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The pore-forming antibacterial peptide magainin 2 was made divalent, tetravalent, and octavalent via a copper(I)-mediated 1-3 dipolar cycloaddition reaction ("click" chemistry). This series of pore-forming compounds was tested in vitro for their ability to form pores in large unilamillar vesicles (LUVs). A large increase in the pore-forming capability was especially observed with the tetravalent and octavalent magainin compounds in the LUVs consisting of DOPC, and the octavalent magainin compound showed a marked increase with the DOPC/DOPG LUVs. Activity was observed in the low nanomolar range for these compounds. © 2007 American Chemical Society.

Nisin is an example of type-A lantibiotics that contain cyclic lanthionine rings and unusual dehydrated amino acids. Among the numerous pore-forming antimicrobial peptides, type-A lantibiotics form an unique family of post-translationally modified peptides. Via the recognition of cell wall precursor lipid II, nisin has the capacity to form pores against Gram-positive bacteria with an extremely high activity in the nanomolar (nM) range. Here we report a high-resolution NMR spectroscopy study of nisin/lipid II interactions in SDS micelles as a model membrane system in order to elucidate the mechanism of molecular recognition at residue level. The binding to lipid II was studied through (15)N-(1)H HSQC titration, backbone amide proton temperature coefficient analysis, and heteronuclear (15)N[(1)H]-NOE relaxation dynamics experiments. Upon the addition of lipid II, significant changes were monitored in the N-terminal part of nisin. An extremely low amide proton temperature coefficient (Delta delta/Delta T) was found for the amide proton of Ala3 (> -0.1 ppb/K) in the complex form. This suggests tight hydrogen bonding and/or isolation from the bulk solvent for this residue. Large chemical shift perturbations were also observed in the first two rings. In contrast, the C-terminal part of nisin was almost unaffected. This part of the molecule remains flexible and solvent-exposed. On the basis of our results, a multistep pore-forming mechanism is proposed. The N-terminal part of nisin first binds to lipid II, and a subsequent structural rearrangement takes place. The C-terminal part of nisin is possibly responsible for the activation of the pore formation. In light of the emerging antibiotic resistance problems, an understanding of the specific recognition mechanism of nisin with lipid II at the residue specific level may therefore aid in the development of novel antibiotics.

The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics.

Mersacidin belongs to the type B lantibiotics (lanthionine-containing antibiotics) that contain post-translationally modified amino acids and cyclic ring structures. It targets the cell wall precursor lipid II and thereby inhibits cell wall synthesis. In light of the emerging antibiotics resistance problem, the understanding of the antibacterial activity on a structural basis provides a key to circumvent this issue. Here we present solution NMR studies of mersacidin-lipid II interaction in dodecylphosphocholine (DPC) micelles. Distinct solution structures of mersacidin were determined in three different states: in water/methanol solution and in DPC micelles with and without lipid II. The structures in various sample conditions reveal remarkable conformational changes in which the junction between Ala-12 and Abu-13 (where Abu is aminobutyric acid) effectively serves as the hinge for the opening and closure of the ring structures. The DPC micelle-bound form resembles the previously determined NMR and x-ray crystal structures of mersacidin in pure methanol but substantially deviates from the other two states in our current report. The structural changes delineate the large chemical shift perturbations observed during the course of a twostep 15N-1H heteronuclear single quantum coherence titration. They also modulate the surface charge distribution of mersacidin suggesting that electrostatics playa central role in the mersacidin-lipid II interaction. The observed conformational adaptability of mersacidin might be a general feature of lipid II-interacting antibiotics/peptides.

The heat shock protein 90 (Hsp90) family of chaperones are well-known, highly important components of the cellular systems which regulate protein homeostasis. Essential in eukaryotes, Hsp90s is also found in prokaryotes, including archaea. Hsp90 is a dimeric protein, with each monomer consisting of three separate structural domains, and undergoes large conformational changes as part of its functional cycle. This cycle is driven by interactions with nucleotides, cochaperone proteins, client proteins and allosteric effects enacted by these and by posttranslational modifications. All of these influence the rate and degree of the opening and closing of the dimer as well as the relative domain orientations and its overall rigidity. Optical tweezers, which can access many of these functionally important conformational changes, therefore provide a unique tool for the study of this large and complex molecular chaperone. Here, we provide protocols for the design and implementation of different Hsp90 constructs and optical tweezers experiments for addressing the many open questions about the function of this important molecular chaperone.

Although nanopores can be used for single-molecule sequencing of nucleic acids using low-cost portable devices, the characterization of proteins and their modifications has yet to be established. Here, we show that hydrophilic or glycosylated peptides translocate too quickly across FraC nanopores to be recognized. However, high ionic strengths (i.e., 3 M LiCl) and low pH (i.e., pH 3) together with using a nanopore with a phenylalanine at its constriction allows the recognition of hydrophilic peptides, and to distinguish between mono- and diglycosylated peptides. Using these conditions, we devise a nanopore method to detect, characterize, and quantify post-translational modifications in generic proteins, which is one of the pressing challenges in proteomic analysis. ispartof: NANO LETTERS vol:22 issue:13 pages:5357-5364 ispartof: location:United States status: published

Nanopores are label-free single-molecule analytical tools that show great potential for stochastic sensing of proteins. Here, we described a ClyA nanopore functionalized with different nanobodies through a 5-6 nm DNA linker at its periphery. Ty1, 2Rs15d, 2Rb17c, and nb22 nanobodies were employed to specifically recognize the large protein SARS-CoV-2 Spike, a medium-sized HER2 receptor, and the small protein murine urokinase-type plasminogen activator (muPA), respectively. The pores modified with Ty1, 2Rs15d, and 2Rb17c were capable of stochastic sensing of Spike protein and HER2 receptor, respectively, following a model where unbound nanobodies, facilitated by a DNA linker, move inside the nanopore and provoke reversible blockade events, whereas engagement with the large- and medium-sized proteins outside of the pore leads to a reduced dynamic movement of the nanobodies and an increased current through the open pore. Exploiting the multivalent interaction between trimeric Spike protein and multimerized Ty1 nanobodies enabled the detection of picomolar concentrations of Spike protein. In comparison, detection of the smaller muPA proteins follows a different model where muPA, complexing with the nb22, moves into the pore, generating larger blockage signals. Importantly, the components in blood did not affect the sensing performance of the nanobody-functionalized nanopore, which endows the pore with great potential for clinical detection of protein biomarkers.

Biological nanopores are emerging as sensitive single-molecule sensors for proteins and peptides. The heterogeneous charge of a polypeptide chain, however, can complicate or prevent the capture and translocation of peptides and unfolded proteins across nanopores. Here, we show that two β-barrel nanopores, aerolysin and cytotoxin K, cannot efficiently detect proteinogenic peptides from a trypsinated protein under a wide range of conditions. However, the introduction of an acidic-aromatic sensing region in the β-barrel dramatically increased the dwell time and the discrimination of peptides in the nanopore at acidic pH. Surprisingly, despite the fact that the two β-barrel nanopores have a similar diameter and an acidic-aromatic construction, their capture mechanisms differ. The electro-osmotic flow played a dominant role for aerolysin, while the electrophoretic force dominated for cytotoxin K. Nonetheless, both β-barrel nanopores allowed the detection of mixtures of trypsinated peptides, with aerolysin nanopores showing a better resolution for larger peptides and cytotoxin K showing a better resolution for shorter peptides. Therefore, this work provides a generic strategy for modifying nanopores for peptide detection that will be most likely be applicable to other nanopore-forming toxins.

In this thesis, we explore the adaptability of biological nanopores towards the detection of proteins and their modifications. First, beta-barrel nanopores are modified to increase the retention and discrimantion of proteogenic peptides. An acidic-aromatic sensing region allowed the capture and detection of a mixture of peptides in the nanopore at low pH. These acidic-aromatic nanopores are then used to detect protein glycosylation, peptide chirality and isobaric peptide modifications.

The invention relates to the field of genetically engineered nanopores and the use thereof in analyzing biopolymers and other (biological) compounds. Provided is a proteinaceous nanopore comprising a mutant pore-forming toxin, or a pore-forming fragment thereof, wherein the lumen-facing recognition region of the pore-forming protein or fragment thereof comprises one or more substitution(s) of lumen-facing amino acid(s) in the recognition region corresponding to amino acids 10-20 of Fragaceatoxin C (FraC), to a natural or non-natural aromatic amino acid residue.

The ability to measure the concentration of metabolites in biological samples is important, both in the clinic and for home diagnostics. Here we present a nanopore‐based biosensor and automated data analysis for quantification of thiamine in urine in less than a minute, without the need for recalibration. For this we use the Cytolysin A nanopore and equip it with an engineered periplasmic thiamine binding protein (TbpA). To allow fast measurements we tuned the affinity of TbpA for thiamine by redesigning the π‐π stacking interactions between the thiazole group of thiamine and TbpA. This substitution resulted furthermore in a marked difference between unbound and bound state, allowing the reliable discrimination of thiamine from its two phosphorylated forms by residual current only. Using an array of nanopores, this will allow the quantification within seconds, paving the way for next‐generation single‐molecule metabolite detection systems., Quantification of small molecules in biological samples is challenging. Using the concentration‐dependent current fluctuations elicited by a thiamine binding protein inside a ClyA nanopore, we show the automated quantification of thiamine in urine in less than a minute. By swapping the internal biosensor, this principle can be applied for the development of multiple next‐generation home‐diagnostic small‐molecule sensors.

The real-time identification of protein biomarkers is crucial for the development of point-of-care and portable devices. Here, we use a PlyAB biological nanopore to detect haemoglobin (Hb) variants. Adult haemoglobin (HbA) and sickle cell anaemia haemoglobin (HbS), which differ by just one amino acid, were distinguished in a mixture with more than 97 % accuracy based on individual blockades. Foetal Hb, which shows a larger sequence variation, was distinguished with near 100 % accuracy. Continuum and Brownian dynamics simulations revealed that Hb occupies two energy minima, one near the inner constriction and one at the trans entry of the nanopore. Thermal fluctuations, the charge of the protein, and the external bias influence the dynamics of Hb within the nanopore, which in turn generates the unique ionic current signal in the Hb variants. Finally, Hb was counted from blood samples, demonstrating that direct discrimination and quantification of Hb from blood using nanopores, is feasible. ispartof: ANGEWANDTE CHEMIE-INTERNATIONAL EDITION vol:61 issue:34 ispartof: location:Germany status: published

Single-molecule nanopore electrophysiology is an emerging technique for the detection of analytes in aqueous solutions with high sensitivity. These detectors have proven applicable for the enzyme-assisted sequencing of oligonucleotides. There has recently been an increased interest in the use of nanopores for the fingerprinting of peptides and proteins, referred to as single-molecule nanopore spectrometry. However, the analysis of the resulting electrophysiology traces remains complicated due to the fast unassisted translocation of such analytes, usually in the order of micro- to milliseconds, and the small ion current signal produced (in the picoampere range). Here, we present the application of a generalized normal distribution function (gNDF) for the characterization of short-lived ion current signals (blockades). We show that the gNDF can be used to determine if the observed blockades have adequate time to reach their maximum current plateau while also providing a description of each blockade based on the open pore current (I O), the difference caused by the pore blockade (ΔI B), the position in time (μ), the standard deviation (σ), and a shape parameter (β), leaving only the noise component. In addition, this method allows the estimation of an ideal range of low-pass filter frequencies that contains maximum information with minimal noise. In summary, we show a parameter-free and generalized method for the analysis of short-lived ion current blockades, which facilitates single-molecule nanopore spectrometry with minimal user bias. ispartof: ACS OMEGA vol:7 issue:30 pages:26040-26046 ispartof: location:United States status: published

Proteins aggregation into amyloid fibrils has been observed in several pathological conditions and exploited in nanotechnology. It is also key in several biochemical processes. In this work we show that ionic liquids (ILs) – a vast class of organic electrolytes – can finely tune amyloid properties, opening a new playground in basic science and applications. The representative case of ethyl-ammonium nitrate (EAN) and tetramethyl-guanidinium acetate (TMGA) ILs on lysozyme is considered. Firstly, atomic force microscopy has shown that the addition of EAN and TMGA leads to thicker and thinner amyloid fibrils of greater and lower electric potential, respectively, with diameters finely tuneable by IL-concentration. Optical tweezers and neutron scattering have shed a light on their mechanism of action. TMGA interacts with the protein hydration layer only, making the relaxation dynamics of these water molecules faster. EAN interacts directly with the protein instead, making it mechanically unstable and slowing down its relaxation dynamics.

The ionic currents passing through nanopores can be used to sequence DNA and identify molecules at the single-molecule level. Recently, researchers have started using nanopores for the detection and analysis of proteins, providing a new platform for single-molecule enzymology studies and more efficient biomolecular sensing applications. For this approach, the homo-oligomeric Cytolysin A (ClyA) nanopore has been demonstrated as a powerful tool. Here, we describe a simple protocol allowing the production of ClyA nanopores. Monomers of ClyA are expressed in Escherichia coli and oligomerized in the presence of detergent. Subsequently, different oligomer variants are electrophoretically resolved and stored in a gel matrix for long-term use.

We present a synthetic nanoscale piston that uses chemical energy to perform molecular transport against an applied bias. Such a device comprises a 13 by 5 nm protein cylinder, embedded in a biological membrane enclosing a single-stranded DNA (ssDNA) rod. Hybridization with DNA cargo rigidifies the rod, allowing for transport of a selected DNA molecule across the nanopore. A strand displacement reaction from ssDNA fuel on the other side of the membrane then liberates the DNA cargo back into solution and regenerates the initial configuration. The entropic penalty of ssDNA confinement inside the nanopore drives DNA transport regardless of the applied bias. Multiple automated and reciprocating cycles are observed, in which the DNA piston moves through the 10 nm length of the nanopore. In every cycle, a single DNA molecule is transported across the nanopore against an external bias force, which is the hallmark of biological transporters. ispartof: NANO LETTERS vol:21 issue:1 pages:762-768 ispartof: location:United States status: published

This thesis describes experimental and computational analysis for the creation of biological nanopore based single-molecule sensors. In essence, nanopore sensors consist of a non-conductive sheet with a nanometre sized aperture connecting two electrolyte filled compartments. The conductance across this aperture is reduced due to ion exclusion whenever a molecule translocates through it, which can be observed as a reduction in current when a potential is applied. Here, we engineered membrane spanning proteins for the creation of these nanometre sized apertures in order to observe and characterize peptides. By engineering the constriction inside the nanopore to interact with translocating peptides, we were able to increase the residence time of the analyte inside the nanopore.By characterizing the current fluctuations over time, we were able to fingerprint trypsin digested proteins based on their translocating peptides. In addition, we were able to trap substrate binding proteins inside a nanopore, which undergo a change in their ion exclusion when bound to an analyte. We sought to rapidly detect vitamin B1 in human urine, using a single nanopore in combination with a binding protein. We combined a physical understanding of the system with an empirical Bayesian approach, allowing the detection of thiamine in approximately 20 seconds.

Journal of medicinal chemistry 65(6), 4798-4817 (2022). doi:10.1021/acs.jmedchem.1c01962, Published by ACS, Washington, DC

Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness. As a consequence of whole genome duplication, teleost fish have two ridA paralogs, while other extant vertebrates contain a single-copy gene. We investigated the biochemical properties of the products of two paralogs, identified in Salmo salar. SsRidA-1 and SsRidA-2 complemented the growth defect of a Salmonella enterica ridA mutant, an in vivo model of 2AA stress. In vitro, both proteins hydrolyzed 2-imino acids (IA) to keto-acids and ammonia. SsRidA-1 was active on IA derived from nonpolar amino acids and poorly active or inactive on IA derived from other amino acids tested. In contrast, SsRidA-2 had a generally low catalytic efficiency, but showed a relatively higher activity with IA derived from L-Glu and aromatic amino acids. The crystal structures of SsRidA-1 and SsRidA-2 provided hints of the remarkably different conformational stability and substrate specificity. Overall, SsRidA-1 is similar to the mammalian orthologs whereas SsRidA-2 displays unique properties likely generated by functional specialization of a duplicated ancestral gene.

Conformational heterogeneity is emerging as a defining characteristic of enzyme function. However, understanding the role of protein conformations requires their thermodynamic and kinetic characterization at the single-molecule level, which remains extremely challenging. Here we report the ligand-induced conformational changes of dihydrofolate reductase (DHFR) by measuring the modulation of the nanopore currents. The long observation time of the electrical recordings enabled the detection of rare conformational transitions hidden in ensemble measurements. We show that DHFR exists in at least four ground-state configurations or conformers with different affinities for its ligands. Unliganded DHFR adopted low-affinity conformers, whereas the binding of substrates promoted the switch to the high-affinity conformer. Conversion between the conformers was accelerated by molecules that stabilized the transition state of DHFR, which suggests that the reaction lowers the energy barrier for conformer exchange and thus facilitates product release. This mechanism might be a general feature in enzymatic reactions affected by product inhibition or when the release of products is the rate-limiting step. [Figure not available: see fulltext.].

Biological nanopores are emerging as powerful tools for single-molecule analysis and sequencing. Here, we engineered the two-component pleurotolysin (PlyAB) toxin to assemble into 7.2 × 10.5 nm cylindrical nanopores with a low level of electrical noise in lipid bilayers, and we addressed the nanofluidic properties of the nanopore by continuum simulations. Surprisingly, proteins such as human albumin (66.5 kDa) and human transferrin (76-81 kDa) did not enter the nanopore. We found that the precise engineering of the inner surface charge of the PlyAB induced electro-osmotic vortices that allowed the electrophoretic capture of the proteins. Once inside the nanopore, two human plasma proteins could be distinguished by the characteristics of their current blockades. This fundamental understanding of the nanofluidic properties of nanopores provides a practical method to promote the capture and analysis of folded proteins by nanopores. ispartof: NANO LETTERS vol:20 issue:5 pages:3819-3827 ispartof: location:United States status: published

The ability to sample multiple reactions on the same single enzyme is important to link rare intermediates with catalysis and to unravel the role of conformational changes. Despite decades of efforts, however, the single-molecule characterization of nonfluorogenic enzymes during multiple catalytic turnovers has been elusive. Here, we show that nanopore currents allow sampling the dynamic exchange between five structural intermediates during E. coli dihydrofolate reductase (DHFR) catalysis. We found that an endosteric effect promotes the binding of the substrate to the enzyme with a specific hierarchy. The chemical step then switched the enzyme from the closed to the occluded conformation, which in turn promotes the release of the reduced cofactor NADP+. Unexpectedly, only a few reactive complexes lead to catalysis. Furthermore, second-long catalytic pauses were observed, possibly reflecting an off-path conformation generated during the reaction. Finally, the free energy from multiple cofactor binding events were required to release the product and switch DHFR back to the reactive conformer. This catalytic fueled concerted mechanism is likely to have evolved to improve the catalytic efficiency of DHFR under the high concentrations of NADP+ in E. coli and might be a general feature for complex enzymatic reactions where the binding and release of the products must be tightly controlled.

The invention relates generally to the field of nanopores and the use thereof in analyzing biopolymers. In particular, it relates to engineered biological nanopores and their application in single molecule analysis, such as single molecule protein identification.

The detection of analytes and the sequencing of DNA using biological nanopores have seen major advances over recent years. The analysis of proteins and peptides with nanopores, however, is complicated by the complex physicochemical structure of polypeptides and the lack of understanding of the mechanism of capture and recognition of polypeptides by nanopores. In this work, we show that introducing aromatic amino acids at precise positions within the lumen of α-helical fragaceatoxin C (FraC) nanopores increased the capture frequency of peptides and largely improved the discrimination among peptides of similar size. Molecular dynamics simulations determined the sensing region of the nanopore, elucidated the microscopic mechanism enabling accurate characterization of the peptides via ionic current blockades in FraC, and characterized the effect of the pore modification on peptide discrimination. This work provides insights to improve the recognition and to augment the capture of peptides by nanopores, which is important for developing a real-time and single-molecule size analyzer for peptide recognition and identification.

The invention relates to the field of nanopores and the use thereof in analyzing biopolymers, including polypeptides and polynucleotides. Provided is an artificial nanopore comprising a multimeric assembly of subunits, each subunit comprising (i) the transmembrane (TM) sequence of a β-barrel or α-helical pore forming protein fused to the amino acid sequence of (ii) a subunit of a ring-forming protein capable of controlling the transport of a polypeptide or polynucleotide across the TM region of the assembly.

Self-assembly is a fundamental feature of biological systems, and control of such processes offers fascinating opportunities to regulate function. Fragaceatoxin C (FraC) is a toxin that upon binding to the surface of sphingomyelin-rich cells undergoes a structural metamorphosis, leading to the assembly of nanopores at the cell membrane and causing cell death. In this study we attached photoswitchable azobenzene pendants to various locations near the sphingomyelin binding pocket of FraC with the aim of remote controlling the nanopore assembly using light. We found several constructs in which the affinity of the toxin for biological membranes could be activated or deactivated by irradiation, thus enabling reversible photocontrol of pore formation. Notably, one construct was completely inactive in the thermally adapted state; it however induced full lysis of cultured cancer cells upon light irradiation. Selective irradiation also allowed isolation of individual nanopores in artificial lipid membranes. Photocontrolled FraC might find applications in photopharmacology for cancer therapeutics and has potential to be used for the fabrication of nanopore arrays in nanopore sensing devices, where the reconstitution, with high spatiotemporal precision, of single nanopores must be controlled.

Summary To address the question of why more than one myosin-II isoform is expressed in a single cell to drive cytokinesis, we analyzed the roles of the myosin-II isoforms, Myo2 and Myp2, of the fission yeast Schizosaccharomyces pombe, in cytokinesis under normal and stressed conditions. We found that Myp2 controls the disassembly, stability, and constriction initiation of the Myo2 ring in response to high-salt stress. A C-terminal coiled-coil domain of Myp2 is required for its immobility and contractility during cytokinesis, and when fused to the tail of the dynamic Myo2, renders the chimera the low-turnover property. We also found, by following distinct processes in real time at the single-cell level, that Myo2 and Myp2 are differentially required but collectively essential for guiding extracellular matrix remodeling during cytokinesis. These results suggest that the dynamic and immobile myosin-II isoforms are evolved to carry out cytokinesis with robustness under different growth conditions., Graphical Abstract, Highlights • The myosin-II isoforms Myo2 and Myp2 display distinct responses to cellular stress • Myp2 controls the constriction initiation of Myo2 during stress response • A C-terminal region of Myp2 is required for its immobility during cytokinesis • Myo2 and Myp2 are differentially required for guiding ECM remodeling during cytokinesis, Molecular Mechanism of Behavior; Cell Biology; Functional Aspects of Cell Biology

Chemical probes that covalently modify proteins of interest are powerful tools for the research of biological processes. Important in the design of a probe is the choice of reactive group that forms the covalent bond, as it decides the success of a probe. However, choosing the right reactive group is not a simple feat and methodologies for expedient screening of different groups are needed. We herein report a modular approach that allows easy coupling of a reactive group to a ligand. α‐Nucleophile ligands are combined with 2‐formylphenylboronic acid derived reactive groups to form iminoboronate probes that selectively label their target proteins. A transimination reaction on the labeled proteins with an α‐amino hydrazide provides further modification, for example to introduce a fluorophore., Iminoboronate chemistry enables the synthesis of chemical probes that selectively label proteins of interest from 2‐formylphenylboronic acid reactive groups and hydrazide‐functionalized ligands. The reversibility of the iminoboronate linkage is exploited to detect the labeled proteins by ligand‐fluorophore exchange. Remarkably, the fast iminoboronate chemistry even allows probe formation within protein mixtures.