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.

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.

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.

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.

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.

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.

Selective Translocation of Cyclic Sugars through Dynamic Bacterial Transporter.

The selective translocation of molecules through membrane pores is an integral process in cells. We present a bacterial sugar transporter, CymA of unusual structural conformation due to a dynamic N terminus segment in the pore, reducing its diameter. We quantified the translocation kinetics of various cyclic sugars of different charge, size, and symmetry across native and truncated CymA devoid of the N terminus using single-channel recordings. The chemically divergent cyclic hexasaccharides bind to the native and truncated pore with high affinity and translocate effectively. Specifically, these sugars bind and translocate rapidly through truncated CymA compared to native CymA. In contrast, larger cyclic heptasaccharides and octasaccharides do not translocate but bind to native and truncated CymA with distinct binding kinetics highlighting the importance of molecular charge, size and symmetry in translocation consistent with liposome assays. Based on the sugar-binding kinetics, we suggest that the N terminus most likely resides inside the native CymA barrel, regulating the transport rate of cyclic sugars. Finally, we present native CymA as a large nanopore sensor for the simultaneous single-molecule detection of various sugars at high resolution, establishing its functional versatility. This natural pore is expected to have several applications in nanobiotechnology and will help further our understanding of the fundamental mechanism of molecular transport.

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.

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.

Constructing ion channels from water-soluble α-helical barrels.

The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide-lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.

Building Synthetic Transmembrane Peptide Pores.

Membrane protein pores have demonstrated applications in nanopore technology. Previous studies have mostly focused on ß-barrel protein pores, whereas α-helix-based transmembrane protein pores are rarely explored in nanopore applications. Here, we developed a synthetic transmembrane peptide pore built entirely from short synthetic α-helical peptides. We examined the formation of a stable uniform ion-selective pore in single-channel electrical recordings. Furthermore, we show that cyclodextrins (CDs) block the peptide pores and determine the kinetics of CD binding and translocation. We suggest that such designed synthetic transmembrane pores will be useful for several applications in biotechnology, including stochastic sensing.

Nanopore Passport Control for Substrate-Specific Translocation.

Membrane protein pores have demonstrated applications in nanobiotechnology and single-molecule chemistry for effective detection of biomolecules. Here, we define the molecular basis of carbohydrate polymers translocation through a substrate-specific bacterial nanopore, CymA, which has a 15-residue N terminus segment inside the pore, restricting its diameter. Using single-channel recordings, we determined the kinetics of cationic cyclic oligosaccharide binding and elucidated the translocation mechanism across the pore in real-time. The cationic cyclic hexasaccharide binds to the densely packed negatively charged residues at the extracellular side of the pore with high affinity, facilitating its entry into the pore driven by the applied voltage. Further, the dissociation rate constant increased with increasing voltages, indicating unidirectional translocation toward the pore exit. Specifically, a larger cationic cyclic octasaccharide rapidly blocked the pore more effectively, resulting in the complete closure of the pore with increasing voltage, implying only strong binding. Further, we show that uncharged oligosaccharides exclusively bind to the extracellular side of the pore and the electroosmotic flow most likely drives their translocation. We propose that CymA favors selective translocation of cyclic hexasaccharide and linear maltooligosaccharides due to an asymmetrical charge pattern and the N terminus that regulates the substrate transport. We suggest that this substrate-specific nanopore with sophisticated geometry will be useful for complex biopolymer characterization.

Designed alpha-helical barrels for charge-selective peptide translocation.

Synthetic alpha-helix based pores for selective sensing of peptides have not been characterized previously. Here, we report large transmembrane pores, pPorA formed from short synthetic alpha-helical peptides of tunable conductance and selectivity for single-molecule sensing of peptides. We quantified the selective translocation kinetics of differently charged cationic and anionic peptides through these synthetic pores at single-molecule resolution. The charged peptides are electrophoretically pulled into the pores resulting in an increase in the dissociation rate with the voltage indicating successful translocation of peptides. More specifically, we elucidated the charge pattern lining the pore lumen and the orientation of the pores in the membrane based on the asymmetry in the peptide-binding kinetics. The salt and pH-dependent measurements confirm the electrostatic dominance and charge selectivity in controlling target peptide interaction with the pores. Remarkably, we tuned the selectivity of the pores to charged peptides by modifying the charge composition of the pores, thus establishing the molecular and electrostatic basis of peptide translocation. We suggest that these synthetic pores that selectively conduct specific ions and biomolecules are advantageous for nanopore proteomics analysis and synthetic nanobiotechnology applications.

Detecting the structural assembly pathway of human antimicrobial peptide pores at single-channel level.

The pore-forming structures of an anionic human antimicrobial peptide dermcidin (DCD) in a membrane environment has not been demonstrated previously. Using single-channel electrical recordings, we characterized the structural and functional properties of the DCD peptide channel in lipid membranes. We show that a 48-residue, 8 nm long anionic DCD-1L peptide is folded in the right conformation in sodium dodecyl sulfate (SDS) that spontaneously inserts into lipid bilayers to form well-defined channels. However, the DCD-1L peptides are not properly folded in n-dodecyl-ß-d-maltoside (DDM), resulting in unstable channels suggesting the significance of specific detergent in stable channel formation. Furthermore, a 25-residue cationic DCD SSL-25 peptide formed channels both in SDS and DDM micelles as the length of the peptide matches with the thickness of the membrane. Finally, we quantified the permeation of small molecules through the DCD channels in liposome assays. Accordingly, we propose a molecular model demonstrating the structural self-assembly of the DCD channels in the membrane. We suggest that an understanding of the mechanism of action of DCD peptides at single-channel resolution will lead to developing peptide-based therapeutics.

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.

A monodisperse transmembrane α-helical peptide barrel.

The fabrication of monodisperse transmembrane barrels formed from short synthetic peptides has not been demonstrated previously. This is in part because of the complexity of the interactions between peptides and lipids within the hydrophobic environment of a membrane. Here we report the formation of a transmembrane pore through the self-assembly of 35 amino acid α-helical peptides. The design of the peptides is based on the C-terminal D4 domain of the Escherichia coli polysaccharide transporter Wza. By using single-channel current recording, we define discrete assembly intermediates and show that the pore is most probably a helix barrel that contains eight D4 peptides arranged in parallel. We also show that the peptide pore is functional and capable of conducting ions and binding blockers. Such α-helix barrels engineered from peptides could find applications in nanopore technologies such as single-molecule sensing and nucleic-acid sequencing.

Light-Patterned Current Generation in a Droplet Bilayer Array.

We have created a 4 × 4 droplet bilayer array comprising light-activatable aqueous droplet bio-pixels. Aqueous droplets containing bacteriorhodopsin (bR), a light-driven proton pump, were arranged on a common hydrogel surface in lipid-containing oil. A separate lipid bilayer formed at the interface between each droplet and the hydrogel; each bilayer then incorporated bR. Electrodes in each droplet simultaneously measured the light-driven proton-pumping activities of each bio-pixel. The 4 × 4 array derived by this bottom-up synthetic biology approach can detect grey-scale images and patterns of light moving across the device, which are transduced as electrical current generated in each bio-pixel. We propose that synthetic biological light-activatable arrays, produced with soft materials, might be interfaced with living tissues to stimulate neuronal pathways.