Single-molecule sensing inside stereo- and regio-defined hetero-nanopores.

Heteromeric pore-forming proteins often contain recognition patterns or stereospecific selection filters. However, the construction of heteromeric pore-forming proteins for single-molecule sensing is challenging due to the uncontrollability of producing position isomers and difficulties in purification of regio-defined products. To overcome these preparation obstacles, we present an in situ strategy involving single-molecule chemical modification of a heptameric pore-forming protein to build a stereo- and regio-specific heteromeric nanopore (hetero-nanopore) with a subunit stoichiometric ratio of 3:4. The steric hindrance inherent in the homo-nanopore of K238C aerolysin directs the stereo- and regio-selective modification of maleimide derivatives. Our method utilizes real-time ionic current recording to facilitate controlled voltage manipulation for stoichiometric modification and position-based side-isomer removal. Single-molecule experiments and all-atom molecular dynamics simulations revealed that the hetero-nanopore features an asymmetric stereo- and regio-defined residue structure. The hetero-nanopore produced was characterized by mass spectrometry and single-particle cryogenic electron microscopy. In a proof-of-concept single-molecule sensing experiment, the hetero-nanopore exhibited 95% accuracy for label-free discrimination of four peptide stereoisomers with single-amino-acid structural and chiral differences in the mixtures. The customized hetero-nanopores could advance single-molecule sensing.

Conformational flexibility driving charge-selective substrate translocation across a bacterial transporter.

Bacterial membrane porins facilitate the translocation of small molecules while restricting large molecules, and this mechanism remains elusive at the molecular level. Here, we investigate the selective uptake of large cyclic sugars across an unusual passive membrane transporter, CymA, comprising a charged zone and a constricting N terminus segment. Using a combination of electrical recordings, protein mutagenesis and molecular dynamics simulations, we establish substrate translocation across CymA governed by the electrostatic pore properties and conformational dynamics of the constriction segment. Notably, we show that the variation in pH of the environment resulted in reversible modulation of the substrate binding site in the pore, thereby regulating charge-selective transport of cationic, anionic and neutral cyclic sugars. The quantitative kinetics of cyclic sugar translocation across CymA obtained in electrical recordings at different pHs are comparable with molecular dynamics simulations that revealed the transport pathway, energetics and favorable affinity sites in the pore for substrate binding. We further define the molecular basis of cyclic sugar translocation and establish that the constriction segment is flexible and can reside inside or outside the pore, regulating substrate translocation distinct from the ligand-gated transport mechanism. Our study provides novel insights into energy-independent large molecular membrane transport for targeted drug design strategies.

Fosfomycin Uptake in Escherichia coli Is Mediated by the Outer-Membrane Porins OmpF, OmpC, and LamB.

The antibiotic fosfomycin (FOS) is widely recognized for the treatment of lower urinary tract infections with Escherichia coli and has lately gained importance as a therapeutic option to combat multidrug-resistant bacteria. However, resistance to FOS frequently develops through mutations reducing its uptake. Although the inner-membrane transport of FOS has been extensively studied in E. coli, its outer-membrane (OM) transport remains insufficiently understood. While evaluating minimal inhibitory concentrations in OM porin-deficient mutants, we observed that the E. coli ΔompFΔompC strain is four times more resistant to FOS than the wild type and the respective single mutants. Continuous monitoring of FOS-induced lysis of porin-deficient strains additionally highlighted the importance of LamB. The relevance of OmpF, OmpC, and LamB to FOS uptake was confirmed by electrophysiological and transcriptional analysis. Our study gives for the first time in-depth insight into the transport of FOS through the OM in E. coli.

Metallacarborane Cluster Anions of the Cobalt Bisdicarbollide-Type as Chaotropic Carriers for Transmembrane and Intracellular Delivery of Cationic Peptides.

Cobalt bisdicarbollides (COSANs) are inorganic boron-based anions that have been previously reported to permeate by themselves through lipid bilayer membranes, a propensity that is related to their superchaotropic character. We now introduce their use as selective and efficient molecular carriers of otherwise impermeable hydrophilic oligopeptides through both artificial and cellular membranes, without causing membrane lysis or poration at low micromolar carrier concentrations. COSANs transport not only arginine-rich but also lysine-rich peptides, whereas low-molecular-weight analytes such as amino acids as well as neutral and anionic cargos (phalloidin and BSA) are not transported. In addition to the unsubstituted isomers (known as ortho- and meta-COSAN), four derivatives bearing organic substituents or halogen atoms have been evaluated, and all six of them surpass established carriers such as pyrenebutyrate in terms of activity. U-tube experiments and black lipid membrane conductance measurements establish that the transport across model membranes is mediated by a molecular carrier mechanism. Transport experiments in living cells showed that a fluorescent peptide cargo, FITC-Arg8, is delivered into the cytosol.

Influence of Membrane Asymmetry on OmpF Insertion, Orientation and Function.

The effect of asymmetric membranes containing lipopolysaccharides (LPS) on the outer membrane protein F (OmpF) reconstitution, channel orientation, and antibiotic permeation across the outer membrane was investigated. After forming an asymmetric planar lipid bilayer composed of LPS on one and phospholipids on the other side, the membrane channel OmpF was added. The ion current recordings demonstrate that LPS has a strong influence on the OmpF membrane insertion, orientation, and gating. Enrofloxacin was used as an example of an antibiotic interacting with the asymmetric membrane and with OmpF. The enrofloxacin caused the blockage of the ion current through the OmpF, depending on the side of addition, the transmembrane voltage applied, and the composition of the buffer. Furthermore, the enrofloxacin changed the phase behavior of the LPS-containing membranes, demonstrating that its membrane activity influences the function of OmpF and potentially the membrane permeability.

How the physical properties of bacterial porins match environmental conditions.

Transmembrane ß-barrel proteins are key systems for transport phenomena in biology. Based on their broad substrate specificity, they represent good candidates for present and future technological applications, such as DNA/RNA and protein sequencing, sensing of biomedical analytes, and production of blue energy. For a better understanding of the process at the molecular level, we applied parallel tempering simulations in the WTE ensemble to compare two ß-barrel porins from Escherichia coli, OmpF and OmpC. Our analysis showed a different behavior of the two highly homologous porins, where subtle amino acid substitutions can modulate critical properties of mass transport. Interestingly, the differences can be mapped to the respective environmental conditions under which the two porins are expressed. Apart from reporting on the advantages of the enhanced sampling methods in assessing the molecular properties of nanopores, our comparative analysis provided new and key results to better understand biological function and technical applications. Eventually, we showed how results from molecular simulations align well with experimental single-channel measurements, thus demonstrating the mature evolution of numerical methodologies for predicting properties in this field crucial for future biomedical applications.

Editorial: Nanopore Electrochemistry.

This Special Collection highlights the most recent developments in nanopore electrochemistry and applications. In this Editorial, guest Editors Yi-Tao Long, Meni Wanunu, and Mathias Winterhalter briefly introduce the research published in this special collection.

TolC-AcrA complex formation monitored by time dependent single-channel electrophysiology.

Characterizing protein-protein interaction on a single molecular level is a challenge, experimentally as well as interpretation of the data. For example, Gram-negative bacteria contain protein complexes spanning the outer and inner cell wall devoted to efflux effectively cell toxic substances. Recent seminal work revealed the high-resolution structure of such a tripartic composition TolC-AcrA-AcrB suggesting to design inhibitors preventing efflux of antibiotics. To show that electrophysiology can provide supporting information here, we reconstitute single TolC homotrimer into a planar lipid membrane, apply a transmembrane voltage and follow the assembly of AcrA to TolC using the modulation of the ion current through TolC channel during binding. In particular, the presence of AcrA in solution increases the average ionic current through TolC and, moreover, reduces the ion-current fluctuations caused by flickering of TolC. Here, we show that statistical properties of ion-current fluctuations (the power spectral density) provide a complementary measure of the interaction of the TolC-AcrA complex in presence of putative efflux pump inhibitors. Both characteristics, the average ion current across TolC and the current noise, taken into consideration together, point to a stiffening of the tip of TolC which might reduce the formation of the complex.

Conformational Dynamics of Loop L3 in OmpF: Implications toward Antibiotic Translocation and Voltage Gating.

In the present work, we delineate the molecular mechanism of a bulky antibiotic permeating through a bacterial channel and uncover the role of conformational dynamics of the constriction loop in this process. Using the temperature accelerated sliced sampling approach, we shed light onto the dynamics of the L3 loop, in particular the F118 to S125 segment, at the constriction regions of the OmpF porin. We complement the findings with single channel electrophysiology experiments and applied-field simulations, and we demonstrate the role of hydrogen-bond stabilization in the conformational dynamics of the L3 loop. A molecular mechanism of permeation is put forward wherein charged antibiotics perturb the network of stabilizing hydrogen-bond interactions and induce conformational changes in the L3 segment, thereby aiding the accommodation and permeation of bulky antibiotic molecules across the constriction region. We complement the findings with single channel electrophysiology experiments and demonstrate the importance of the hydrogen-bond stabilization in the conformational dynamics of the L3 loop. The generality of the present observations and experimental results regarding the L3 dynamics enables us to identify this L3 segment as the source of gating. We propose a mechanism of OmpF gating that is in agreement with previous experimental data that showed the noninfluence of cysteine double mutants that tethered the L3 tip to the barrel wall on the OmpF gating behavior. The presence of similar loop stabilization networks in porins of other clinically relevant pathogens suggests that the conformational dynamics of the constriction loop is possibly of general importance in the context of antibiotic permeation through porins.

Cephalosporin translocation across enterobacterial OmpF and OmpC channels, a filter across the outer membrane.

Gram-negative porins are the main entry for small hydrophilic molecules. We studied translocation of structurally related cephalosporins, ceftazidime (CAZ), cefotaxime (CTX) and cefepime (FEP). CAZ is highly active on E. coli producing OmpF (Outer membrane protein F) but less efficient on cells expressing OmpC (Outer membrane protein C), whereas FEP and CTX kill bacteria regardless of the porin expressed. This matches with the different capacity of CAZ and FEP to accumulate into bacterial cells as quantified by LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry). Furthermore, porin reconstitution into planar lipid bilayer and zero current assays suggest permeation of ≈1,000 molecules of CAZ per sec and per channel through OmpF versus ≈500 through OmpC. Here, the instant killing is directly correlated to internal drug concentration. We propose that the net negative charge of CAZ represents a key advantage for permeation through OmpF porins that are less cation-selective than OmpC. These data could explain the decreased susceptibility to some cephalosporins of enterobacteria that exclusively express OmpC porins.

Identification of Single Amino Acid Chiral and Positional Isomers Using an Electrostatically Asymmetric Nanopore.

Chirality is essential in nearly all biological organizations and chemical reactions but is rarely considered due to technical limitations in identifying L/D isomerization. Using OmpF, a membrane channel from Escherichia coli with an electrostatically asymmetric constriction zone, allows discriminating chiral amino acids in a single peptide. The heterogeneous distribution of charged residues in OmpF causes a strong lateral electrostatic field at the constriction. This laterally asymmetric constriction zone forces the sidechains of the peptides to specific orientations within OmpF, causing distinct ionic current fluctuations. Using statistical analysis of the respective ionic current variations allows distinguishing the presence and position of a single amino acid with different chiralities. To explore potential applications, the disease-related peptide ß-Amyloid and its d-Asp1 isoform and a mixture of the icatibant peptide drug (HOE 140) and its d-Ser7 mutant have been discriminated. Both chiral isomers were not applicable to be distinguished by mass spectroscopy approaches. These findings highlight a novel sensing mechanism for identifying single amino acids in single peptides and even for achieving single-molecule protein sequencing.

The cryo-EM structure of the S-layer deinoxanthin-binding complex of Deinococcus radiodurans informs properties of its environmental interactions.

The radiation-resistant bacterium Deinococcus radiodurans is known as the world's toughest bacterium. The S-layer of D. radiodurans, consisting of several proteins on the surface of the cellular envelope and intimately associated with the outer membrane, has therefore been useful as a model for structural and functional studies. Its main proteinaceous unit, the S-layer deinoxanthin-binding complex (SDBC), is a hetero-oligomeric assembly known to contribute to the resistance against environmental stress and have porin functional features; however, its precise structure is unknown. Here, we resolved the structure of the SDBC at ∼2.5 Å resolution by cryo-EM and assigned the sequence of its main subunit, the protein DR_2577. This structure is characterized by a pore region, a massive ß-barrel organization, a stalk region consisting of a trimeric coiled coil, and a collar region at the base of the stalk. We show that each monomer binds three Cu ions and one Fe ion and retains one deinoxanthin molecule and two phosphoglycolipids, all exclusive to D. radiodurans. Finally, electrophysiological characterization of the SDBC shows that it exhibits transport properties with several amino acids. Taken together, these results highlight the SDBC as a robust structure displaying both protection and sieving functions that facilitates exchanges with the environment.

Changes in Salt Concentration Modify the Translocation of Neutral Molecules through a ΔCymA Nanopore in a Non-monotonic Manner.

The voltage-dependent transport through biological and artificial nanopores is being used in many applications such as DNA or protein sequencing and sensing. The primary approach to determine the transport has been to measure the temporal ion current fluctuations caused by solutes when applying external voltages. Crossing the nanoscale confinement in the presence of an applied electric field primarily relies on two factors, i.e., the electrophoretic drag and electroosmosis. The electroosmotic flow (EOF) is a voltage-dependent ion-associated flow of solvent molecules, i.e., usually water, and depends on many factors, such as pH, temperature, pore diameter, and also the concentration of ions. The exact interplay between these factors is so far poorly understood. In this joint experimental and computational study, we have investigated the dependence of the EOF on the concentration of the buffer salt by probing the transport of α-cyclodextrin molecules through the ΔCymA channel. For five different KCl concentrations in the range between 0.125 and 2 M, we performed applied-field molecular dynamics simulations and analyzed the ionic flow and the EOF across the ΔCymA pore. To our surprise, the concentration-dependent net ionic flux changes non-monotonically and nonlinearly and the EOF is seen to follow the same pattern. On the basis of these findings, we were able to correlate the concentration-dependent EOF with experimental kinetic constants for the translocation of α-cyclodextrin through the ΔCymA nanopore. Overall, the results further improve our understanding of the EOF-mediated transport through nanopores and show that the EOF needs to seriously be taken into consideration when analyzing the permeation of (neutral) substrates through nanopores.

Silicon Nitride-Based Micro-Apertures Coated with Parylene for the Investigation of Pore Proteins Fused in Free-Standing Lipid Bilayers.

In this work, we present a microsystem setup for performing sensitive biological membrane translocation measurements. Thin free-standing synthetic bilayer lipid membranes (BLM) were constructed in microfabricated silicon nitride apertures (<100 µm in diameter), conformal coated with Parylene (Parylene-C or Parylene-AF4). Within these BLMs, electrophysiological measurements were conducted to monitor the behavior of different pore proteins. Two approaches to integrate pore-forming proteins into the membrane were applied: direct reconstitution and reconstitution via outer membrane vesicles (OMVs) released from Gram-negative bacteria. The advantage of utilizing OMVs is that the pore proteins remain in their native lipid and lipopolysaccharide (LPS) environment, representing a more natural state compared to the usage of fused purified pore proteins. Multiple aperture chips can be easily assembled in the 3d-printed holder to conduct parallel membrane transport investigations. Moreover, well defined microfabricated apertures are achievable with very high reproducibility. The presented microsystem allows the investigation of fast gating events (down to 1 ms), pore blocking by an antibiotic, and gating events of small pores (amplitude of approx. 3 pA).

The C2 entity of chitosugars is crucial in molecular selectivity of the Vibrio campbellii chitoporin.

The marine bacterium Vibrio campbellii expresses a chitooligosaccharide-specific outer-membrane channel (chitoporin) for the efficient uptake of nutritional chitosugars that are externally produced through enzymic degradation of environmental host shell chitin. However, the principles behind the distinct substrate selectivity of chitoporins are unclear. Here, we employed black lipid membrane (BLM) electrophysiology, which handles the measurement of the flow of ionic current through porins in phospholipid bilayers for the assessment of porin conductivities, to investigate the pH dependency of chitosugar-chitoporin interactions for the bacterium's natural substrate chitohexaose and its deacetylated form, chitosan hexaose. We show that efficient passage of the N-acetylated chitohexaose through the chitoporin is facilitated by its strong affinity for the pore. In contrast, the deacetylated chitosan hexaose is impermeant; however, protonation of the C2 amino entities of chitosan hexaose allows it to be pulled through the channel in the presence of a transmembrane electric field. We concluded from this the crucial role of C2-substitution as the determining factor for chitoporin entry. A change from N-acetylamino- to amino-substitution effectively abolished the ability of approaching molecules to enter the chitoporin, with deacetylation leading to loss of the distinctive structural features of nanopore opening and pore access of chitosugars. These findings provide further understanding of the multistep pathway of chitin utilization by marine Vibrio bacteria and may guide the development of solid-state or genetically engineered biological nanopores for relevant technological applications.

A general approach to protein folding using thermostable exoshells.

In vitro protein folding is a complex process which often results in protein aggregation, low yields and low specific activity. Here we report the use of nanoscale exoshells (tES) to provide complementary nanoenvironments for the folding and release of 12 highly diverse protein substrates ranging from small protein toxins to human albumin, a dimeric protein (alkaline phosphatase), a trimeric ion channel (Omp2a) and the tetrameric tumor suppressor, p53. These proteins represent a unique diversity in size, volume, disulfide linkages, isoelectric point and multi versus monomeric nature of their functional units. Protein encapsulation within tES increased crude soluble yield (3-fold to >100-fold), functional yield (2-fold to >100-fold) and specific activity (3-fold to >100-fold) for all the proteins tested. The average soluble yield was 6.5 mg/100 mg of tES with charge complementation between the tES internal cavity and the protein substrate being the primary determinant of functional folding. Our results confirm the importance of nanoscale electrostatic effects and provide a solution for folding proteins in vitro.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Structural analysis of the architecture and in situ localization of the main S-layer complex in Deinococcus radiodurans.

Bacterial surface layers are paracrystalline assemblies of proteins that provide the first line of defense against environmental shocks. Here, we report the 3D structure, in situ localization, and orientation of the S-layer deinoxanthin-binding complex (SDBC), a hetero-oligomeric assembly of proteins that in Deinococcus radiodurans represents the main S-layer unit. The SDBC is resolved at 11-Å resolution by single-particle analysis, while its in situ localization is determined by cryo-electron crystallography on intact cell-wall fragments leading to a projection map at 4.5-Å resolution. The SDBC exhibits a triangular base with three comma-shaped pores, and a stalk departing orthogonally from the center of the base and oriented toward the intracellular space. Combining state-of-the-art techniques, results show the organization of this S-layer and its connection within the underlying membranes, demonstrating the potential for applications from nanotechnologies to medicine.

How to Enter a Bacterium: Bacterial Porins and the Permeation of 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.

Large-Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca*.

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter-pair-based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate-limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times, in agreement with the liposome study. Applied-field and steered molecular dynamics simulations added an atomistic view of the permeation events. It can be concluded that a concentration gradient of 1 µm Ptm leads to a translocation rate of about one molecule per second and per channel.