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Hypothesis: We hypothesize that simultaneous incorporation of ion channel peptides (in this case, potassium channel as a model) and hydrophobic magnetite Fe 3 O 4 nanoparticles (hFe 3 O 4 NPs) within lipidic hexagonal mesophases, and aligning them using an external magnetic field can significantly enhance ion transport through lipid membranes., Experiments: In this study, we successfully characterized the incorporation of gramicidin membrane ion channels and hFe 3 O 4 NPs in the lipidic hexagonal structure using SAXS and cryo-TEM methods. Additionally, we thoroughly investigated the conductive characteristics of freestanding films of lipidic hexagonal mesophases, both with and without gramicidin potassium channels, utilizing a range of electrochemical techniques, including impedance spectroscopy, normal pulse voltammetry, and chronoamperometry., Findings: Our research reveals a state-of-the-art breakthrough in enhancing ion transport in lyotropic liquid crystals as matrices for integral proteins and peptides. We demonstrate the remarkable efficacy of membranes composed of hexagonal lipid mesophases embedded with K + transporting peptides. This enhancement is achieved through doping with hFe 3 O 4 NPs and exposure to a magnetic field. We investigate the intricate interplay between the conductive properties of the lipidic hexagonal structure, hFe 3 O 4 NPs, gramicidin incorporation, and the influence of Ca 2+ on K + channels. Furthermore, our study unveils a new direction in ion channel studies and biomimetic membrane investigations, presenting a versatile model for biomimetic membranes with unprecedented ion transport capabilities under an appropriately oriented magnetic field. These findings hold promise for advancing membrane technology and various biotechnological and biomedical applications of membrane proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The utilization of biomimetic membranes supported by advanced self-assembled monolayers is gaining attraction as a promising sensing tool. Biomimetic membranes offer exceptional biocompatibility and adsorption capacity upon degradation, transcending their role as mere research instruments to open new avenues in biosensing. This study focused on anchoring a sparsely tethered bilayer lipid membrane onto a self-assembled monolayer composed of a biodegradable polymer, functionalized with poly(ethylene glycol)-cholesterol moieties, for lipid membrane integration. Real-time monitoring via quartz crystal microbalance, coupled with characterization using surface-enhanced infrared absorption spectroscopy and electrochemical impedance spectroscopy, provided comprehensive insights into each manufacturing phase. The resulting lipid layer, along with transmembrane pores formed by gramicidin A, exhibited robust stability. Electrochemical impedance spectroscopy analysis confirmed membrane integrity, successful pore formation, and consistent channel density. Notably, gramicidin A demonstrated sustained functionality as an ion channel upon reconstitution, with its functionality being effectively blocked and inhibited in the presence of calcium ions. These findings mark significant strides in developing intricate biodegradable nanomaterials with promising applications in biomedicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Proton transport across lipid membranes is one of the most fundamental reactions that make up living organisms. In vitro, however, the study of proton transport reactions can be very challenging due to limitations imposed by proton concentrations, compartment size, and unstirred layers as well as buffer exchange and buffer capacity. In this study, we have developed a proton permeation assay based on the microfluidic trapping of giant vesicles enclosing the pH-sensitive dye pyranine to address some of these challenges. Time-resolved fluorescence imaging upon a rapid pH shift enabled us to investigate the facilitated H + permeation mediated by either a channel or a carrier. Specifically, we compared the proton transport rates as a function of different proton gradients of the channel gramicidin D and the proton carrier carbonyl cyanide-m-chlorophenyl hydrazone. Our results demonstrate the efficacy of the assay in monitoring proton transport reactions and distinguishing between a channel-like and a carrier-like mechanism. This groundbreaking result enabled us to elucidate the enigmatic mode of the proton permeation mechanism of the recently discovered natural fibupeptide lugdunin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gramicidin S (GS), one of the first discovered antimicrobial peptides, still shows strong antibiotic activity after decades of clinical use, with no evidence of resistance. The relatively high hemolytic activity and narrow therapeutic window of GS limit its use in topical applications. Encapsulation and targeted delivery may be the way to develop the internal administration of this drug. The lipid composition of membranes and non-covalent interactions affect GS's affinity for and partitioning into lipid bilayers as monomers or oligomers, which are crucial for GS activity. Using both differential scanning calorimetry (DSC) and FTIR methods, the impact of GS on dipalmitoylphosphatidylcholine (DPPC) membranes was tested. Additionally, the combined effect of GS and cholesterol on membrane characteristics was observed; while dipalmitoylphosphatydylglycerol (DPPG) and cerebrosides did not affect GS binding to DPPC membranes, cholesterol significantly altered the membrane, with 30% mol concentration being most effective in enhancing GS binding. The effect of star-like dextran-polyacrylamide D-g-PAA(PE) on GS binding to the membrane was tested, revealing that it interacted with GS in the membrane and significantly increased the proportion of GS oligomers. Instead, calcium ions affected GS binding to the membrane differently, with independent binding of calcium and GS and no interaction between them. This study shows how GS interactions with lipid membranes can be effectively modulated, potentially leading to new formulations for internal GS administration. Modified liposomes or polymer nanocarriers for targeted GS delivery could be used to treat protein misfolding disorders and inflammatory conditions associated with free-radical processes in cell membranes.

Spectrally-resolved single-molecule localization microscopy (srSMLM) has emerged as a powerful tool for exploring the spectral properties of single emitters in localization microscopy. By simultaneously capturing the spatial positions and spectroscopic signatures of individual fluorescent molecules, srSMLM opens up the possibility of investigating an additional dimension in super-resolution imaging. However, appropriate and dedicated tools are required to fully capitalize on the spectral dimension. Here, we propose the application of the spectral phasor analysis as an effective method for summarizing and analyzing the spectral information obtained from srSMLM experiments. The spectral phasor condenses the complete spectrum of a single emitter into a two-dimensional space, preserving key spectral characteristics for single-molecule spectral exploration. We demonstrate the effectiveness of spectral phasor in efficiently classifying single Nile Red fluorescence emissions from largely overlapping cyanine fluorescence signals in dual-color PAINT experiments. Additionally, we employed spectral phasor with srSMLM to reveal subtle alterations occurring in the membrane of Gram-positive Enterococcus hirae in response to gramicidin exposure, a membrane-perturbing antibiotic treatment. Spectral phasor provides a robust, model-free analytic tool for the detailed analysis of the spectral component of srSMLM, enhancing the capabilities of multi-color spectrally-resolved single-molecule imaging., (© 2024 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)

Gramicidin A (gA) is a short hydrophobic β-helical peptide that forms cation-selective channels in lipid membranes in the course of transbilayer dimerization. The length of the gA helix is smaller than the thickness of a typical lipid monolayer. Consequently, elastic deformations of the membrane arise in the configurations of gA monomers, conducting dimer, and the intermediate state of coaxial pair, where gA monomers from opposing membrane monolayers are located one on top of the other. The gA channel is characterized by the average lifetime of the conducting state. The elastic properties of the membrane influence the average lifetime, thus making gA a convenient sensor of membrane elasticity. However, the utilization of gA to investigate the elastic properties of mixed membranes comprising two or more components frequently relies on the assumption of ideality, namely that the elastic parameters of mixed-lipid bilayers depend linearly on the concentrations of the components. Here, we developed a general approach that does not rely on the aforementioned assumption. Instead, we explicitly accounted for the possibility of inhomogeneous lateral distribution of all lipid components, as well as for membrane-mediated lateral interactions of gA monomers, dimer, coaxial pair, and minor lipid components. This approach enabled us to derive unknown elastic parameters of lipid monolayer from experimentally determined lifetimes of gA channel in mixed-lipid bilayers. A general algorithm was formulated that allows the unknown elastic parameters of a lipid monolayer to be obtained using gA as a mechanical sensor.

Ion channels are membrane proteins that allow ionic signals to pass through channel pores for biofunctional modulations. However, biodevices that integrate bidirectional biological signal transmission between a device and biological converter through supported lipid bilayers (SLBs) while simultaneously controlling the process are lacking. Therefore, in this study, we aimed to develop a hybrid biotransducer composed of ATP synthase and proton channel gramicidin A (gA), controlled by a sulfonated polyaniline (SPA) conducting polymer layer deposited on a microelectrode, and to simulate a model circuit for this system. We controlled proton transport across the gA channel using both electrical and chemical input signals by applying voltage to the SPA or introducing calcium ions (inhibitor) and ethylenediaminetetraacetic acid molecules (inhibitor remover). The insertion of gA and ATP synthase into SLBs on microelectrodes resulted in an integrated biotransducer, in which the proton current was controlled by the flux of adenosine diphosphate molecules and calcium ions. Lastly, we created an XOR logic gate as an enzymatic logic system where the output proton current was controlled by Input A (ATP synthase) and Input B (calcium ions), making use of the unidirectional and bidirectional transmission of protons in ATP synthase and gA, respectively. We combined gA, ATP synthase, and SPA as a hybrid bioiontronics system to control bidirectional or unidirectional ion transport across SLBs in biotransducers. Thus, our findings are potentially relevant for a range of advanced biological and medical applications.

Using the gramicidin A channel as a molecular probe, we show that tubulin binding to planar lipid membranes changes the channel kinetics-seen as an increase in the lifetime of the channel dimer-and thus points towards modification of the membrane's mechanical properties. The effect is more pronounced in the presence of non-lamellar lipids in the lipid mixture used for membrane formation. To interpret these findings, we propose that tubulin binding redistributes the lateral pressure of lipid packing along the membrane depth, making it closer to the profile expected for lamellar lipids. This redistribution happens because tubulin perturbs the lipid headgroup spacing to reach the membrane's hydrophobic core via its amphiphilic α-helical domain. Specifically, it increases the forces of repulsion between the lipid headgroups and reduces such forces in the hydrophobic region. We suggest that the effect is reciprocal, meaning that alterations in lipid bilayer mechanics caused by membrane remodeling during cell proliferation in disease and development may also modulate tubulin membrane binding, thus exerting regulatory functions. One of those functions includes the regulation of protein-protein interactions at the membrane surface, as exemplified by VDAC complexation with tubulin.

Antimicrobial peptides (AMPs) are promising therapeutic agents against bacteria. We have previously reported an amphipathic AMP Stripe composed of cationic L-Lys and hydrophobic L-Leu/L-Ala residues, and Stripe exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria. Gramicidin A (GA), composed of repeating sequences of L- and D-amino acids, has a unique β 6.3 -helix structure and exhibits broad antimicrobial activity. Inspired by the structural properties and antimicrobial activities of LD-alternating peptides such as GA, in this study, we designed Stripe derivatives with LD-alternating sequences. We found that simply alternating L- and D-amino acids in the Stripe sequence to give StripeLD caused a reduction in antimicrobial activity. In contrast, AltStripeLD, with cationic and hydrophobic amino acids rearranged to yield an amphipathic distribution when the peptide adopts a β 6.3 -helix, displayed higher antimicrobial activity than AltStripe. These results suggest that alternating L-/D-cationic and L-/D-hydrophobic amino acids in accordance with the helical structure of an AMP may be a useful way to improve antimicrobial activity and develop new AMP drugs.

Gramicidin S, natural antimicrobial peptide is used commercially in medicinal lozenges for sore throat and Gram-negative and Gram-positive bacterial infections. However, its clinical potential is limited to topical applications because of its high red blood cells (RBC) cytotoxicity. Given the importance of developing potential antibiotics and inspired by the cyclic structure and druggable features of Gramicidin S, we edited proline α-carbon with stereodynamic nitrogen to examine the direct impact on biological activity and cytotoxicity with respect to prolyl counterpart. Natural Gramicidin S (12), proline-edited peptides 13-16 and wild-type d-Phe-d-Pro β-turn mimetics (17 and 18) were synthesized using solid phase peptide synthesis and investigated their activity against clinically relevant bacterial pathogens. Interestingly, mono-proline edited analogous peptide 13 showed moderate improvement in antimicrobial activity against E. coli ATCC 25922 and K.pneumoniae BAA 1705 as compared to Gramicidin S. Furthermore, proline edited peptide 13 exhibited equipotent antimicrobial effect against MDR S. aureus and Enterococcus spp. Analysis of cytotoxicity against VERO cells and RBC, reveals that proline edited peptides showed two-fivefold lesser cytotoxicity than the counterpart Gramicidin S. Our study suggests that introducing single azPro/Pro mutation in Gramicidin S marginally improved the activity and lessens the cytotoxicity as compared with the parent peptide., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

A novel strategy to treat Staphylococcus aureus (S. aureus) skin infections is presented, where UV light is used to facilitate concomitant light-controlled activation and delivery of an antimicrobial therapeutic agent. Specifically, a new photoswitchable gramicidin S analogue was immobilized onto a polymeric wearable patch via a photocleavable linker that undergoes photolysis at the same wavelength of light required for activation of the peptide. Unlike toxic gramicidin S, the liberated active photoswitchable peptide exhibits antimicrobial activity against S. aureus while being ostensibly non-haemolytic to red blood cells. Moreover, irradiation with visible light switches off the antimicrobial properties of the peptide within seconds, presenting an ideal strategy to regulate antibiotic activity for localized bacterial infections with the potential to mitigate resistance., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Membranes are important sites of intermolecular interactions in biological systems. However, they present significant analytical challenges as they contain multiple analytes and are dynamic in nature. In this work, we show how a Jasco J-1500 circular dichroism spectropolarimeter can be used with a microvolume Couette flow cell and appropriate cut-off filters to measure excitation fluorescence detected linear dichroism (FDLD) of fluorophores embedded in liposomal membranes. The result is a spectrum that selectively probes the fluorophore(s) and eliminates the scattering that is apparent in the corresponding flow linear dichroism (LD) spectrum. The FDLD spectrum is opposite in sign from the LD spectrum with relative magnitudes modified by the quantum yields of the transitions. FDLD thus enables analyte orientations to be identified in a membrane. Data for a membrane peptide, gramicidin, and two aromatic analytes, anthracene and pyrene, are presented. Issues with the "leakage" of photons by the long pass filters used is also discussed., (© 2023 The Authors. Chirality published by Wiley Periodicals LLC.)

We report on the structure of Gramicidin S (GS) in a model membrane mimetic environment represented by the amphipathic solvent 1-octanol using one-dimensional (1D) and two-dimensional (2D) IR spectroscopy. To explore potential structural changes of GS, we also performed a series of spectroscopic measurements at differing temperatures. By analyzing the amide I band and using 2D-IR spectral changes, results could be associated to the disruption of aggregates/oligomers, as well as structural and conformational changes happening in the concentrated solution of GS. The ability of 2D-IR to enable differentiation in melting transitions of oligomerized GS structures is attributed to the sensitivity of the technique to vibrational coupling. Two melting transition temperatures were identified; at T m1 in the range 41-47 °C where the GS aggregates/oligomers disassemble and at T m2 = 57 ± 2 °C where there is significant change involving GS β-sheet-type hydrogen bonds, whereby it is proposed that there is loss of interpeptide hydrogen bonds and we are left with mainly intrapeptide β-sheet and β-turn hydrogen bonds of the smaller oligomers. Further analysis with quantum mechanical/molecular mechanics (QM/MM) simulations and second derivative results highlighted the participation of active GS side chains. Ultimately, this work contributes toward understanding the GS structure and the formulation of GS analogues with improved bioactivity.

Identification of the most stable structure(s) of a system is a prerequisite for the calculation of any of its properties from first-principles. However, even for relatively small molecules, exhaustive explorations of the potential energy surface (PES) are severely hampered by the dimensionality bottleneck. In this work, we address the challenging task of efficiently sampling realistic low-lying peptide coordinates by resorting to a surrogate based genetic algorithm (GA)/density functional theory (DFT) approach (sGADFT) in which promising candidates provided by the GA are ultimately optimized with DFT. We provide a benchmark of several computational methods (GAFF, AMOEBApro13, PM6, PM7, DFTB3-D3(BJ)) as possible prescanning surrogates and apply sGADFT to two test case systems that are (i) two isomer families of the protonated Gly-Pro-Gly-Gly tetrapeptide (Masson, A.; J. Am. Soc. Mass Spectrom. 2015, 26, 1444-1454) and (ii) the doubly protonated cyclic decapeptide gramicidin S (Nagornova, N. S.; J. Am. Chem. Soc. 2010, 132, 4040-4041). We show that our GA procedure can correctly identify low-energy minima in as little as a few hours. Subsequent refinement of surrogate low-energy structures within a given energy threshold (≤10 kcal/mol (i), ≤5 kcal/mol (ii)) via DFT relaxation invariably led to the identification of the most stable structures as determined from high-resolution infrared (IR) spectroscopy at low temperature. The sGADFT method therefore constitutes a highly efficient route for the screening of realistic low-lying peptide structures in the gas phase as needed for instance for the interpretation and assignment of experimental IR spectra.

The chase toward endowing chemical compounds with machine-like functions mimicking those of biological molecular machineries has yielded a variety of artificial molecular motors (AMMs). Pharmaceutical applications of photoexcited monomolecular unidirectionally-rotating AMMs have been envisioned in view of their ability to permeabilize biological membranes. Nonetheless, the mechanical properties of lipid membranes render the proposed drilling activity of AMMs doubtful. Here, we show that singlet oxygen released by a photoexcited "molecular drill" oxidized unsaturated lipids composing giant unilamellar vesicles. In contrast, giant liposomes built of saturated lipids were inert to AMM photoactuation. The AMM did not mechanically destroy gramicidin A ion channels in planar bilayer lipid membranes but instead photoinactivated them. Sodium azide, a singlet oxygen quencher, reduced both AMM-mediated light-induced dye release from unsaturated large unilamellar vesicles and protected gramicidin A from photoinactivation. Upon additional consideration of the underlying bilayer mechanics, we conclude that AMMs' envisioned therapeutic and pharmaceutical applications rely on their photodynamic activity rather than their nanomechanical drilling abilities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The linear 15-mer peptide gramicidin A (gA) produced by Bacillus brevis is known to form the simplest natural ion channel in lipid membranes representing a head-to-head transmembrane dimer. Its incorporation into a planar lipid bilayer manifests itself in regular electrical current transitions. If two gA subunits are tightly connected by a water-soluble, flexible linker of a certain length, the current transitions become heterogeneous: in a part of them, the amplitude is almost twofold higher than that of a single channel, thereby demonstrating the synchronous opening of two single channels. The lifetime, i.e. the open-state duration, of this dual channel is by several orders of magnitude longer than that of the single channel. Here, we used the ideas of the theory of excitons to hypothesize about the mechanism of synchronous opening and closing of two adjacent channels. Two independent (uncoupled) single channels can be described by two independent conformational coordinates q 1 and q 2, while two closely located channels can exhibit collective behavior, if the coupling between them produces mixing of the individual states ( q 1,0) and (0, q 2). We suppose that a similar phenomenon can occur not only with synthetic derivatives of gA, but also with such natural channel-forming peptide antibiotics and toxins as alamethicin and syringomycin. In particular, channel clustering observed with these peptides may be also associated with formation of collective conductance states, resulting from mixing of their monomeric states, which allows us to explain the fact that clusters of these channels transmit ions and nonelectrolytes of the same size as the original single channels.

Gramicidin A (gA) is a linear antimicrobial peptide that can form a channel and specifically conduct monovalent cations such as H + across the lipid membrane. The antimicrobial activity of gA is associated with the formation of hydroxyl free radicals and the imbalance of NADH metabolism, possibly a consequence caused by the conductance of cations. The ion conductivity of gramicidin A can be blocked by Ca 2+ ions. However, the effect of Ca 2+ ions on the antimicrobial activity of gA is unclear. To unveil the role of Ca 2+ ions, we examined the effect of Ca 2+ ions on the antimicrobial activity of gramicidin A against Staphylococcus aureus ( S. aureus ). Results showed that the antimicrobial mechanism of gA and antimicrobial activity by Ca 2+ ions are concentration-dependent. At the low gA concentration (≤1 μM), the antimicrobial mechanism of gA is mainly associated with the hydroxyl free radical formation and NADH metabolic imbalance. Under this mode, Ca 2+ ions can significantly inhibit the hydroxyl free radical formation and NADH metabolic imbalance. On the other hand, at high gA concentration (≥5 μM), gramicidin A acts more likely as a detergent. Gramicidin A not only causes an increase in hydroxyl free radical levels and NAD + /NADH ratios but also induces the destruction of the lipid membrane composition. At this condition, Ca 2+ ions can no longer reduce the gA antimicrobial activity but rather enhance the bacterial killing ability of gramicidin A., Competing Interests: The authors declare no conflicts of interest.

Many sophisticated approaches for analyzing properties of chiral matter have been developed in recent years. But in general, the available chiroptical methods are limited to either solvated or small gaseous molecules. Studying the chirality of large biopolymers in the gas phase, including aspects of the secondary structure, becomes accessible by combining the electrospray ionization technique with chiroptical detection protocols. Here, laser-induced photodetachment from gramicidin anions, a peptide consisting of 15 amino acids has been investigated. The angular distribution of photoelectrons is demonstrated to be sensitive to the substitution of protons by cesium ions, which is accompanied by a conformational change. The photoelectron circular dichroism (PECD) is -0.5% for bare gramicidin, whereas gramicidin with several Cs + ions attached exhibits a PECD of +0.5%. The results are complemented and supported by ion mobility studies. The presented approach offers the prospect of studying chirality and the secondary structure of various biopolymers.

The lipid cubic phase (LCP) is a nanomaterial composed of water channels surrounded by lipid bilayers. LCPs are stable at room temperature and are biocompatible. These features make the lipid cubic phases similar to biological membranes, and hence, are favorable for embedding membrane proteins. We show that the monoolein cubic phase deposited on the electrode forms a 3D lipid bilayer film convenient for electrochemical investigations of membrane proteins. In this research, we studied the effect of embedding an ionophoric peptide, gramicidin A (gA), on the structure and properties of the LCP film. The phase identity and structural parameters of the gramicidin-doped phase were characterized by small-angle X-ray scattering (SAXS). The potassium ion transport through the film were studied by electroanalytical methods: alternating current voltammetry (ACV), chronoamperometry (CA) and electrochemical impedance spectroscopy (EIS). Increased values for the current of the gramicidin-doped cubic phase compared to the empty cubic phase and changes of the EIS parameters confirmed that the peptide remained in the film in its active dimeric form. Our results show that the LCP can be considered a suitable 3D biomimetic film for the investigation of ion channels and other transporting membrane proteins, and for their application in electrochemical sensors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Natural protein channels have evolved with exquisite structures to transport ions selectively and rapidly. Learning from nature to construct biomimetic artificial channels is always challenging. Herein we present a unimolecular transmembrane proton channel by quinoline-derived helix, which exhibited highly selective and ultrafast proton transport behaviors. This helix-based channel possesses a small luminal cavity of 1 Å in diameter, which could efficiently reject the permeation of cations, anions or water molecules but only permits the translocation of protons owing to the size effect. The proton flow rate exceeded 10 7 H + s -1 channel -1 and reached the same magnitude with gramicidin A. Mechanism investigation revealed that the directionally arrayed NH-chain inside the synthetic channel played a pivotal role during the proton flux. This work not only presented a helix-based channel with the smallest observable nanopore, but also unveiled an unexplored pathway for realizing efficient transport of protons via the consecutive NH-chain.

Membrane proteins can be regulated by alterations in material properties intrinsic to the hosting lipid bilayer. Here, we investigated whether the reversible photoisomerization of bilayer-embedded diacylglycerols (OptoDArG) with two azobenzene-containing acyl chains may trigger such regulatory events. We observed an augmented open probability of the mechanosensitive model channel gramicidin A (gA) upon photoisomerizing OptoDArG's acyl chains from trans to cis: integral planar bilayer conductance brought forth by hundreds of simultaneously conducting gA dimers increased by typically >50% - in good agreement with the observed increase in single-channel lifetime. Further, (i) increments in the electrical capacitance of planar lipid bilayers and protrusion length of aspirated giant unilamellar vesicles into suction pipettes, as well as (ii) changes of small-angle X-ray scattering of multilamellar vesicles indicated that spontaneous curvature, hydrophobic thickness, and bending elasticity decreased upon switching from trans- to cis-OptoDArG. Our bilayer elasticity model for gA supports the causal relationship between changes in gA activity and bilayer material properties upon photoisomerization. Thus, we conclude that photolipids are deployable for converting bilayers of potentially diverse origins into light-gated actuators for mechanosensitive proteins., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The demand for more efficient methods of establishing the undetermined stereochemistries of peptidic natural products continues unabated. A new method for microscale stereochemical determination was devised by integrating solid-phase synthesis, split-and-mix randomization, 18 O/ 16 O-encoding of d/l-configurations, tandem mass spectrometry, and biological evaluation. Here we applied gramicidin A as the molecule for a blind test. Gramicidin A and its 31 diastereomers were randomly prepared in microgram scale with 18 O/ 16 O-stereochemical encoding and subjected to MS/MS-structural determination and cytotoxicity assay. Only the parent gramicidin A was selected from among the 32 stereoisomers, validating the high reliability of the present strategy., (© 2021 Wiley-VCH GmbH.)

We use the quantum-classical path integral (QCPI) methodology to investigate the relaxation dynamics of an excess proton that has been inserted in a water dimer embedded in the gramicidin A channel at room temperature. We obtain one-dimensional potential slices for the quantum degree of freedom through a proper transformation to internal coordinates. Our results indicate that the proton transfer is driven by the oscillation of the oxygen pair, and that the transfer occurs primarily at single-well or nearby low-barrier configurations. Yet, we find that tunneling and zero-point energy lead to a significant acceleration of the proton transfer dynamics.

A series of glycoside-peptide conjugates were prepared by engineering at the N-terminus of the natural peptide gramicidin A. The conjugate containing galactose moiety formed a unimolecular transmembrane channel and mediated ion transport to induce apoptosis of cancer cells. More importantly, it exhibited liver cancer cell-targeting behavior due to the galactose-asialoglycoprotein receptor recognition.

Antimicrobial peptides (AMPs) are promising antibacterial agents often hindered by their undesired hemolytic activity. Inspired by gramicidin S (GS), a well-known cyclodecapeptide, we synthesized a panel of antibacterial cyclopeptidomimetics using β,γ-diamino acids (β,γ-DiAAs). We observed that peptidomimetic CP-2 displays a bactericidal activity similar to that of GS while possessing lower side-effects. Moreover, extensive studies revealed that CP-2 likely kills bacteria through membrane disruption. Altogether, CP-2 is a promising membrane-active antibiotic with therapeutic potential., (© 2020 Wiley-VCH GmbH.)

We investigated gramicidin A (gA) subunit dimerization in lipid bilayers using microsecond-long replica-exchange umbrella sampling simulations, millisecond-long unbiased molecular dynamics simulations, and machine learning. Our simulations led to a dimer structure that is indistinguishable from the experimentally determined gA channel structures, with the two gA subunits joined by six hydrogen bonds (6HB). The simulations also uncovered two additional dimer structures, with different gA-gA stacking orientations that were stabilized by four or two hydrogen bonds (4HB or 2HB). When examining the temporal evolution of the dimerization, we found that two bilayer-inserted gA subunits can form the 6HB dimer directly, with no discernible intermediate states, as well as through paths that involve the 2HB and 4HB dimers.

The structure of an ornithine (Orn)-free Gramicidin S (GS) analogue, cyclo(Val-Nle-Leu-D-Phe-Pro) 2 (NGS), was studied. Its circular dichroism (CD) spectrum showed that NGS has a structure similar to GS, though the value of [θ] indicated smaller β-turn and sheet populations. This is probably because the Nle side chain could not form intramolecular hydrogen bonds stabilizing the sheet structure. The chemical shift perturbation of αH and J NH-αH were similar in GS and NGS. Three independent NGS molecules formed intramolecular β-sheet structures in crystal. The turn structures of D-Phe-Pro moieties were classed as type II' β-turns, but one part was unclassed. The molecules were arranged in a twisting manner, which resulted in the formation of a helical sheet. Similar structural characteristics were observed previously in a Leu-type, Orn-free GS analogue and in GS trifluoroacetic acid salt.

A class of unimolecular channels formed by pillararene-gramicidin hybrid molecules are presented. The charge status of the peptide domain in these channels has a significant impact on their ion transport and antimicrobial activity. These channels exhibited different membrane-association abilities between microbial cells and mammalian cells. One of the channels displayed a higher antimicrobial activity towards S. aureus (IC 50 = 0.55 μM) and negligible hemolytic toxicity, showing potential to serve as a systemic antibiotic.

Understanding the mechanisms of nanoparticle interaction with cell membranes is essential for designing materials for applications such as bioimaging and drug delivery, as well as for assessing engineered nanomaterial safety. Much attention has focused on nanoparticles that bind strongly to biological membranes or induce membrane damage, leading to adverse impacts on cells. More subtle effects on membrane function mediated via changes in biophysical properties of the phospholipid bilayer have received little study. Here, we combine electrophysiology measurements, infrared spectroscopy, and molecular dynamics simulations to obtain insight into a mode of nanoparticle-mediated modulation of membrane protein function that was previously only hinted at in prior work. Electrophysiology measurements on gramicidin A (gA) ion channels embedded in planar suspended lipid bilayers demonstrate that anionic gold nanoparticles (AuNPs) reduce channel activity and extend channel lifetimes without disrupting membrane integrity, in a manner consistent with changes in membrane mechanical properties. Vibrational spectroscopy indicates that AuNP interaction with the bilayer does not perturb the conformation of membrane-embedded gA. Molecular dynamics simulations reinforce the experimental findings, showing that anionic AuNPs do not directly interact with embedded gA channels but perturb the local properties of lipid bilayers. Our results are most consistent with a mechanism in which anionic AuNPs disrupt ion channel function in an indirect manner by altering the mechanical properties of the surrounding bilayer. Alteration of membrane mechanical properties represents a potentially important mechanism by which nanoparticles induce biological effects, as the function of many embedded membrane proteins depends on phospholipid bilayer biophysical properties., Competing Interests: The authors declare no competing interest.

Partitioning of bioactive molecules, including drugs, into cell membranes may produce indiscriminate changes in membrane protein function. As a guide to safe drug development, it therefore becomes important to be able to predict the bilayer-perturbing potency of hydrophobic/amphiphilic drugs candidates. Toward this end, we exploited gramicidin channels as molecular force probes and developed in silico and in vitro assays to measure drugs' bilayer-modifying potency. We examined eight drug-like molecules that were found to enhance or suppress gramicidin channel function in a thick 1,2-dierucoyl- sn -glycero-3-phosphocholine (DC 22:1 PC) but not in thin 1,2-dioleoyl- sn -glycero-3-phosphocholine (DC 18:1 PC) lipid bilayer. The mechanism underlying this difference was attributable to the changes in gramicidin dimerization free energy by drug-induced perturbations of lipid bilayer physical properties and bilayer-gramicidin interactions. The combined in silico and in vitro approaches, which allow for predicting the perturbing effects of drug candidates on membrane protein function, have implications for preclinical drug safety assessment.

Human African trypanosomiasis (HAT) is caused by Trypanosoma brucei parasites. The T. brucei aquaglyceroporin isoform 2, TbAQP2, has been linked to the uptake of pentamidine. Negative membrane potentials and transmembrane pH gradients were suggested to promote transport of the dicationic antitrypanosomal drug. Application of ionophores to trypanosomes further hinted at direct inhibition of TbAQP2 by carbonyl cyanide m -chlorophenyl hydrazone (CCCP). Here, we tested for direct effects of three classical ionophores (CCCP, nigericin, gramicidin) on the functionality of TbAQP2 and the related TbAQP3 at conditions that are independent from the membrane potential or a proton gradient. We expressed TbAQP2 and TbAQP3 in yeast, and determined permeability of uncharged glycerol at neutral pH using stopped-flow light scattering. The mobile proton carrier CCCP directly inhibited TbAQP2 glycerol permeability at an IC 50 of 2 µM, and TbAQP3 to a much lesser extent (IC 50 around 1 mM) likely due to different selectivity filter layouts. Nigericin, another mobile carrier, left both isoforms unaffected. The membrane-integral pore-forming gramicidin evenly inhibited TbAQP2 and TbAQP2 in the double-digit micromolar range. Our data exemplify the need for suitable controls to detect unwanted ionophore side effects even when used at concentrations that are typically recommended to disturb the transmembrane ion distribution.

Gramicidin A (1) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of 1 with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of 1. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of 1 that selectively induce toxicity toward target organisms.

Plasmon resonance frequency irradiated gold nanoparticles (GNPs) have gained interest as a laser-targeted treatment for infections, tumors and for the controlled release of drugs in situ. Questions still remain, however, as to the efficiency of heat delivery within biological tissues and how this can be reliably determined. Here, we demonstrate how a nanomaterial-electrode interface that mimics cell membranes can detect the localized heat transfer characteristics arising from plasmon resonance frequency-matched laser excitation of GNPs. We demonstrate that the lipid bilayer membrane can be affected by conjugated GNP induced hyperthermia when irradiated with a laser power output as low as 135 nW/μm 2 . This is four orders of magnitude lower power than previously reported. By restricting the lateral movement of the lipids in the bilayer membrane, it was shown that the change in membrane conductance as a result of the heat transfer was due to the creation of transient lipidic toroidal pores within the membrane. We further demonstrate that the heat transfer from the GNPs alters diffusion rates of monomers of the gramicidin-A peptide within the lipid leaflets. This work highlights how targeted low laser power GNP hyperthermia treatments, in vivo, could play a dual role of interfering with both cell membrane morphology and dynamics, along with membrane protein function., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prof Bruce Cornell is Director – Science and Technology at Surgical Diagnostics P/L., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Gramicidin A (gA) forms several convertible conformations in different environments. In this study, we investigated the effect of calcium halides on the molecular state and antimicrobial activity of gramicidin A. The molecular state of gramicidin A is highly affected by the concentration of calcium salt and the type of halide anion. Gramicidin A can exist in two states that can be characterized by circular dichroism (CD), mass, nuclear magnetic resonance (NMR) and fluorescence spectroscopy. In State 1, the main molecular state of gramicidin A is as a dimer, and the addition of calcium salt can convert a mixture of four species into a single species, which is possibly a left-handed parallel double helix. In State 2, the addition of calcium halides drives gramicidin A dissociation and denaturation from a structured dimer into a rapid equilibrium of structured/unstructured monomer. We found that the abilities of dissociation and denaturation were highly dependent on the type of halide anion. The dissociation ability of calcium halides may play a vital role in the antimicrobial activity, as the structured monomeric form had the highest antimicrobial activity. Herein, our study demonstrated that the molecular state was correlated with the antimicrobial activity.

We report the parallel synthesis of gramicidin S derivatives featuring backbone N-amino substituents. Analogues were prepared by incorporation of N-amino dipeptide subunits on solid support. Nine backbone-aminated macrocycles were evaluated for growth inhibitory activity against ESKAPE pathogens and hemolytic activity against human red blood cells. Diamination of the Orn residues in the β-strand region of gramicidin S was found to enhance broad-spectrum antimicrobial activity without a corresponding increase in hemolytic activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Water wires are critical for the functioning of many membrane proteins, as in channels that conduct water, protons, and other ions. Here, in liquid crystalline lipid bilayers under symmetric environmental conditions, the selective hydrogen bonding interactions between eight waters comprising a water wire and a subset of 26 carbonyl oxygens lining the antiparallel dimeric gramicidin A channel are characterized by 17 O NMR spectroscopy at 35.2 T (or 1,500 MHz for 1 H) and computational studies. While backbone 15 N spectra clearly indicate structural symmetry between the two subunits, single site 17 O labels of the pore-lining carbonyls report two resonances, implying a break in dimer symmetry caused by the selective interactions with the water wire. The 17 O shifts document selective water hydrogen bonding with carbonyl oxygens that are stable on the millisecond timescale. Such interactions are supported by density functional theory calculations on snapshots taken from molecular dynamics simulations. Water hydrogen bonding in the pore is restricted to just three simultaneous interactions, unlike bulk water environs. The stability of the water wire orientation and its electric dipole leads to opposite charge-dipole interactions for K + ions bound at the two ends of the pore, thereby providing a simple explanation for an ∼20-fold difference in K + affinity between two binding sites that are ∼24 Å apart. The 17 O NMR spectroscopy reported here represents a breakthrough in high field NMR technology that will have applications throughout molecular biophysics, because of the acute sensitivity of the 17 O nucleus to its chemical environment., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The de Broglie wave nature of matter is a paradigmatic example of quantum physics and it has been exploited in precision measurements of forces and fundamental constants. However, matter-wave interferometry has remained an outstanding challenge for natural polypeptides, building blocks of life, which are fragile and difficult to handle. Here, we demonstrate the wave nature of gramicidin, a natural antibiotic composed of 15 amino acids. Its center of mass is delocalized over more than 20 times the molecular size in our time-domain Talbot-Lau interferometer. We compare the observed interference fringes with a model that includes both a rigorous treatment of the peptide's quantum wave nature as well as a quantum chemical assessment of its optical properties to distinguish our result from classical predictions. The realization of quantum optics with this prototypical biomolecule paves the way for quantum-assisted measurements on a large class of biologically relevant molecules.

Discovering and developing the antimicrobial peptides are recently focused on pharmaceutical firm, since they serve as complementary to antibiotics in prevailing over drug resistance by eliciting the disruption of microbial membrane. Still, there are lots of challenges to bring up the structurally stable and functionally efficient antimicrobial peptides. It is well known that gramicidin D is the prominent antimicrobial peptide that exists as g-AB, g-BC, and g-AC. This study analyzes the structural stability and the functional activity of hetero-dimeric double-stranded gramicidin-D peptides, thereby demonstrating its potent antimicrobial activity against antibiotic-resistant micro-organisms. To investigate the structural stability and functionality of gramicidin D, we performed static and dynamic analysis. Initially, we observed a maximum number of intermolecular interactions and membrane penetration in g-AB as compared to g-BC and g-AC. To substantiate further, the geometrical and thermodynamic parameters revealed the retention of maximum stability in g-AB than g-AC and g-BC. Thus, the conformational free energy and the binding free energy showed the variation among gramicidin-D peptides for the prediction of increased stability and functionality. In conclusion, g-AB peptide has definitely demonstrated adequate structural stability and functionality and this work will need to be considered in peptide-based drug discovery.

Prerequisite for chemical shift (CS) and CS tensor calculations are highly refined structures defining the molecular surroundings of the nuclei under study. Here, we present geometry optimizations with 13 C and 15 N CS constraints for large bio-molecules like peptides and proteins. The method discussed here provides both, refined structures and chemical shift tensors. Furthermore, since the experimental resonances of aligned systems are related to CS tensors, they strongly depend on the orientation and motion of molecules, their fragments, functional groups and moieties. For efficient CS calculations we apply a semi-empirical approach-the bond polarization theory (BPT). The BPT relies on linear bond polarization parameters and we present a new set of parameters based on ab initio second-order Møller-Plesset perturbation theory calculations. The new parametrization extends the applicability of the BPT approach to a wide range of organic molecules and bio-polymers. Here, the method has been applied to the protein ubiquitin and the membrane-active peptide gramicidin A (dimer) in oriented bilayers. The calculated 13 C and 15 N CS values of best-refined structures published until now gave a large scatter with respect to the experiment. It will be shown that BPT CS optimizations can reduce these errors to values near the experimental uncertainty. In combination with molecular dynamics with orientational constraints it is possible to study motional dynamics and BPT calculations can provide residual chemical shift anisotropies.

Membrane protein functions can be altered by subtle changes in the host lipid bilayer physical properties. Gramicidin channels have emerged as a powerful system for elucidating the underlying mechanisms of membrane protein function regulation through changes in bilayer properties, which are reflected in the thermodynamic equilibrium distribution between nonconducting gramicidin monomers and conducting bilayer-spanning dimers. To improve our understanding of how subtle changes in bilayer thickness alter the gramicidin monomer and dimer distributions, we performed extensive atomistic molecular dynamics simulations and fluorescence-quenching experiments on gramicidin A (gA). The free-energy calculations predicted a nonlinear coupling between the bilayer thickness and channel formation. The energetic barrier inhibiting gA channel formation was sharply increased in the thickest bilayer (1,2-dierucoyl-sn-glycero-3-phosphocholine). This prediction was corroborated by experimental results on gramicidin channel activity in bilayers of different thickness. To further explore the mechanism of channel formation, we performed extensive unbiased molecular dynamics simulations, which allowed us to observe spontaneous gA dimer formation in lipid bilayers. The simulations revealed structural rearrangements in the gA subunits and changes in lipid packing, as well as water reorganization, that occur during the dimerization process. Together, the simulations and experiments provide new, to our knowledge, insights into the process and mechanism of gramicidin channel formation, as a prototypical example of the bilayer regulation of membrane protein function., (Copyright © 2019 Biophysical Society. All rights reserved.)

Lipid bilayer membranes formed from the artificial 1,3-diamidophospholipid Pad-PC-Pad have the remarkable property that their hydrophobic thickness can be modified in situ: the particular arrangement of the fatty acid chains in Pad-PC-Pad allows them to fully interdigitate below 37 °C, substantially thinning the membrane with respect to the noninterdigitated state. Two stimuli, traversing the main phase transition temperature of the lipid or addition of cholesterol, have previously been shown to disable the interdigitated state. Both manipulations cause an increase in hydrophobic thickness of about 25 Å due to enhanced conformational entropy of the lipids. Here, we characterize the interdigitated state using electrophysiological recordings from free-standing lipid-membranes formed on micro structured electrode cavity arrays. Compared to standard membranes made from 1,2-diphytanoyl- sn -glycero-3-phosphocholin (DPhPC), pure Pad-PC-Pad membranes at room temperature had lowered electroporation threshold and higher capacitance. Ion channel formation by the peptide Gramicidin A was clearly facilitated in pure Pad-PC-Pad membranes at room temperature, with activity occurring at significantly lower peptide concentrations and channel dwell times increased by 2 orders of magnitude with respect to DPhPC-membranes. Both elevation of temperature beyond the phase transition and addition of cholesterol reduced channel dwell times, as expected if the reduced membrane thickness stabilized channel formation due to decreased hydrophobic mismatch.

Using a recently developed binary bilayer system (BBS) consisting of two patches of laterally contacting bilayers, umbrella sampling molecular dynamics (MD) simulations were performed for quantitative characterization of protein-lipid interactions. The BBS is composed of 1,2-dilauroyl- sn -glycero-3-phosphocholine (DLPC) and 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) with an embedded model membrane protein, a gramicidin A (gA) channel. The calculated free energy difference for the transfer of a gA channel from DLPC (hydrophobic thickness ≈ 21.5 Å) to DMPC (hydrophobic thickness ≈ 25.5 Å) bilayers, Δ G (DLPC → DMPC), is -2.2 ± 0.7 kcal/mol. This value appears at odds with the traditional view that the hydrophobic length of the gA channel is ∼22 Å. To understand this discrepancy, we first note that recent MD simulations by different groups have shown that lipid bilayer thickness profiles in the vicinity of a gA channel differ qualitatively from the deformation profile predicted from continuum elastic bilayer models. Our MD simulations at low and high gA:lipid molar ratios and different membrane compositions indicate that the gA channel's effective hydrophobic length is ∼26 Å. Using this effective hydrophobic length, Δ G (DLPC → DMPC) determined here is in excellent agreement with predictions based on continuum elastic models (-3.0 to -2.2 kcal/mol) where the bilayer deformation energy is approximated as a harmonic function of the mismatch between the channel's effective hydrophobic length and the hydrophobic thickness of the bilayer. The free energy profile for gA in the BBS includes a barrier at the interface between the two bilayers which can be attributed to the line tension at the interface between two bilayers with different hydrophobic thicknesses. This observation implies that translation of a peptide between two different regions of a cell membrane (such as between the liquid ordered and disordered phases) may include effects of a barrier at the interface in addition to the relative free energies of the species far from the interface. The BBS allows for direct transfer free energy calculations between bilayers without a need of a reference medium, such as bulk water, and thus provides an efficient simulation protocol for the quantitative characterization of protein-lipid interactions at all-atom resolution.

It is now known that mammalian brains leverage plasticity of both chemical and electrical synapses (ES) for collocating memory and processing. Unlike chemical synapses, ES join neurons via gap junction ion channels that permit fast, threshold-independent, and bidirectional ion transport. Like chemical synapses, ES exhibit activity-dependent plasticity, which modulates the ionic conductance between neurons and, thereby, enables adaptive synchronization of action potentials. Many types of adaptive computing devices that display discrete, threshold-dependent changes in conductance have been developed, yet far less effort has been devoted to emulating the continuously variable conductance and activity-dependent plasticity of ES. Here, we describe an artificial electrical synapse (AES) that exhibits voltage-dependent, analog changes in ionic conductance at biologically relevant voltages. AES plasticity is achieved at the nanoscale by linking dynamical geometrical changes of a host lipid bilayer to ion transport via gramicidin transmembrane ion channels. As a result, the AES uniquely mimics the composition, biophysical properties, bidirectional and threshold-independent ion transport, and plasticity of ES. Through experiments and modeling, we classify our AES as a volatile memristor, where the voltage-controlled conductance is governed by reversible changes in membrane geometry and gramicidin channel density. Simulations show that AES plasticity can adaptively synchronize Hodgkin-Huxley neurons. Finally, by modulating the molecular constituents of the AES, we show that the amplitude, direction, and speed of conductance changes can be tuned. This work motivates the development and integration of ES-inspired computing devices for achieving more capable neuromorphic hardware.

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.

We demonstrate a bionanoelectronic platform for a supported lipid bilayer formed on an MoS 2 film for biosensing, biomolecule recognition, and bioelectronic applications. A large-area MoS 2 film was synthesized on a sapphire substrate and treated with O 2 plasma or Al 2 O 3 deposition to change the surface from hydrophobic to hydrophilic. Measurements of fluorescence and fluorescence recovery after photobleaching confirmed the physical properties of the lipid bilayer on the treated surfaces. We fabricated an electronic device using the treated MoS 2 film and characterized the influence of the lipid bilayer on its electrical properties. Furthermore, transmembrane ion channels peptide (gramicidin A) were incorporated into the lipid bilayer and modulations of the electrical properties of the device under various pH conditions and calcium ion were observed. This sensitive and stable platform has strong potential for housing artificial channels and transmembrane ion channels for advanced bioapplications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A solid-phase approach including on-resin Ugi reactions was developed for the construction of β-hairpins. Various N -alkylated dipeptide fragments proved capable of aligning antiparallel β-sheets in a macrocyclic scaffold, thus serving as β-hairpin templates. Gramicidin S was used as the model β-hairpin to compare the Ugi-derived β-turns with the type-II' β-turn. The results show that the multicomponent incorporation of such N -alkylated residues allows for the simultaneous stabilization and exo-cyclic functionalization of cyclic β-hairpins.

Gramicidin S (GS), one of the oldest commercially used peptide antibiotics, is known for its robust antibacterial activity against both Gram-positive and Gram-negative bacterial strains. Although it was discovered well over 70 years ago, its clinical potential was limited to topical applications because of its high hemolytic activity. To overcome this side effect, significant efforts have been invested in the chase for GS analogues with high therapeutic index (e.g., high antimicrobial activity and low hemolytic activity) in the past decades. In this Perspective, the structural properties and biological profiles (including the recently discovered activities) of representative GS analogues designed by different approaches are described and analyzed. We also present how the general structure-activity relationships were established and how they could help in the design of more efficient GS analogues.

The comprehension and control of the interactions between nanoparticles and proteins at a molecular level are crucial to improve biomedical applications of nanomaterials and to develop nanosystems able to influence and regulate the conformational changes in proteins. In this work, we explore the interactions between Gramicidin A peptide (GramA) and dodecanethiol-stabilized small silver nanoparticles (D-AgNPs), paying particular attention to the effect on GramA conformation in POPC bilayers. D-AgNPs have been prepared to have dimensions (5 nm) and a hydrophobic nature compatible with the POPC lipid bilayer. Fluorescence, Raman and IR spectroscopies have been used to investigate both peptide conformation and its position inside the phospholipid bilayer. Results are discussed in terms of solvent exposure and conformation of GramA peptide.

Antimicrobial peptides (AMPs) are generally cationic and amphipathic peptides that show potential applications to combat the growing threat of antibiotic resistant infections. AMPs are known to interact with bacterial membranes, but their mechanisms of toxicity and selectivity are poorly understood, in part because it is challenging to characterize AMP oligomeric complexes within lipid bilayers. Here, we used native mass spectrometry to measure the stoichiometry of AMPs inserted into lipoprotein nanodiscs with different lipid components. Titrations of increasing peptide concentration and collisional activation experiments reveal that AMPs can exhibit a range of behaviors from nonspecific incorporation into the nanodisc to formation of specific complexes. This new approach to characterizing formation of AMP complexes within lipid membranes will provide unique insights into AMP mechanisms.

Microbial colonization can be detrimental to the integrity of metal surfaces and lead to microbiologically influenced corrosion (MIC). Biocorrosion is a serious problem for aquatic and marine industries in the world. In Minnesota (USA), where this study was conducted, biocorrosion severely affects the maritime transportation industry. The anticorrosion activity of a variety of compounds, including chemical (magnesium peroxide) and biological (surfactin, capsaicin, and gramicidin) molecules were investigated as coating additives. We also evaluated a previously engineered, extremely stable, non-biocidal enzyme known to interfere in bacterial signaling, SsoPox (a quorum quenching lactonase). Experimental steel coupons were submerged in water from the Duluth Superior Harbor (DSH) for 8 weeks in the laboratory. Biocorrosion was evaluated by counting the number and the coverage of corrosion tubercles on coupons and also by ESEM imaging of the coupon surface. Three experimental coating additives significantly reduced the formation of corrosion tubercles: surfactin, magnesium peroxide and the quorum quenching lactonase by 31%, 36% and 50%, respectively. DNA sequence analysis of the V4 region of the bacterial 16S rRNA gene revealed that these decreases in corrosion were associated with significant changes in the composition of bacterial communities on the steel surfaces. These results demonstrate the potential of highly stable quorum quenching lactonases to provide a reliable, cost-effective method to treat steel structures and prevent biocorrosion., Competing Interests: The authors have declared that no competing interests exist.