Nanoscale potassium sensing based on valinomycin-anchored fluorescent gold nanoclusters.

Gold nanoclusters are a smart platform for sensing potassium ions (K+). They have been synthesized using bovine serum albumin (BSA) and valinomycin (Val) to protect and cap the nanoclusters. The nanoclusters (Val-AuNCs) produced have a red emission at 616 nm under excitation with 470 nm. In the presence of K+, the valinomycin polar groups switch to the molecule's interior by complexing with K+, forming a bracelet structure, and being surrounded by the hydrophobic exterior conformation. This structure allows a proposed fluorometric method for detecting K+ by switching between the Val-AuNCs' hydrophilicity and hydrophobicity, which induces the aggregation of gold nanoclusters. As a result, significant quenching is seen in fluorescence after adding K+. The quenching in fluorescence in the presence of K+ is attributed to the aggregation mechanism. This sensing technique provides a highly precise and selective sensing method for K+ in the range 0.78 to 8 µM with LOD equal to 233 nM. The selectivity of Val-AuNCs toward K+ ions was investigated compared to other ions. Furthermore, the Val-AuNCs have novel possibilities as favorable sensor candidates for various imaging applications. Our detection technique was validated by determining K+ ions in postmortem vitreous humor samples, which yielded promising results.

Theoretical investigation of hydroxylated analogues of valinomycin as potassium transporter.

Valinomycin is a potent ionophore known for its ability to transport potassium ions across biological membranes. The study focuses on the hydroxylated analogues of valinomycin (HyVLMs) and compares their energy profiles and capabilities for transporting potassium ions across phospholipid membranes. Using metadynamics, we investigated the energy profiles of wildtype valinomycin (VLM_1) and its three hydroxylated analogues (VLM_2, VLM_3, and VLM_4). We observed that all analogues exhibited energy maxima in the centre of the membrane and preferred positions below the phospholipid heads. Furthermore, the entry barriers for membrane penetration were similar among the analogues, suggesting that the hydroxyl group did not significantly affect their passage through the membrane. Transition state calculations provided insights into the ability of valinomycin analogues to capture potassium ions, with VLM_4 showing the lowest activation energy and VLM_2 displaying the highest. Our findings contribute to understanding the mechanisms of potassium transport by valinomycin analogues and highlight their potential as ionophores. The presence of the hydroxyl group is of particular importance because it paves the way for subsequent chemical modifications and the synthesis of new antiviral agents with reduced intrinsic toxicity.

Cation-responsive cavity expansion of valinomycin revealed by cryogenic ion trap infrared spectroscopy.

Valinomycin (VM) is a natural K+-selective ionophore that transports K+ through the cell membrane. VM captures K+ in its central cavity with a C3-symmetric ß-turn-like backbone. Although the binding affinity is drastically decreased for the VM-sodium (Na+VM) complex with respect to K+VM, VM holds relatively high affinity to Rb+ and Cs+. The high affinity for larger ions irrespective of ionic size seems to conflict with the expected optimal size matching model and raises questions on what factors determine ion selectivity. A combination of infrared spectroscopy with supporting computational calculations reveals that VM can accommodate larger Rb+ and Cs+ by flexibly changing its cavity size with the elongation of its folded ß-turn-like backbone. The high affinity to Rb+ and Cs+ can be ascribed to a size-dependent cavity expansion. These findings provide a new perspective on molecular recognition and selectivity beyond the conventional size matching model.

Discovery of Novel Cyclic Ethers with Synergistic Antiplasmodial Activity in Combination with Valinomycin.

With drug resistance threatening our first line antimalarial treatments, novel chemotherapeutics need to be developed. Ionophores have garnered interest as novel antimalarials due to their theorized ability to target unique systems found in the Plasmodium-infected erythrocyte. In this study, during the bioassay-guided fractionation of the crude extract of Streptomyces strain PR3, a group of cyclodepsipeptides, including valinomycin, and a novel class of cyclic ethers were identified and elucidated. Further study revealed that the ethers were cyclic polypropylene glycol (cPPG) oligomers that had leached into the bacterial culture from an extraction resin. Molecular dynamics analysis suggests that these ethers are able to bind cations such as K+, NH4+ and Na+. Combination studies using the fixed ratio isobologram method revealed that the cPPGs synergistically improved the antiplasmodial activity of valinomycin and reduced its cytotoxicity in vitro. The IC50 of valinomycin against P. falciparum NF54 improved by 4-5-fold when valinomycin was combined with the cPPGs. Precisely, it was improved from 3.75 ± 0.77 ng/mL to 0.90 ± 0.2 ng/mL and 0.75 ± 0.08 ng/mL when dosed in the fixed ratios of 3:2 and 2:3 of valinomycin to cPPGs, respectively. Each fixed ratio combination displayed cytotoxicity (IC50) against the Chinese Hamster Ovary cell line of 57-65 µg/mL, which was lower than that of valinomycin (12.4 µg/mL). These results indicate that combinations with these novel ethers may be useful in repurposing valinomycin into a suitable and effective antimalarial.

Rethinking Ion Transport by Ionophores: Experimental and Computational Investigation of Single Water Hydration in Valinomycin-K+ Complexes.

Valinomycin is a macrocyclic ionophore that transports K+ across hydrophobic membranes. Its function depends on selectivity, capture, transport, and release of the ion. While thermodynamics clearly indicate that valinomycin binds K+ preferentially over all other alkali ions, characterizing the capture/transport/release of K+ by valinomycin at the molecular level remains a challenge. The bracelet-like structure of valinomycin-K+ (K+VM) has the ion completely enveloped, facilitating transport through the cell membrane. We report that hydration by a single water molecule, (K+VM)(H2O), produces three different conformers, identified by infrared spectroscopy and supporting computational studies. For two minor conformers, the water prevents the ionophore from closing, a conformation that would inhibit diffusion through the membrane. However, the dominant conformer encloses both the ion and the water, replicating the bracelet-like K+VM and arguably enhancing diffusion through the membrane. This potential for active participation of water in transport through the hydrophobic cellular membrane has never been previously considered.

Effects of valinomycin doping on the electrical and structural properties of planar lipid bilayers supported on polyelectrolyte multilayers.

Supported Lipid Bilayers (SLBs) on Polyelectrolyte Multilayers (PEMs) have large potential as models for developing sensor devices. SLBs can be designed with receptors and channels, which benefit from the biological environment of the lipid layers, to create a sensing interface for ions and biomarkers. PEMs assembled by the Layer-by-Layer (LBL) technique and used as supports for a lipid bilayer enable an easy integration of the bilayer on almost any surface and device. For electrochemical sensors, LBL assembly enables nanoscale tunable separation of the lipid bilayer from the electrode surface, avoiding undesired effects of the electrode surface on the lipid bilayers. We study the fabrication of valinomycin-doped SLBs on PEMs as a model system for biophysical studies and for selective ion sensing. SLBs are fabricated from dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS) 50:50 vesicles doped with valinomycin, as a K+-selective carrier. SLBs were deposited on electrodes coated with poly(allyl amine hydrochloride) (PAH) and poly(styrene sodium sulfonate) (PSS) multilayers. Lipid bilayer formation was monitored by using Quartz Crystal Microbalance with Dissipation (QCMD) technique and Atomic Force Microscopy (AFM). Electrochemical impedance spectroscopy (EIS) and potentiometric measurements were performed to assess K+ selectivity over other ions and the potential of valinomycin-doped SLBs for K+-sensing.

Multipodal coordination and mobility of molecular cations inside the macrocycle valinomycin.

The macrocycle valinomycin displays an outstanding ability in cation binding and carriage across hydrophobic environments (e.g., cell membranes) and constitutes a central landmark for the design of novel ionophores for the regulation of biochemical processes. Most previous investigations have focused on the capture of metal cations (primarily K+). Here, we address the versatility of valinomycin in the encapsulation of molecular ions of small and moderate size, with NH4+ and H4PO4+ as case studies. A combination of infrared action vibrational spectroscopy and quantum chemical computations of molecular structure and dynamics is employed with the two-fold aim of assessing the dominant H-bonding coordination networks in the complexes and of characterizing the positional and rotational freedom of the guest cations inside the cavity of the macrocycle. Valinomycin binds NH4+ with only moderate distortion of the C3 configuration adopted in the complexes with the metal cations. The ammonium cation occupies the center of the cavity and displays two low-energy coordination arrangements that are dynamically connected through a facile rotation of the cation. The inclusion of the bulkier phosphoric acid cation demands significant stretching of the valinomycin backbone. Interestingly, the H4PO4+ cation achieves ample positional and rotational mobility inside valinomycin. The valinomycin backbone is capable of adopting barrel-like configurations when the cation occupies a region close to the center of the cavity, and funnel-like configurations when it diffuses to positions close to the exit face. This can accommodate the cation in varying coordination arrangements, characterized by different H-bonding between the four POH arms and the ester carbonyl groups of the macrocycle.

Polymorphic Forms of Valinomycin Investigated by NMR Crystallography.

A dodecadepsipeptide valinomycin (VLM) has been most recently reported to be a potential anti-coronavirus drug that could be efficiently produced on a large scale. It is thus of importance to study solid-phase forms of VLM in order to be able to ensure its polymorphic purity in drug formulations. The previously available solid-state NMR (SSNMR) data are combined with the plane-wave DFT computations in the NMR crystallography framework. Structural/spectroscopical predictions (the PBE functional/GIPAW method) are obtained to characterize four polymorphs of VLM. Interactions which confer a conformational stability to VLM molecules in these crystalline forms are described in detail. The way how various structural factors affect the values of SSNMR parameters is thoroughly analyzed, and several SSNMR markers of the respective VLM polymorphs are identified. The markers are connected to hydrogen bonding effects upon the corresponding (13C/15N/1H) isotropic chemical shifts of (CO, Namid, Hamid, Hα) VLM backbone nuclei. These results are expected to be crucial for polymorph control of VLM and in probing its interactions in dosage forms.

Ionophore-based pH independent detection of ions utilizing aggregation-induced effects.

Ionophores have been integrated into various electrochemical and optical sensing platforms for the selective detection of ions. Previous ionophore-based optical sensors rely on a H+ chromoionophore as the signal transducer and consequently, suffered from a pH cross-response. pH independent methods were proposed very recently by utilizing the solvatochromic dyes or the exhaustive mode. Here, we report a pH independent sensing principle based on nanospheres containing ionophores. As the ion-exchange occurs, the signal transducer undergoes aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ), leading to a dramatic change in fluorescence intensity. The principle was evaluated on different ionophores including those selective for K+, Na+, Ca2+, and Pb2+. The nanospheres were also introduced into microfluidic chips and successfully applied for the determination of sodium and potassium ion concentrations in diluted blood serum and urine samples.

How Valinomycin Ionophores Enter and Transport K+ across Model Lipid Bilayer Membranes.

Valinomycin, a cyclic peptide, was incorporated into a biomimetic lipid membrane tethered to the surface of a gold (111) electrode. Electrochemical impedance spectroscopy was used to study the ionophore properties of the peptide, and polarization modulation infrared reflection absorption spectroscopy was employed to determine the conformation and orientation of valinomycin in the membrane. The combination of these two techniques provided unique information about the ionophore mechanism where valinomycin transports ions across the membrane by creating a complex with potassium ions and forming an ion pair with a counter anion. The ion pair resides within the hydrophobic fragment of the membrane and adopts a small angle of ∼22° with respect to the surface normal. This novel study provides new insights explaining the valinomycin ion transport mechanism in model biological membranes.

Single-stranded DNA designed lipophilic G-quadruplexes as transmembrane channels for switchable potassium transport.

Single-stranded DNA designed G-quadruplexes, modified with lipophilic 12-carbon spacers and cholesterol to span lipid membranes, were developed as smart transmembrane channels for selective and switchable potassium ion (K+) transport across membranes.

Graphene Quantum Dots Integrated in Ionophore-Based Fluorescent Nanosensors for Na+ and K.

To enrich the recipes of ion-selective nanosensors, graphene quantum dots (GQDs) were integrated into ionophore-based fluorescent nanosensors with exquisite selectivity and high sensitivity for Na+ and K+. The unique property of GQDs gave the nanosensors ultrasmall size (ca. 10 nm), high brightness, good biocompatibility, and potential pH sensing possibility. At pH 7.4, the sensors exhibited a detection range from 0.1 mM to 1 M for Na+ and from 3 µM to 1 mM for K+. The nanosensors were successfully applied to blood serum and urine samples. Chemically induced intracellular sodium concentration change in HeLa cells was also qualitatively monitored.

Single-molecule insights into surface-mediated homochirality in hierarchical peptide assembly.

Homochirality is very important in the formation of advanced biological structures, but the origin and evolution mechanisms of homochiral biological structures in complex hierarchical process is not clear at the single-molecule level. Here we demonstrate the single-molecule investigation of biological homochirality in the hierarchical peptide assembly, regarding symmetry break, chirality amplification, and chirality transmission. We find that homochirality can be triggered by the chirality unbalance of two adsorption configuration monomers. Co-assembly between these two adsorption configuration monomers is very critical for the formation of homochiral assemblies. The site-specific recognition is responsible for the subsequent homochirality amplification and transmission in their hierarchical assembly. These single-molecule insights open up inspired thoughts for understanding biological homochirality and have general implications for designing and fabricating artificial biomimetic hierarchical chiral materials.

Fabrication of a solution-gated transistor based on valinomycin modified iron oxide nanoparticles decorated zinc oxide nanorods for potassium detection.

There are considerable interests to detect and monitor the abnormal level of minerals in water for avoiding/preventing any toxic effects after consumption. Herein, we report the fabrication of solution-gated field-effect-transistor (FET) based potassium sensor using iron oxide nanoparticles (Fe2O3 NPs) modified directly grown zinc oxide nanorods (ZnO NRs). The Fe2O3 NPs modification of ZnO NRs provided stability to nanorods surface and improved surface area for valinomycin immobilization. As-fabricated potassium sensor (valinomycin-Fe2O3 NPs-ZnO NRs/SiO2/Si) provided enhanced current response with increasing potassium concentration. During sensing measurements, FET sensor showed high sensitivity (4.65 µA/µM/cm2) in the linear range of 0.1 µM to 125 µM, low limit of detection (∼0.04 µM), good stability, excellent reproducibility, and favorable selectivity. Thus, good sensing performance of the FET based potassium sensor presents it as simple, low-cost, and convenient device for selective detection of potassium in solution.

Synthetic Ion Channels and DNA Logic Gates as Components of Molecular Robots.

A molecular robot is a next-generation biochemical machine that imitates the actions of microorganisms. It is made of biomaterials such as DNA, proteins, and lipids. Three prerequisites have been proposed for the construction of such a robot: sensors, intelligence, and actuators. This Minireview focuses on recent research on synthetic ion channels and DNA computing technologies, which are viewed as potential candidate components of molecular robots. Synthetic ion channels, which are embedded in artificial cell membranes (lipid bilayers), sense ambient ions or chemicals and import them. These artificial sensors are useful components for molecular robots with bodies consisting of a lipid bilayer because they enable the interface between the inside and outside of the molecular robot to function as gates. After the signal molecules arrive inside the molecular robot, they can operate DNA logic gates, which perform computations. These functions will be integrated into the intelligence and sensor sections of molecular robots. Soon, these molecular machines will be able to be assembled to operate as a mass microrobot and play an active role in environmental monitoring and in vivo diagnosis or therapy.

Metal-Assisted Folding of Prolinomycin Allows Facile Design of Functional Peptides.

Cyclic peptides have been proposed as privileged scaffolds that might mimic the folding and function of natural proteins. However, simple cyclic peptides typically cannot fold into well-defined structures. Herein, we describe a foldable cyclic peptide scaffold on which functional side chains can be displayed for targeted recognition of biomolecules. The foldable scaffold is based on prolinomycin, a proline-rich analogue of valinomycin. We report synthetic mutants of prolinomycin that retain the metal-assisted folding behavior under physiological conditions. The predictable structure formation of prolinomycin makes it a powerful platform to enable the development of synthetic receptors for biomolecules of interest. We demonstrate the potential of this scaffold by creating prolinomycin mutants that selectively bind anionic vesicles and bacterial cells.

The structure of a valinomycin-hexaaquamagnesium trifluoromethanesulfonate compound.

Valinomycin is a naturally occurring cyclic dodecadepsipeptide with the formula cyclo-[D-HiVA→L-Val →L-LA→L-Val]3 (D-HiVA is D-α-hydroxyisovaleic acid, Val is valine and LA is lactic acid), which binds a K(+) ion with high selectively. In the past, several cation-binding modes have been revealed by X-ray crystallography. In the K(+), Rb(+) and Cs(+) complexes, the ester O atoms coordinate the cation with a trigonal antiprismatic geometry, while the six amide groups form intramolecular hydrogen bonds and the network that is formed has a bracelet-like conformation (Type 1 binding). Type 2 binding is seen with the Na(+) cation, in which the valinomycin molecule retains the bracelet conformation but the cations are coordinated by only three ester carbonyl groups and are not centrally located. In addition, a picrate counter-ion and a water molecule is found at the center of the valinomycin bracelet. Type 3 binding is observed with divalent Ba(2+), in which two cations are incorporated, bridged by two anions, and coordinated by amide carbonyl groups, and there are no intramolecular amide hydrogen bonds. In this paper, we present a new Type 4 cation-binding mode, observed in valinomycin hexaaquamagnesium bis(trifluoromethanesulfonate) trihydrate, C54H90N6O18·[Mg(H2O)6](CF3SO3)2·3H2O, in which the valinomycin molecule incorporates a whole hexaaquamagnesium ion, [Mg(H2O)6](2+), via hydrogen bonding between the amide carbonyl groups and the hydrate water H atoms. In this complex, valinomycin retains the threefold symmetry observed in Type 1 binding, but the amide hydrogen-bond network is lost; the hexaaquamagnesium cation is hydrogen bonded by six amide carbonyl groups. (1)H NMR titration data is consistent with the 1:1 binding stoichiometry in acetonitrile solution. This new cation-binding mode of binding a whole hexaaquamagnesium ion by a cyclic polypeptide is likely to have important implications for the study of metal binding with biological models under physiological conditions.

Affinity Capillary Electrophoresis Applied to Investigation of Valinomycin Complexes with Ammonium and Alkali Metal Ions.

This chapter deals with the application of affinity capillary electrophoresis (ACE) to investigation of noncovalent interactions (complexes) of valinomycin, a macrocyclic dodecadepsipeptide antibiotic ionophore, with ammonium and alkali metal ions (lithium, sodium, potassium, rubidium, and cesium). The strength of these interactions was characterized by the apparent binding (stability, association) constants (K b) of the above valinomycin complexes using the mobility shift assay mode of ACE. The study involved measurements of effective electrophoretic mobility of valinomycin at variable concentrations of ammonium or alkali metal ions in the background electrolyte (BGE). The effective electrophoretic mobilities of valinomycin measured at ambient temperature and variable ionic strength were first corrected to the reference temperature 25 °C and constant ionic strength (10 or 25 mM). Then, from the dependence of the corrected valinomycin effective mobility on the ammonium or alkali metal ion concentration in the BGE, the apparent binding constants of the valinomycin-ammonium or valinomycin-alkali metal ion complexes were determined using a nonlinear regression analysis. Logarithmic form of the binding constants (log K b) were found to be in the range of 1.50-4.63, decreasing in the order Rb(+) > K(+) > Cs(+) > > Na(+) > NH4 (+) ~ Li(+).

Effects of Lipid Tethering in Extremophile-Inspired Membranes on H(+)/OH(-) Flux at Room Temperature.

This work explores the proton/hydroxide permeability (PH+/OH-) of membranes that were made of synthetic extremophile-inspired phospholipids with systematically varied structural elements. A fluorescence-based permeability assay was optimized to determine the effects on the PH+/OH- through liposome membranes with variations in the following lipid attributes: transmembrane tethering, tether length, and the presence of isoprenoid methyl groups on one or both lipid tails. All permeability assays were performed in the presence of a low concentration of valinomycin (10 nM) to prevent buildup of a membrane potential without artificially increasing the measured PH+/OH-. Surprisingly, the presence of a transmembrane tether did not impact PH+/OH- at room temperature. Among tethered lipid monolayers, PH+/OH- increased with increasing tether length if the number of carbons in the untethered acyl tail was constant. Untethered lipids with two isoprenoid methyl tails led to lower PH+/OH- values than lipids with only one or no isoprenoid tails. Molecular dynamics simulations revealed a strong positive correlation between the probability of observing water molecules in the hydrophobic core of these lipid membranes and their proton permeability. We propose that water penetration as revealed by molecular dynamics may provide a general strategy for predicting proton permeability through various lipid membranes without the need for experimentation.

Synthesis and Biological Evaluation of a Valinomycin Analog Bearing a Pentafluorophenyl Active Ester Moiety.

A valuable analog of the K(+)-ionophore valinomycin (1), bearing a pentafluorophenyl ester moiety, has been obtained by selective reaction between the tertiary hydroxyl moiety of analog 2 (available from valinomycin hydroxylation) and the isocyanate group of pentafluorophenyl N-carbonyl glycinate (3) catalyzed by bis(N,N-dimethylformamide)dichlorodioxomolybdenum(VI). LC-HRMS studies show that analog 4 undergoes easy derivatization under mild conditions by reaction with OH- and NH2-containing compounds. Mitochondrial depolarization assays suggest that 4 acts as a K(+)-ionophore, provided that the glycine carboxyl group is appropriately masked.