Teaching an old dog new tricks: A lipid membrane-based electric immunosensor for real-time probing of the spike S1 protein subunit from SARS-CoV-2.

Fast, cheap, and easy to implement point-of-care testing for various pathogens constituted a game changer in past years due to its potential for early disease diagnosis. Herein, we report on the proof-of-concept of a simple method enabling in vitro detection of a structural spike protein subunit from the SARS-CoV-2 (S1 ) in aqueous samples. At the core of this discovery lies the well-known paradigm of monitoring the capacitive current across a reconstituted zwitterionic lipid membrane subjected to a periodic transmembrane potential, followed by the real-time spectral analysis enabling the extraction of the second harmonic of the capacitive current. Subsequent changes in the amplitude of this harmonic recorded during lipid membrane-S1 interactions were correlated with alterations induced in the inner membrane potential profile by the S1 protein subunit adsorption, and were shown to be augmented by ionic strength, the presence of a specific monoclonal antibody designed against the S1 subunit and the angiotensin-converting enzyme 2 (ACE2) protein receptor, and uninhibited by the presence of other human serum proteins.

Single-molecule, hybridization-based strategies for short nucleic acids detection and recognition with nanopores.

DNA nanotechnology has seen large developments over the last 30 years through the combination of detection and discovery of DNAs, and solid phase synthesis to increase the chemical functionalities on nucleic acids, leading to the emergence of novel and sophisticated in features, nucleic acids-based biopolymers. Arguably, nanopores developed for fast and direct detection of a large variety of molecules, are part of a revolutionary technological evolution which led to cheaper, smaller and considerably easier to use devices enabling DNA detection and sequencing at the single-molecule level. Through their versatility, the nanopore-based tools proved useful biomedicine, nanoscale chemistry, biology and physics, as well as other disciplines spanning materials science to ecology and anthropology. This mini-review discusses the progress of nanopore- and hybridization-based DNA detection, and explores a range of state-of-the-art applications afforded through the combination of certain synthetically-derived polymers mimicking nucleic acids and nanopores, for the single-molecule biophysics on short DNA structures.

A Nanopore Sensor for Multiplexed Detection of Short Polynucleotides Based on Length-Variable, Poly-Arginine-Conjugated Peptide Nucleic Acids.

Real-time and easy-to-use detection of nucleic acids is crucial for many applications, including medical diagnostics, genetic screening, forensic science, or monitoring the onset and progression of various diseases. Herein, an exploratory single-molecule approach for multiplexed discrimination among similar-sized single-stranded DNAs (ssDNA) is presented. The underlying strategy combined (i) a method based on length-variable, short arginine (poly-Arg) tags appended to peptide nucleic acid (PNA) probes, designed to hybridize with selected regions from complementary ssDNA targets (cDNA) in solution and (ii) formation and subsequent detection with the α-hemolysin nanopore of (poly-Arg)-PNA-cDNA duplexes containing two overhangs associated with the poly-Arg tail and the non-hybridized segment from ssDNA. We discovered that the length-variable poly-Arg tail marked distinctly the molecular processes associated with the nanopore-mediated duplexes capture, trapping and unzipping. This enabled the detection of ssDNA targets via the signatures of (poly-Arg)-PNA-cDNA blockade events, rendered most efficient from the ß-barrel entrance of the nanopore, and scaled proportional in efficacy with a larger poly-Arg moiety. We illustrate the approach by sensing synthetic ssDNAs designed to emulate fragments from two regions of SARS-CoV-2 nucleocapsid phosphoprotein N-gene.

Unzipping Mechanism of Free and Polyarginine-Conjugated DNA-PNA Duplexes, Preconfined Inside the α-Hemolysin Nanopore.

In this work, comparative studies on DNA-PNA and polyarginine-conjugated DNA-PNA duplexes unzipping inside the α-hemolysin nanopore (α-HL) are presented. We identified significant differences in the blockade currents, as the applied voltage across the nanopore facilitated the duplex capture inside the nanopore's vestibule against the constriction region, subsequent cDNA strand insertion inside the nanopore's ß-barrel past the constriction site, its complete unzip from the duplex, and translocation. We observed that inside the voltage-biased nanopore, polyarginine-conjugated DNA-PNA duplexes dehybridize faster than their DNA-PNA counterparts and proposed a model to describe the duplex unzipping. This study identifies key particularities of DNA-PNA duplex unzipping as it takes place inside the nanopore and being preceded by entrapment in the vestibule domain of the α-HL. Our results are a crucial step toward understanding the nucleic acids duplexes unzipping kinetics variability, in confined, variable geometries.

Nanoscale Probing of Informational Polymers with Nanopores. Applications to Amyloidogenic Fragments, Peptides, and DNA-PNA Hybrids.

The decades long advances in nanotechnology, biomolecular sciences, and protein engineering ushered the introduction of groundbreaking technologies devoted to understanding how matter behaves at single particle level. Arguably, one of the simplest in concept is the nanopore-based paradigm, with deep roots in what is originally known as the Coulter counter, resistive-pulse technique. Historically, a nanopore system comprising the oligomeric protein generated by Staphylococcus aureus toxin α-hemolysin (α-HL) was first applied to detecting polynucleotides, as revealed in 1996 by John J. Kasianowicz, Eric Brandin, Daniel Branton, and David W. Deamer, in the Proceedings of the National Academy of Sciences. Nowadays, a wide variety of other solid-state or protein-based nanopores have emerged as efficient tools for stochastic sensing of analytes as small as single metal ions, handling single molecules, or real-time, label-free probing of chemical reactions at single-molecule level. In this Account, we demonstrate the usefulness of the α-HL nanopore on probing metal-induced folding of peptides, and to investigating the reversible binding of various metals to physiologically relevant amyloid fragments. The widely recognized Achilles heel of the approach, is the relatively short dwell time of the analytes inside the nanopore. This hinders the collection of sufficient data required to infer statistically meaningful conclusions about the physical or chemical state of the studied analyte. To mitigate this, various approaches were successfully applied in particular experiments, including but not restricted to altering physical parameters of the aqueous solution, downsizing the nanopore geometry, the controlled tuning of the balance between the electrostatic and electro-osmotic forces, coating nanopores with a fluid lipid bilayer, employing a pressure-voltage biased pore. From our perspective, in this Account, we will present two strategies aimed at controlling the analyte passage across the α-HL. First, we will reveal how the electroosmotic flow can be harnessed to control residence time, direction, and the sequence of spatiotemporal dynamics of a single peptide along the nanopore. This also allows one to identify the mesoscopic trajectory of a peptide exiting the nanopore through either the vestibule or ß-barrel moiety. Second, we lay out the principles of an approach dubbed "nanopore tweezing", enabling simultaneous capture rate increase and escape rate decrease of a peptide from the α-HL, with the applied voltage. At its core, this method requires the creation of an electrical dipole on the peptide under study, via engineering positive and negative amino acid residues at the two ends of the peptide. Concise applications of this approach are being demonstrated, as in proof-of-concept experiments we probed the primary structure exploration of polypeptides, via discrimination between selected neutral amino acid residues. Another useful venue provided by the nanopores is represented by single-molecule force experiments on captured analytes inside the nanopore, which proved useful in exploring force-induced rupture of nucleic acids duplexes, hairpins, or various nucleic acids-ligand conjugates. We will show that when applied to oppositely charged, polypeptide-functionalized PNA-DNA duplexes, the nanopore tweezing introduces a new generation of force-spectroscopy nanopore-based platforms, facilitating unzipping of a captured duplex and enabling the duplex hybridization energy estimation.

Nanopore-Assisted, Sequence-Specific Detection, and Single-Molecule Hybridization Analysis of Short, Single-Stranded DNAs.

We report here on the ability of the α-hemolysin (α-HL) nanopore to achieve label-free, selective, and real-time detection of 15 nt long ssDNA fragments in solution, by exploiting their hybridization with freely added, polycationic peptides-functionalized PNAs. At the core of our work lies the paradigm that when PNAs and ssDNA are mixed together, the bulk concentration of free PNA decreases, depending upon the (mis)match degree between complementary strands and their relative concentrations. We demonstrate that the ssDNA sensing principle and throughput of the method are determined by the rate at which nonhybridized, polycationic peptides-functionalized PNA molecules arrive at the α-HL's vestibule entrance and thread into the nanopore. We found that with the application of a 30-fold salt gradient across the nanopore, the method enhances single-molecule detection sensitivity in the nanomolar range of ssDNA concentrations. This study demonstrates that the transmembrane potential-dependent unzip of single PNA-DNA duplexes at the α-HL's ß-barrel entry permits discrimination between sequences that differ by one base pair.

Pse-T2, an Antimicrobial Peptide with High-Level, Broad-Spectrum Antimicrobial Potency and Skin Biocompatibility against Multidrug-Resistant Pseudomonas aeruginosa Infection.

Pseudin-2, isolated from the frog Pseudis paradoxa, exhibits potent antibacterial activity but also cytotoxicity. In an effort to develop clinically applicable antimicrobial peptides (AMPs), we designed pseudin-2 analogs with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. In addition, truncated analogs of pseudin-2 and Lys-substituted peptides were synthesized to produce linear 18-residue amphipathic α-helices, which were further investigated for their mechanism and functions. These truncated analogs exhibited higher antimicrobial activity and lower cytotoxicity than pseudin-2. In particular, Pse-T2 showed marked pore formation, permeabilization of the outer/inner bacterial membranes, and DNA binding. Fluorescence spectroscopy and scanning electron microscopy showed that Pse-T2 kills bacterial cells by disrupting membrane integrity. In vivo, wounds infected with multidrug-resistant (MDR) Pseudomonas aeruginosa healed significantly faster when treated with Pse-T2 than did untreated wounds or wounds treated with ciprofloxacin. Moreover, Pse-T2 facilitated infected-wound closure by reducing inflammation through suppression of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α). These data suggest that the small antimicrobial peptide Pse-T2 could be useful for future development of therapeutic agents effective against MDR bacterial strains.

Single-Molecule, Real-Time Dissecting of Peptide Nucleic Acid-DNA Duplexes with a Protein Nanopore Tweezer.

Peptide nucleic acids (PNAs) are artificial, oligonucleotides analogues, where the sugar-phosphate backbone has been substituted with a peptide-like N-(2-aminoethyl)glycine backbone. Because of their inherent benefits, such as increased stability and enhanced binding affinity toward DNA or RNA substrates, PNAs are intensively studied and considered beneficial for the fields of materials and nanotechnology science. Herein, we designed cationic polypeptide-functionalized, 10-mer PNAs, and demonstrated the feasible detection of hybridization with short, complementary DNA substrates, following analytes interaction with the vestibule entry of an α-hemolysin (α-HL) nanopore. The opposite charged state at the polypeptide-functionalized PNA-DNA duplex extremities, facilitated unzipping of a captured duplex at the lumen entry of a voltage-biased nanopore, followed by monomers threading. These processes were resolvable and identifiable in real-time, from the temporal profile of the ionic current through a nanopore accompanying conformational changes of a single PNA-DNA duplex inside the α-HL nanopore. By employing a kinetic description within the discrete Markov chains theory, we proposed a minimalist kinetic model to successfully describe the electric force-induced strand separation in the duplex. The distinct interactions of the duplex at either end of the nanopore present powerful opportunities for introducing new generations of force-spectroscopy nanopore-based platforms, enabling from the same experiment duplex detection and assessment of interstrand base pairing energy.

If Squeezed, a Camel Passes Through the Eye of a Needle: Voltage-Mediated Stretching of Dendrimers Facilitates Passage Through a Nanopore.

Herein, we report uni-molecular observations of electric potential- and electrolyte-dependent elasticity of poly(amidoamine) (PAMAM)-G1.5 dendrimers containing sodium carboxylate surface groups, using the electric field-assisted migration through the α-hemolysin nanopore (α-HL). Although at moderate transmembrane potentials the dendrimer (~ 2.5 nm in diameter) is sterically excluded from translocation across the constriction region of the nanopore (~ 1.5 nm in diameter), we found a threshold for its translocation that depends on both the electrolyte pH and ionic strength. We posit that the decreased repulsive intramolecular interactions among dendrimer's branches at low when compared to neutral pH, caused mainly by the protonation of surface groups on the dendrimer, determine a larger propensity of the dendrimer to collapse and deform. This in turns enables the dendrimer to adopt more favorably conformations that facilitate its optimal squeezing through the α-HL's constriction region at low pH, despite the fact that the estimated net force acting on it becomes approximately one order of magnitude lower than at neutral pH. Experiments performed in a low ionic strength buffer, which decreases Coulombic screening, enhance the intramolecular forces on the dendrimer and renders the dendrimer stiffer than in high ionic strength buffer, confirming the dendrimer elastic properties-dependent threshold for deformation inside the nanopore.

Protein Nanopore-Based Discrimination between Selected Neutral Amino Acids from Polypeptides.

Nanopore probing of biological polymers has the potential to achieve single-molecule sequencing at low cost, high throughput, portability, and minimal sample preparation and apparatus. In this article, we explore the possibility of discrimination between neutral amino acid residues from the primary structure of 30 amino acids long, engineered peptides, through the analysis of single-molecule ionic current fluctuations accompanying their slowed-down translocation across the wild type α-hemolysin (α-HL) nanopore, and molecular dynamics simulations. We found that the transient presence inside the α-HL of alanine or tryptophan residues from the primary sequence of engineered peptides results in distinct features of the ionic current fluctuation pattern associated with the peptide reversibly blocking the nanopore. We propose that α-HL sensitivity to the molecular exclusion at the most constricted region mediates ionic current blockade events correlated with the volumes that are occluded by at least three alanine or tryptophan residues, and provides the specificity needed to discriminate between groups of neutral amino acids. Further, we find that the pattern of current fluctuations depends on the orientation of the threaded amino acid residues, suggestive of a conformational anisotropy of the ensemble of conformations of the peptide on the restricted nanopore region, related to its relative axial orientation inside the nanopore.

Antimicrobial Peptide CMA3 Derived from the CA-MA Hybrid Peptide: Antibacterial and Anti-inflammatory Activities with Low Cytotoxicity and Mechanism of Action in Escherichia coli.

CA-MA is a hybrid antimicrobial peptide (AMP) derived from two naturally occurring AMPs, cecropin A and magainin 2. CA-MA shows strong antimicrobial activity against Gram-negative and Gram-positive bacteria but also exhibits cytotoxicity toward mammalian cells. Our objective was to identify CA-MA analogues with reduced cytotoxicity by systematic replacement of amino acids with positively charged R groups (His and Lys), aliphatic R groups (Leu), or polar R groups (Glu). Among the CA-MA analogues studied (CMA1 to -6), CMA3 showed the strongest antimicrobial activity, including against drug-resistant Escherichia coli and Pseudomonas aeruginosa strains isolated from hospital patients. CMA3 appeared to act by inducing pore formation (toroidal model) in the bacterial membrane. In cytotoxicity assays, CMA3 showed little cytotoxicity toward human red blood cells (hRBCs) or HaCaT cells. Additionally, no fluorescence was released from small or giant unilamellar vesicles exposed to 60 µM CMA3 for 80 s, whereas fluorescence was released within 35 s upon exposure to CA-MA. CMA3 also exerted strong lipopolysaccharide (LPS)-neutralizing activity in RAW 264.7 cells, and BALB/c mice exposed to LPS after infection by Escherichia coli showed improved survival after administration of one 0.5-mg/kg of body weight or 1-mg/kg dose of CMA3. Finally, in a mouse model of septic shock, CMA3 reduced the levels of proinflammatory factors, including both nitric oxide and white blood cells, and correspondingly reduced lung tissue damage. This study suggests that CMA3 is an antimicrobial/antiendotoxin peptide that could serve as the basis for the development of anti-inflammatory and/or antimicrobial agents with low cytotoxicity.

Nanopore investigation of the stereoselective interactions between Cu(2+) and D,L-histidine amino acids engineered into an amyloidic fragment analogue.

Stereochemistry is an essential theme for a number of industries and applications, constructed around discriminating various chiral enantiomers, including amino acids, chiral metal complexes, and drugs. In this work, we designed a set of peptide mutants of the human amyloidic Aß1-16 sequence, known to display an effective Cu(2+) coordinating pocket provided mainly by the intramolecular His-6, His-13, and His-14 residues, that were engineered to contain L- and D-His enantiomers in positions 6 and 13 and provide a local coordination environment with distinct Cu(2+) binding geometries and affinities. We examined the mechanism of selective chiral recognition of Cu(2+) by such mutant peptides, by quantifying their stochastic sensing in real time with a single α-hemolysin (α-HL) protein immobilized in a planar lipid membrane, while incubated in various concentrations of Cu(2+). Our data reveal that the Cu(2+)-binding affinity lies within the micromolar range, and decreases by orders of magnitude as L-His is replaced with its Denantiomer, with the effect being prevalent when such changes were inflicted on the His-6 residue. The presented results demonstrate the feasibility of tuning the metal selectivity in a relatively simple peptide substrate by enantiomeric replacement of key metal binding residues and illustrates the potential of the protein nanopores as a promising approach to quantify the chiral recognition of l/d amino acids by metals.

Probing of various physiologically relevant metals-amyloid-ß peptide interactions with a lipid membrane-immobilized protein nanopore [corrected].

One of the prevailing paradigms regarding the onset of Alzheimer's disease endows metal ions with key roles in certain steps of the amyloid-ß (Aß) peptide aggregation cascade, through peptide conformational changes induced by metal binding. Herein, we focused on the truncated, more soluble Aß1-16 peptide fragment from the human Aß1-40, and demonstrated the utility of a sensing element based on the α-hemolysin (α-HL) protein to examine and compare at single-molecule level the interactions between such peptides and various metals. By using the same approach, we quantified Cu(2+) and Zn(2+) binding affinities to the Aß1-16 fragment, whereas the statistical analysis of blockages induced by a single Aß1-16 peptide on the current flow through an open α-HL pore show that the metal propensity to interacting with the peptide and entailing conformational changes obey the following order: Cu(2+) > Zn(2+) > Fe(3+) > Al(3+).

Electrophysiology investigation of Trichogin GA IV activity in planar lipid membranes reveals ion channels of well-defined size.

Trichogin GA IV, an antimicrobial peptaibol, exerts its function by augmenting membrane permeability, but the molecular aspects of its pore-forming mechanism are still debated. Several lines of evidence indicate a 'barrel-stave' channel structure, similar to that of alamethicin, but the length of a trichogin helix is too short to span a normal bilayer. Herein, we present electrophysiology measurements in planar bilayers, showing that trichogin does form channels of a well-defined size (R=4.2⋅10(9)  Ω; corresponding at least to a trimeric aggregate) that span the membrane and allow ion diffusion, but do not exhibit voltage-dependent rectification, unlike those of alamethicin.

Controlling DNA Fragments Translocation across Nanopores with the Synergic Use of Site-Directed Mutagenesis, pH-Dependent Charge Tuning, and Electroosmotic Flow.

Biological and solid-state nanopores are at the core of transformative techniques and nanodevices, democratizing the examination of matter and biochemical reactions at the single-molecule level, with low cost, portability, and simplicity in operation. One of the crucial hurdles in such endeavors is the fast analyte translocation, which limits characterization, and a rich number of strategies have been explored over the years to overcome this. Here, by site-directed mutagenesis on the α-hemolysin protein nanopore (α-HL), sought to replace selected amino acids with glycine, electrostatic binding sites were induced on the nanopore's vestibule and constriction region and achieved in the most favorable case a 20-fold increase in the translocation time of short single-stranded DNA (ssDNA) at neutral pH, with respect to the wild-type (WT) nanopore. We demonstrated an efficient tool of controlling the ssDNA translocation time, via the interplay between the nanopore-ssDNA surface electrostatic interactions and electroosmotic flow, all mediated by the pH-dependent ionization of amino acids lining the nanopore's translocation pathway. Our data also reveal the nonmonotonic, pH-induced alteration of ssDNA average translocation time. Unlike mildly acidic conditions (pH ∼ 4.7), at a pH ∼ 2.8 maintained symmetrically or asymmetrically across the WT α-HL, we evidenced the manifestation of a dominant electroosmotic flow, determining the speeding up of the ssDNA translocation across the nanopore by counteracting the ssDNA-nanopore attractive electrostatic interactions. We envision potential applications of the presented approach by enabling easy-to-use, real-time detection of short ssDNA sequences, without the need for complex biochemical modifications to the nanopore to mitigate the fast translocation of such sequences.

Investigation of Cu2+ binding to human and rat amyloid fragments Aß (1-16) with a protein nanopore.

Recent evidence shows that metal coordination by amyloid beta peptides (Aß) determines structural alterations of peptides, and His-13 from Aß is crucial for Cu(2+) binding. This study used the truncated, more soluble Aß1-16 isoforms derived from human and rat amyloid peptides to explore their interaction with Cu(2+) by employing the membrane-immobilized α-hemolysin (α-HL) protein as a nanoscopic probe in conjunction with single-molecule electrophysiology techniques. Unexpectedly, the experimental data suggest that unlike the case of the human Aß1-16 peptide, Cu(2+) complexation by its rat counterpart leads to an augmented association and dissociation kinetics of the peptide reversible interaction with the protein pore, as compared to the Cu(2+)-free peptide. Single-molecule electrophysiology data reveal that both human and rat Cu(2+)-complexed Aß peptides induce a higher degree of current flow obstruction through the α-HL pore, as compared to the Cu(2+)-free peptides. It is suggested that morphology changes brought by Cu(2+) binding to such amyloidic fragments depend crucially upon the presence of the His-13 residue on the primary sequence of such peptide fragments, and the α-HL protein-based approach provides unique opportunities and challenges to probing metal-induced folding of peptides.

Considerable slowdown of short DNA fragment translocation across a protein nanopore using pH-induced generation of enthalpic traps inside the permeation pathway.

A pressing challenge in the realm of nanopore-based sensing technologies for nucleic acid characterization has been the cheap and efficient control of analyte translocation. To address this, a plethora of methods were tested, including mutagenesis, molecular motors, enzymes, or the optimization of experimental conditions. Herein, we present a paradigm exploiting the manipulation of electrostatic interactions between 22-mer single-stranded DNAs (22_ssDNA) and low pH-induced charges in the alpha-hemolysin (α-HL) nanopore, to efficiently control the passage of captured molecules. We discovered that in electrolytes buffered at pH = 5 and pH = 4.5 where the nanopore's vestibule and lumen become oppositely charged as compared to that at neutral pH, the electrostatic anchoring at these regions of a 22_ssDNA fragment leads to a dramatic increase of the translocation time, orders of magnitude larger compared to that at neutral pH. This pH-dependent tethering effect is reversible, side invariant, and sensitive to the ionic strength and ssDNA contour length. In the long run, our discovery has the potential to provide a simple read-out of the sequence of bases pertaining to short nucleotide sequences, thus extending the efficacy of current nanopore-based sequencers.

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection.

Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially protein-based nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channel-forming alamethicin monomers. We found that the resulting chimeric molecules successfully coassemble in a voltage-dependent manner in planar lipid membranes generating diameter-variable oligomers. The subsequent interaction at the flexible extramembrane segment of such formed dynamic nanopores with aqueously added complementary ssDNA fragments leads to overall conformational alterations affecting the peptide assembly state kinetics and mediated ionic current. Such recognition events were found specific to the primary structure of target ssDNA and uninhibited the presence of serum. Our platform demonstrates the feasibility of designing an entirely new class of versatile chimeric biosensors, for which, dependent upon the nature of the attached receptor moiety and underlying recognition chemistry, the applicability area may extend to other analytes.

Correction: Considerable slowdown of short DNA fragment translocation across a protein nanopore using pH-induced generation of enthalpic traps inside the permeation pathway.

Correction for 'Considerable slowdown of short DNA fragment translocation across a protein nanopore using pH-induced generation of enthalpic traps inside the permeation pathway' by Loredana Mereuta et al., Nanoscale, 2023, 15, 14754-14763, https://doi.org/10.1039/D3NR03344A.

Protein nanopore-based, single-molecule exploration of copper binding to an antimicrobial-derived, histidine-containing chimera peptide.

Metal ions binding exert a crucial influence upon the aggregation properties and stability of peptides, and the propensity of folding in various substates. Herein, we demonstrate the use of the α-HL protein as a powerful nanoscopic tool to probe Cu(2+)-triggered physicochemical changes of a 20 aminoacids long, antimicrobial-derived chimera peptide with a His residue as metal-binding site, and simultaneously dissect the kinetics of the free- and Cu(2+)-bound peptide interaction to the α-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the α-HL pore and a Cu(2+)-free peptide is higher than that of a Cu(2+)-complexed peptide. We posit that mainly due to conformational changes induced by the bound Cu(2+) on the peptide, the resulting complex encounters a higher energy barrier toward its association with the protein pore, stemming most likely from an extra entropy cost needed to fit the Cu(2+)-complexed peptide within the α-HL lumen region. The lower dissociation rate constant of the Cu(2+)-complexed peptide from α-HL pore, as compared to that of Cu(2+)-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu(2+)-complexed peptide, which may be indicative of specific Cu(2+)-mediated contributions to the binding of the Cu(2+)-complexed peptide within the pore lumen.