Functionally Active Synthetic α-Helical Pores.

ConspectusTransmembrane pores are currently at the forefront of nanobiotechnology, nanopore chemistry, and synthetic chemical biology research. Over the past few decades, significant studies in protein engineering have paved the way for redesigning membrane protein pores tailored for specific applications in nanobiotechnology. Most previous efforts predominantly centered on natural ß-barrel pores designed with atomic precision for nucleic acid sequencing and sensing of biomacromolecules, including protein fragments. The requirement for a more efficient single-molecule detection system has driven the development of synthetic nanopores. For example, engineering channels to conduct ions and biomolecules selectively could lead to sophisticated nanopore sensors. Also, there has been an increased interest in synthetic pores, which can be fabricated to provide more control in designing architecture and diameter for single-molecule sensing of complex biomacromolecules. There have been impressive advancements in developing synthetic DNA-based pores, although their application in nanopore technology is limited. This has prompted a significant shift toward building synthetic transmembrane α-helical pores, a relatively underexplored field offering novel opportunities. Recently, computational tools have been employed to design and construct α-helical barrels of defined structure and functionality.We focus on building synthetic α-helical pores using naturally occurring transmembrane motifs of membrane protein pores. Our laboratory has developed synthetic α-helical transmembrane pores based on the natural porin PorACj (Porin A derived from Corynebacterium jeikeium) that function as nanopore sensors for single-molecule sensing of cationic cyclodextrins and polypeptides. Our breakthrough lies in being the first to create a functional and large stable synthetic transmembrane pore composed of short synthetic α-helical peptides. The key highlight of our work is that these pores can be synthesized using easy chemical synthesis, which permits its easy modification to include a variety of functional groups to build charge-selective sophisticated pores. Additionally, we have demonstrated that stable functional pores can be constructed from D-amino acid peptides. The analysis of pores composed of D- and L-amino acids in the presence of protease showed that only the D pores are highly functional and stable. The structural models of these pores revealed distinct surface charge conformation and geometry. These new classes of synthetic α-helical pores are highly original systems of general interest due to their unique architecture, functionality, and potential applications in nanopore technology and chemical biology. We emphasize that these simplified transmembrane pores have the potential to be components of functional nanodevices and therapeutic tools. We also suggest that such designed peptides might be valuable as antimicrobial agents and can be targeted to cancer cells. This article will focus on the evolutions in assembling α-helical transmembrane pores and highlight their advantages, including structural and functional versatility.

Sensing PEGylated Peptide Conformations Using a Protein Nanopore.

Membrane pores are exploited for the stochastic sensing of various analytes, and here, we use electrical recordings to explore the interaction of PEGylated peptides of different sizes with a protein pore, CymA. This wide-diameter natural pore comprises densely filled charged residues, facilitating electrophoretic binding of polyethylene glycol (PEG) tagged with a nonaarginine peptide. The small PEG 200 peptide conjugates produced monodisperse blockages and exhibited voltage-dependent translocation across the pores. Notably, the larger PEG 1000 and 2000 peptide conjugates yielded heterogeneous blockages, indicating a multitude of PEG conformations hindering their translocation through the pore. Furthermore, a much larger PEG 5000 peptide occludes the pore entrance, resulting in complete closure. The competitive binding of different PEGylated peptides with the same pore produced specific blockage signals reflecting their identity, size, and conformation. Our proposed model of sensing distinct polypeptide conformations corresponds to disordered protein unfolding, suggesting that this pore can find applications in proteomics.

Membrane Disruptive Action of Cationic Antibacterial Peptide B1CTcu3.

A twenty-two-residue peptide Brevinin1 Clinotarsus curtipus-3 (B1CTcu3), identified from the skin secretion of frog Clinotarsus curtipes of the Western Ghats, exhibited a broad range of antibacterial activity against Gram-negative and Gram-positive bacteria, including the methicillin-resistant Staphylococcus aureus (MRSA). It showed anti-biofilm activity even at sub-minimum inhibitory concentration (sub-MIC) against Pseudomonas aeruginosa and Staphylococcus aureus. Analysis of the scanning electron microscopic (SEM) images, confocal images, flow cytometric data and the effect of salt concentration on antibacterial potency suggests that the killing action of the peptide is through the membranolytic process. Single channel electric recording confirmed that the peptide elicited pores on the bacterial cell membrane as it induces a heterogeneous channel in the lipid bilayer. It also showed cytotoxicity against MDA-MB-231 breast cancer cell with IC50 of 25 µM. B1CTcu3 peptide could serve as the template for next-generation antibacterial agents, particularly against antibiotic resistant pathogenic bacteria.

Autonomously Assembled Synthetic Transmembrane Peptide Pore.

The porinACj is an α-helical porin that spans the mycolic acid outer membrane of Gram-positive mycolate, Corynebacterium jeikeium. Here, we report that a 40-amino acid, synthetic peptide, pPorA corresponding to porin PorACj, inserts into the lipid bilayers and forms well-defined pores. By electrical recordings, we measured the single-channel properties that revealed the autonomous assembly of large conductance ion-selective synthetic pores. Further, we characterized the functional properties by blocking the peptide pores by cyclodextrins of different charge and symmetry. We deduced the subunit stoichiometry and putative structure of the pore by site-specific chemical modification in single-channel electrical recordings and gel electrophoresis. On the basis of these findings, we suggest that this is a large functional uniform transmembrane pore built entirely from short synthetic α-helical peptides. Accordingly, we propose a model demonstrating structural assembly of large α-helix-based peptide pores for understanding the action of antimicrobial peptides and for the design of pores with applications in biotechnology.

Controlling Interactions of Cyclic Oligosaccharides with Hetero-Oligomeric Nanopores: Kinetics of Binding and Release at the Single-Molecule Level.

Controlling the molecular interactions through protein nanopores is crucial for effectively detecting single molecules. Here, the development of a hetero-oligomeric nanopore derived from Nocardia farcinica porin AB (NfpAB) is discussed for single-molecule sensing of biopolymers. Using single-channel recording, the interaction of cyclic oligosaccharides such as cationic cyclodextrins (CDs) of different symmetries and charges with NfpAB is measured. Studies of the transport kinetics of CDs reveal asymmetric geometry and charge distribution of NfpAB. The applied potential promotes the attachment of the cationic CDs to the negatively charged pore surface due to electrostatic interaction. Further, the attached CDs are released from the pore by reversing the applied potential in time-resolved blockages. Release of CDs from the pore depends on its charge, size, and magnitude of the applied potential. The kinetics of CD attachment and release is controlled by fine-tuning the applied potential demonstrating the successful molecular transport across these nanopores. It is suggested that such controlled molecular interactions with protein nanopores using organic templates can be useful for several applications in nanopore technology and single-molecule chemistry.

Transport across the outer membrane porin of mycolic acid containing actinomycetales: Nocardia farcinica.

The role of the outer-membrane channel from a mycolic acid containing Gram-positive bacteria Nocardia farcinica, which forms a hydrophilic pathway across the cell wall, was characterized. Single channel electrophysiology measurements and liposome swelling assays revealed the permeation of hydrophilic solutes including sugars, amino acids and antibiotics. The cation selective N. farcinica channel exhibited strong interaction with the positively charged antibiotics; amikacin and kanamycin, and surprisingly also with the negatively charged ertapenem. Voltage dependent kinetics of amikacin and kanamycin interactions were studied to distinguish binding from translocation. Moreover, the importance of charged residues inside the channel was investigated using mutational studies that revealed rate limiting interactions during the permeation.

Chitoporin from Vibrio harveyi, a channel with exceptional sugar specificity.

Chitoporin (VhChiP) is a sugar-specific channel responsible for the transport of chitooligosaccharides through the outer membrane of the marine bacterium Vibrio harveyi. Single channel reconstitution into black lipid membrane allowed single chitosugar binding events in the channel to be resolved. VhChiP has an exceptionally high substrate affinity, with a binding constant of K = 5.0 × 10(6) M(-1) for its best substrate (chitohexaose). The on-rates of chitosugars depend on applied voltages, as well as the side of the sugar addition, clearly indicating the inherent asymmetry of the VhChiP lumen. The binding affinity of VhChiP for chitohexaose is 1-5 orders of magnitude larger than that of other known sugar-specific porins for their preferred substrates. Thus, VhChiP is the most potent sugar-specific channel reported to date, with its high efficiency presumably reflecting the need for the bacterium to take up chitin-containing nutrients promptly under turbulent aquatic conditions to exploit them efficiently as its sole source of energy.

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.

Structural and mechanistic insights into Quinolone Synthase to address its functional promiscuity.

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS's steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes.

Differential detergent extraction of mycobacterium marinum cell envelope proteins identifies an extensively modified threonine-rich outer membrane protein with channel activity.

A striking characteristic of mycobacteria is the presence of an unusual outer membrane which forms a thick permeability barrier and provides resistance to many antibiotics. Although specialized proteins must reside in this layer, only few mycolate outer membrane (MOM) proteins have been identified to date. Their discovery is complicated by difficulties in obtaining good separation of mycobacterial inner and outer membranes. During our efforts to identify novel mycobacterial outer membrane proteins (MOMPs), we discovered that we can enrich for MOMPs using differential solubilization of mycobacterial cell envelopes. Subsequently, these different fractions were analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). This proteomic analysis confirmed that our marker proteins for inner membrane and MOM were found in their expected fractions and revealed a few interesting candidate MOMPs. A number of these putative MOMPs were further analyzed for their expression and localization in the cell envelope. One identified MOMP, MMAR_0617 of Mycobacterium marinum, was purified and demonstrated to form a large oligomeric complex. Importantly, this protein showed a clear single-channel conductance of 0.8 ± 0.1 ns upon reconstitution into artificial planar lipid bilayers. The most surprising feature of MMAR_0617 is a long C-terminal threonine-rich domain with extensive modifications. In summary, we have identified a novel mycobacterial outer membrane porin with unusual properties.

Peptide translocation through the mesoscopic channel: binding kinetics at the single molecule level.

Single channel electrophysiological studies have been carried out to elucidate the underlying interactions during the translocation of polypeptides through protein channels. For this we used OmpF from the outer cell membrane of E. coli and arginine-based peptides of different charges, lengths and covalently linked polyethylene glycol as a model system. In order to reveal the fast kinetics of peptide binding, we performed a temperature scan. Together with the voltage-dependent single-channel conductance, we quantify peptide binding and translocation.

Synthetic α-Helical Nanopore Reactor for Chemical Sensing.

The use of nanopores for the single-molecule sensing of folded proteins and biomacromolecules has recently gained attention. Here, we introduce a simplified synthetic α-helical transmembrane pore, pPorA, as a nanoreactor and sensor that exhibits functional versatility comparable to that of engineered protein and DNA nanopores. The pore, built from the assembly of synthetic 40-amino-acid-long peptides, is designed to contain cysteine residues within the lumen and at the pore terminus for site-specific chemical modification probed using single-channel electrical recordings. The reaction of the pore with differently charged activated thiol reagents was studied, wherein positively charged reagents electrophoretically driven into the pore resulted in pore blocking in discrete steps upon covalent bond formation. The asymmetric blockage patterns resulting from cis and trans-side addition of reagents reveal the pore orientation in the lipid membrane. Furthermore, activated PEG thiols covalently blocked the pores over a longer duration in a charge-independent manner, establishing the large diameter and orientation of the formed pores. While the covalent binding of thiol reagents caused a drop in the pore conductance, cationic cyclic octasaccharides produced time-resolved translocation events, confirming the structural flexibility and tunability of the pores. The ability of the pore to accommodate large analytes and the considerable current amplitude variation following bond formation events are promising for developing platforms to resolve multistep chemical reactions at the single-molecule level for applications in synthetic nanobiotechnology.

Protein translocation through Tom40: kinetics of peptide release.

Mitochondrial proteins are almost exclusively imported into mitochondria from the cytosol in an unfolded or partially folded conformation. Regardless of whether they are destined for the outer or inner membrane, the intermembrane space, or the matrix, proteins begin the importation process by crossing the mitochondrial outer membrane via a specialized protein import machinery whose main component is the Tom40 channel. High-resolution ion conductance measurements through the Tom40 channel in the presence of the mitochondrial presequence peptide pF(1)ß revealed the kinetics of peptide binding. Here we show that the rates for association k(on) and dissociation k(off) strongly depend on the applied transmembrane voltage. Both kinetic constants increase with an increase in the applied voltage. The increase of k(off) with voltage provides strong evidence of peptide translocation. This allows us to distinguish quantitatively between substrate blocking and permeation.

Antibiotic uptake through membrane channels: role of Providencia stuartii OmpPst1 porin in carbapenem resistance.

The role of major porin OmpPst1 of Providencia stuartii in antibiotic susceptibility for two carbapenems is investigated by combining high-resolution conductance measurements, liposome swelling, and microbiological assays. Reconstitution of a single OmpPst1 into a planar lipid bilayer and measuring the ion current, in the presence of imipenem, revealed a concentration-dependent decrease in conductance, whereas meropenem produced well-resolved short ion current blockages. Liposome swelling assays suggested a small flux of imipenem in contrast to a rapid permeation of meropenem. The lower antibiotic susceptibility of P. stuartii to imipenem compared to meropenem correlated well with the decreased level of permeation of the former through the OmpPst1 channel.

Molecular analysis of antimicrobial agent translocation through the membrane porin BpsOmp38 from an ultraresistant Burkholderia pseudomallei strain.

Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes melioidosis, an infectious disease of animals and humans common in northern and north-eastern parts of Thailand. Successful treatment of melioidosis is difficult due to intrinsic resistance of Bps to various antibacterial agents. It has been suggested that the antimicrobial resistance of this organism may result from poor permeability of the active compounds through porin channels located in the outer membrane (OM) of the bacterium. In previous work, a 38-kDa protein, named "BpsOmp38", was isolated from the OM of Bps. A topology prediction and liposome-swelling assay suggested that BpsOmp38 comprises a ß-barrel structure and acts as a general diffusion porin. The present study employed black lipid membrane (BLM) reconstitution to demonstrate the single-channel conductance of the trimeric BpsOmp38 to be 2.7±0.3 nS in 1M KCl. High-time resolution BLM measurements displayed ion current blockages of seven antimicrobial agents in a concentration-dependent manner with the translocation on-rate (kon) following the order: norfloxacin≫ertapenem>ceftazidime>cefepime>imipenem>meropenem>penicillin G. The dwell time of a selected antimicrobial agent (ertapenem) decayed exponentially with increasing temperature. The energy barrier for the ertapenem binding to the affinity site inside the BpsOmp38 channel was estimated from the Arrhenius plot to be 12 kT and for the ertapenem release to be 13 kT at +100 mV. The BLM data obtained from this study provide the first insight into antimicrobial agent translocation through the BpsOmp38 channel.

Simple reconstitution of protein pores in nano lipid bilayers.

We developed a new, simple and robust approach for rapid screening of single molecule interactions with protein channels. Our glass nanopipets can be fabricated simply by drawing glass capillaries in a standard pipet puller, in a matter of minutes, and do not require further modification before use. Giant unilamellar vesicles break when in contact with the tip of the glass pipet and form a supported bilayer with typical seal resistances of ∼140 GΩ, which is stable for hours and at applied potentials up to 900 mV. Bilayers can be formed, broken, and re-formed more than 50 times using the same pipet enabling rapid screening of bilayers for single protein channels. The stability of the lipid bilayer is significantly superior to that of traditionally built bilayers supported by Teflon membranes, particularly against perturbation by electrical and mechanical forces. We demonstrate the functional reconstitution of the E. coli porin OmpF and α-hemolysin in a glass nanopipet supported bilayer. Interactions of the antibiotic enrofloxacin with the OmpF channel have been studied at the single-molecule level, demonstrating the ability of this method to detect single molecule interactions with protein channels. High-resolution conductance measurements of protein channels can be performed with low sample and buffer consumption. Glass nanopipet supported bilayers are uniquely suited for single-molecule studies as they are more rigid and the lifetime of a stable membrane is on the scale of hours, closer to that of natural cell membranes.

Assembly of alpha-helical transmembrane pores through an intermediate state.

Pore-forming alpha-helical proteins are well known for their dynamic assembly mechanism and it has been challenging to delineate the pore-forming structures in membranes. Previously, attempts have been made to elucidate their assembly mechanism and there is a large gap due to complex pathways by which these membrane-active pores impart their effect. Here we demonstrate a multi-step structural assembly pathway of alpha-helical peptide pores formed by a 37 amino acid synthetic peptide, pPorU, based on the natural porin from Corynebacterium urealyticum using single-channel electrical recordings. More specifically, we report detectable intermediate states during the membrane insertion and pore formation of pPorU. The fully assembled pore exhibited unusually large stable conductance, voltage-dependent gating, and functional blockage by cyclic sugars generally applicable to a range of transmembrane pores. Furthermore, we used rationally designed mutants to understand the role of specific amino acids in the assembly of these peptide pores. Mutant peptides that differ from wild-type peptides produced noisy and unstable intermediate states and low conductance pores, demonstrating sequence specificity in the pore-formation process supported by molecular dynamics simulations. We suggest that our study contributes to understanding the mechanism of action of naturally occurring alpha-helical pore-forming proteins and should be of broad interest to build peptide-based nanopore sensors.

Electrostatic Filtering of Polypeptides Through Membrane Protein Pores.

Naturally-occurring membrane proteins have been engineered as nanopore sensors for the single-molecule detection of various biochemical molecules. Here, we present a natural bacterial porin, CymA containing a dynamic component and densely packed charged residues in the pore, shaping a unique structural conformation and charge feature. Using single-channel recordings, we investigated the translocation of charged polypeptides through native CymA and truncated CymA lacking the dynamic element. Cationic polypeptides bind to the pore with high affinity, specifically at low salt conditions indicating an electrostatic charge and voltage-dependent translocation. Anionic peptides did not bind to the pore, confirming the selective binding of polypeptides with the pore due to their specific charge distribution. Further, the distinct peptide translocation kinetics between native and truncated indicated the role of the dynamic segment in molecular transport. We suggest that these natural membrane pores that permit the selective translocation of cationic polypeptides are advantageous for nanopore proteomics applications.

Assembly of transmembrane pores from mirror-image peptides.

Tailored transmembrane alpha-helical pores with desired structural and functional versatility have promising applications in nanobiotechnology. Herein, we present a transmembrane pore DpPorA, based on the natural pore PorACj, built from D-amino acid α-helical peptides. Using single-channel current recordings, we show that DpPorA peptides self-assemble into uniform cation-selective pores in lipid membranes and exhibit properties distinct from their L-amino acid counterparts. DpPorA shows resistance to protease and acts as a functional nanopore sensor to detect cyclic sugars, polypeptides, and polymers. Fluorescence imaging reveals that DpPorA forms well-defined pores in giant unilamellar vesicles facilitating the transport of hydrophilic molecules. A second D-amino acid peptide based on the polysaccharide transporter Wza forms transient pores confirming sequence specificity in stable, functional pore formation. Finally, molecular dynamics simulations reveal the specific alpha-helical packing and surface charge conformation of the D-pores consistent with experimental observations. Our findings will aid the design of sophisticated pores for single-molecule sensing related technologies.