

Angew Chem Int Ed Engl 2020 05 24;59(22):8517-8521. Epub 2020 Mar 24.

Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759, Bremen, Germany.

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we present an approach using fusion of native outer membrane vesicles (OMVs) into a planar lipid bilayer, allowing characterization of membrane protein channels in their native environment. Two major membrane channels from E. coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion surprisingly revealed only single or few channel activities. The asymmetry of the OMVs translates after fusion into the lipid membrane with the lipopolysaccharides (LPS) dominantly present at the side of OMV addition. Compared to the conventional reconstitution method, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution and significantly lower permeation. We suggest using outer membrane vesicles for functional and structural studies of membrane channels in the native membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201913618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317717PMC
May 2020

Manipulation of charge distribution in the arginine and glutamate clusters of the OmpG pore alters sugar specificity and ion selectivity.

Biochim Biophys Acta Biomembr 2019 10 13;1861(10):183021. Epub 2019 Jul 13.

Division of Biochemistry and Applied Protein Center Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, D-91058 Erlangen, Germany; Department Biology, Division of Plant Physiology, Philipps-University Marburg, D-35043 Marburg, Germany. Electronic address:

OmpG is a general diffusion pore in the E. coli outer membrane with a molecular architecture comprising a 14-stranded β-barrel scaffold and unique structural features. In contrast to other non-specific porins, OmpG lacks a central constriction zone and has an exceptionally wide pore diameter of about 13 Å. The equatorial plane of OmpG harbors an annulus of four alternating basic and acidic patches whose function is only poorly characterized. We have investigated the role of charge distribution for ion selectivity and sugar transport with the help of OmpG variants mutated in the annulus. Substituting the glutamate residues of the annulus for histidines or alanines led to a strong reduction in cation selectivity. Replacement of the glutamates in the annulus by histidine residues also disfavored the passage of pentoses and hexoses relative to disaccharides. Our results demonstrate that despite the wide pore diameter, an annulus only consisting of two opposing basic patches confers reduced cation and monosaccharide transport compared to OmpG wild type. Furthermore, randomization of charged residues in the annulus had the potential to abolish pH-dependency of sugar transport. Our results indicate that E, E, R, R and R in the annulus form electrostatic interactions with R, E and D in loop L6 that influence pH-dependency of sugar transport.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamem.2019.07.009DOI Listing
October 2019

Small-Molecule Permeation across Membrane Channels: Chemical Modification to Quantify Transport across OmpF.

Angew Chem Int Ed Engl 2019 03 27;58(14):4737-4741. Epub 2019 Feb 27.

Department of Life Sciences and Chemistry, Jacobs University, Campus Ring 1, 28759, Bremen, Germany.

Biological channels facilitate the exchange of molecules across membranes, but general tools to quantify transport are missing. Electrophysiology is the method of choice to study the functional properties of channels. However, analyzing the current fluctuation of channels typically does not identify successful transport, that is, distinguishing translocation from binding. To distinguish both processes, we added an additional barrier at the channel exit acting as a molecular counter. To identify permeation, we compare the molecule residence time in the native channel with one that is chemically modified at the exit. We use the well-studied outer membrane channel from E. coli, OmpF. Position 181, which is below the constriction region, was subsequently mutated into cysteine (E181C) in an otherwise cysteine-free system, then functionalized by covalent binding with one of the two blockers MTSES or GLT. We measured the passage of model peptides, mono-, tri-, hepta-arginine and of norfloxacin, as an example for antibiotic permeation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201814489DOI Listing
March 2019

Fosfomycin Permeation through the Outer Membrane Porin OmpF.

Biophys J 2019 01 8;116(2):258-269. Epub 2018 Dec 8.

Department of Physics and Earth Sciences, Jacobs University Bremen, Bremen, Germany. Electronic address:

Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2018.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350074PMC
January 2019

Ampicillin permeation across OmpF, the major outer-membrane channel in .

J Biol Chem 2018 05 14;293(18):7030-7037. Epub 2018 Mar 14.

From the Department of Life Sciences and Chemistry, Jacobs University Bremen, 28719 Bremen, Germany

The outer cell wall of the Gram-negative bacteria is a crucial barrier for antibiotics to reach their target. Here, we show that the chemical stability of the widely used antibiotic ampicillin is a major factor in the permeation across OmpF to reach the target in the periplasm. Using planar lipid bilayers we investigated the interactions and permeation of OmpF with ampicillin, its basic pH-induced primary degradation product (penicilloic acid), and the chemically more stable benzylpenicillin. We found that the solute-induced ion current fluctuation is 10 times higher with penicilloic acid than with ampicillin. Furthermore, we also found that ampicillin can easily permeate through OmpF, at an ampicillin gradient of 10 μm and a conductance of ≅ 3.8 fS, with a flux rate of roughly 237 molecules/s of ampicillin at = 10 mV. The structurally related benzylpenicillin yields a lower conductance of ≅ 2 fS, corresponding to a flux rate of ≈120 molecules/s. In contrast, the similar sized penicilloic acid was nearly unable to permeate through OmpF. MD calculations show that, besides their charge difference, the main differences between ampicillin and penicilloic acid are the shape of the molecules, and the strength and direction of the dipole vector. Our results show that OmpF can impose selective permeation on similar sized molecules based on their structure and their dipolar properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA117.000705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936826PMC
May 2018