A Calibration-Free Optical Emission Spectroscopic Method to Determine the Composition of a Spark Discharge Plasma Used for AuAg Binary Nanoparticle Synthesis.

Spark discharge generators (SDGs) employ controlled gaseous environments to induce spark ablation of non-insulating electrodes, resulting in the formation of various nanostructures in the gas phase. The method offers technological advantages such as continuous particle production, scalable yield, and minimal waste. Additionally, the versatility of the process enables the generation of alloy nanoparticles from various material combinations, including immiscible ones. In order to fully exploit its potential, understanding the atomic mixing process during electrode ablation, particularly in the case of dissimilar electrodes, is crucial. Temporally and spatially resolved optical emission spectroscopy (OES) has been previously demonstrated as an effective characterization tool for spark plasmas in SDGs. However, to gain a deeper insight into the vapor mixing process, it is essential to quantitatively determine the plasma composition in both space and time. This paper introduces a calibration-free OES-based method tailored for spark plasmas utilized in binary nanoparticle generation. The method introduces the so-called multi-element combinatory Boltzmann plots, which use intensity ratios of emission atomic lines from different materials, allowing for the direct estimation of total number concentration ratios. The approach is tested using synthetic spectra and validated with experimental spark spectra obtained near an alloyed gold-silver (AuAg) electrode with a known composition. The study demonstrates the capabilities and robustness of the proposed method, with a focus on the AuAg system due to its significance in plasmonic research and frequent synthesis using spark ablation.

Host cell targeting of novel antimycobacterial 4-aminosalicylic acid derivatives with tuftsin carrier peptides.

Mycobacterium tuberculosis is an intracellular pathogen and the uptake of the antimycobacterial compounds by host cells is limited. Novel antimycobacterials effective against intracellular bacteria are needed. New N-substituted derivatives of 4-aminosalicylic acid have been designed and evaluated. To achieve intracellular efficacy and selectivity, these compounds were conjugated to tuftsin peptides via oxime or amide bonds. These delivery peptides can target tuftsin- and neuropilin receptor-bearing cells, such as macrophages and various other cells of lung origin. We have demonstrated that the in vitro antimycobacterial activity of the 4-aminosalicylic derivatives against M. tuberculosis H37Rv was preserved in the peptide conjugates. The free drugs were ineffective on infected cells, but the conjugates were active against the intracellular bacteria and have the selectivity on various types of host cells. The intracellular distribution of the carrier peptides was assessed, and the peptides internalize and display mainly in the cytosol in a concentration-dependent manner. The penetration ability of the most promising carrier peptide OT5 was evaluated using Transwell-inserts and spheroids. The pentapeptide exhibited time- and concentration-dependent penetration across the non-contact monolayers. Also, the pentapeptide has a fair penetration rate towards the center of spheroids formed of EBC-1 cells.

Membrane Association Modes of Natural Anticancer Peptides: Mechanistic Details on Helicity, Orientation, and Surface Coverage.

Anticancer peptides (ACPs) could potentially offer many advantages over other cancer therapies. ACPs often target cell membranes, where their surface mechanism is coupled to a conformational change into helical structures. However, details on their binding are still unclear, which would be crucial to reach progress in connecting structural aspects to ACP action and to therapeutic developments. Here we investigated natural helical ACPs, Lasioglossin LL-III, Macropin 1, Temporin-La, FK-16, and LL-37, on model liposomes, and also on extracellular vesicles (EVs), with an outer leaflet composition similar to cancer cells. The combined simulations and experiments identified three distinct binding modes to the membranes. Firstly, a highly helical structure, lying mainly on the membrane surface; secondly, a similar, yet only partially helical structure with disordered regions; and thirdly, a helical monomeric form with a non-inserted perpendicular orientation relative to the membrane surface. The latter allows large swings of the helix while the N-terminal is anchored to the headgroup region. These results indicate that subtle differences in sequence and charge can result in altered binding modes. The first two modes could be part of the well-known carpet model mechanism, whereas the newly identified third mode could be an intermediate state, existing prior to membrane insertion.

Controlling Peptide Function by Directed Assembly Formation: Mechanistic Insights Using Multiscale Modeling on an Antimicrobial Peptide-Drug-Membrane System.

Owing to their potential applicability against multidrug-resistant bacteria, antimicrobial peptides (AMPs) or host defense peptides (HDPs) gain increased attention. Besides diverse immunomodulatory roles, their classical mechanism of action mostly involves membrane disruption of microbes. Notably, their unbalanced overexpression has also been associated with host cell cytotoxicity in various diseases. Relatedly, AMPs can be subject to aggregate formation, either via self-assembly or together with other compounds, which has demonstrated a modulation effect on their biological functions, thus highly relevant both for drug targeting projects and understanding their in vivo actions. However, the molecular aspects of the related assembly formation are not understood. Here, we focused in detail on an experimentally studied AMP-drug system, i.e., CM15-suramin, and performed all-atom and coarse-grain (CG) simulations. Results obtained for all systems were in close line with experimental observations and indicate that the CM15-suramin aggregation is an energetically favorable and dynamic process. In the presence of bilayers, the peptide-drug assembly formation was highly dependent on lipid composition, and peptide aggregates themselves were also capable of binding to the membranes. Interestingly, longer CG simulations with zwitterionic membranes indicated an intermediate state in the presence of both AMP-drug assemblies and monomeric peptides located on the membrane surface. In sharp contrast, larger AMP-drug aggregates could not be detected with a negatively charged membrane, rather the AMPs penetrated its surface in a monomeric form, in line with previous in vitro observations. Considering experimental and theoretical results, it is promoted that in biological systems, cationic AMPs may often form associates with anionic compounds in a reversible manner, resulting in lower bioactivity. This is only mildly affected by zwitterionic membranes; however, membranes with a negative charge strongly alter the energetic preference of AMP assemblies, resulting in the dissolution of the complexes into the membrane. The phenomenon observed here at a molecular level can be followed in several experimental systems studied recently, where peptides interact with food colors, drug molecules, or endogenous compounds, which strongly indicates that reversible associate formation is a general phenomenon for these complexes. These results are hoped to be exploited in novel therapeutic strategies aiming to use peptides as drug targets and control AMP bioactivity by directed assembly formation.

Membrane active Janus-oligomers of ß3-peptides.

Self-assembling peptides offer a versatile set of tools for bottom-up construction of supramolecular biomaterials. Among these compounds, non-natural peptidic foldamers experience increased focus due to their structural variability and lower sensitivity to enzymatic degradation. However, very little is known about their membrane properties and complex oligomeric assemblies - key areas for biomedical and technological applications. Here we designed short, acyclic ß3-peptide sequences with alternating amino acid stereoisomers to obtain non-helical molecules having hydrophilic charged residues on one side, and hydrophobic residues on the other side, with the N-terminus preventing formation of infinite fibrils. Our results indicate that these ß-peptides form small oligomers both in water and in lipid bilayers and are stabilized by intermolecular hydrogen bonds. In the presence of model membranes, they either prefer the headgroup regions or they insert between the lipid chains. Molecular dynamics (MD) simulations suggest the formation of two-layered bundles with their side chains facing opposite directions when compared in water and in model membranes. Analysis of the MD calculations showed hydrogen bonds inside each layer, however, not between the layers, indicating a dynamic assembly. Moreover, the aqueous form of these oligomers can host fluorescent probes as well as a hydrophobic molecule similarly to e.g. lipid transfer proteins. For the tested, peptides the mixed chirality pattern resulted in similar assemblies despite sequential differences. Based on this, it is hoped that the presented molecular framework will inspire similar oligomers with diverse functionality.

Analysis of Procollagen C-Proteinase Enhancer-1/Glycosaminoglycan Binding Sites and of the Potential Role of Calcium Ions in the Interaction.

In this study, we characterize the interactions between the extracellular matrix protein, procollagen C-proteinase enhancer-1 (PCPE-1), and glycosaminoglycans (GAGs), which are linear anionic periodic polysaccharides. We applied molecular modeling approaches to build a structural model of full-length PCPE-1, which is not experimentally available, to predict GAG binding poses for various GAG lengths, types and sulfation patterns, and to determine the effect of calcium ions on the binding. The computational data are analyzed and discussed in the context of the experimental results previously obtained using surface plasmon resonance binding assays. We also provide experimental data on PCPE-1/GAG interactions obtained using inhibition assays with GAG oligosaccharides ranging from disaccharides to octadecasaccharides. Our results predict the localization of GAG-binding sites at the amino acid residue level onto PCPE-1 and is the first attempt to describe the effects of ions on protein-GAG binding using modeling approaches. In addition, this study allows us to get deeper insights into the in silico methodology challenges and limitations when applied to GAG-protein interactions.

The molecular mechanism of structural changes in the antimicrobial peptide CM15 upon complex formation with drug molecule suramin: a computational analysis.

Dynamic increase of resistant bacterial infectious diseases continuously requires development of novel compounds against them. The molecular level understanding of the mechanism and interactions of natural host-defense peptides or antimicrobial peptides (AMPs) is an important step towards rational design and development of compounds inspired by their function. A particular set of these peptides have disordered structure, the ordering of which may modify their antimicrobial properties. Recent experiments demonstrate that such conformational transitions of AMPs could be mediated by the presence of small organic compounds, such as approved drug molecules. However, the molecular mechanisms underlying these structural changes are unclear. In this study, we apply molecular docking and molecular dynamics-based approaches to rigorously analyze the interactions between the drug suramin and the AMP CM15, a synthetic unstructured hybrid peptide. We characterize the energetic properties of putative CM15-suramin complexes revealing particular impacts of CM15 residues as well as the parts of suramin on these interactions. We find that α-helical content of the peptide is increased in the presence of suramin, which is in agreement with the experimental data. Kinetics analysis from canonical molecular dynamics and metadynamics simulations suggest that the effect of suramin does not promote the formation of α-helix but rather results from its ability to stabilize the α-helical population in the conformational pool of the peptide. Potentially, understanding the physico-chemical basis underlying the interactions between drug molecules and disordered AMPs will prove useful in strategies for antimicrobial compound development. Further on, the given computational protocol for the analysis of such flexible systems provide a basis for future theoretical investigation of similar biomolecular complexes.

Disorder-to-helix conformational conversion of the human immunomodulatory peptide LL-37 induced by antiinflammatory drugs, food dyes and some metabolites.

The human antimicrobial and immunomodulatory peptide LL-37 is ubiquitously expressed and secreted by epithelial cells of mucosal surfaces including the gastrointestinal tract, the primary absorption site of orally administered drugs and food components. Besides antimicrobial properties, LL-37 also contributes to the pathophysiology of various diseases such as ulcerative colitis, Crohn's disease and cancer. Non-covalent association of antiinflammatory drugs, porphyrin pigments, bile salts and food dyes to the peptide was uncovered and evaluated by circular dichroism (CD) spectroscopy. These agents induce the disorder-to-order conformational transition of the natively unstructured LL-37 leading to its helical folding. Even in the presence of chloride ions, when LL-37 is partially folded, the inducers were able to rise the α-helix content. CD titration data indicated positive cooperativity between the ligand molecules accommodated to the peptide chain resulting in multimeric complexes with apparent dissociation constants ranged from 2 to 500 µM. Computational docking suggested the prominent role of the Lys8-Arg19 segment in the accommodation of small molecules, governed principally by salt bridges and H-bonding. Since pleiotropic biological functions of LL-37 are strongly conformation-dependent, it could be anticipated that folding inducer compounds may modulate its in vivo actions and also of related cationic peptides.

The lipid mediator lysophosphatidic acid induces folding of disordered peptides with basic amphipathic character into rare conformations.

Membrane-active, basic amphipathic peptides represent a class of biomolecules with diverse functions. Sequentially close protein segments also show similar behaviour in several ways. Here we investigated the effect of the lipid mediator lysophosphatidic acid (LPA) on the conformation of structurally disordered peptides including extracellular antimicrobial peptides (AMPs), and calmodulin-binding motifs derived from cytosolic and membrane target proteins. The interaction with associated LPA resulted in gain of ordered secondary structure elements, which for most cases were previously uncharacteristic of the particular peptide. Results revealed mechanism of the LPA-peptide interactions with regulation of the lipid on peptide conformation and oligomerization in a concentration-dependent manner involving (1) relocation of tryptophan residues into the lipid cluster, (2) multiple contacts between the binding partners dictated by complex driving forces, (3) multiple peptide binding to LPA associates with an affinity in the low micromolar range, and (4) selectivity for LPA compared with structurally related lipids. In line with recent findings showing endogenous molecules inducing structural changes in AMPs, we propose that accumulation of LPA in signalling or pathological processes might modulate host-defense activity or trigger certain processes by direct interaction with cationic amphipathic peptide sequences.

Protein-Ligand Interaction Energy-Based Entropy Calculations: Fundamental Challenges For Flexible Systems.

Entropy calculations represent one of the most challenging steps in obtaining the binding free energy in biomolecular systems. A novel computationally effective approach (IE) was recently proposed to calculate the entropy based on the computation of protein-ligand interaction energy directly from molecular dynamics (MD) simulations. We present a study focused on the application of this method to flexible molecular systems and compare its performance with well-established normal mode (NM) and quasiharmonic (QH) entropy calculation approaches. Our results demonstrated that the IE method is intended for calculating entropy change for binding partners in fixed conformations, as by the original definition of IE, and is not applicable to the molecular complexes in which the interacting partners undergo significant conformational changes during the binding process.

Heparin and Heparan Sulfate Binding of the Antiparasitic Drug Imidocarb: Circular Dichroism Spectroscopy, Isothermal Titration Calorimetry, and Computational Studies.

This study is aimed to assess the binding interaction between the antiparasitic cationic drug imidocarb (IMD) and sulfated glycosaminoglycans (GAGs), the ubiquitious nonprotein macromolecules of living organisms. These complex, heterogeneous polyanions are the integral constituents of cell membranes and the extracellular matrix and display affinity toward basic compounds, the binding of which may affect their biological functions. Exciton-type circular dichroism (CD) spectroscopic features measured at low salt concentration verify the heparin and heparan sulfate binding of IMD, which occurs in a cooperative manner by association of several drug molecules to a disaccharide unit. Isothermal titration calorimetry (ITC) measurements reassured the heparin interaction, resulting in a Kd value in the low micromolar range. In contrast, when considering high molar excess of the heparin-binding sites, closer resembling in vivo conditions, an entirely different CD signature was induced, suggesting a shift from the oligo- to monomeric binding mode. This observation was also supported by ITC measurements using an identical sample setup. To better mimic in vivo conditions, several measurements were performed in physiological salt concentration ranges. On the basis of these, the inter- and intramolecular origin of CD activity observed under low- and high-salt conditions refer to electrostatically held oligomeric and intermolecular H-bonded monomeric drug-GAG adducts, respectively. To complement the experimental data, quantum chemical calculations were performed to assess the photophysical and conformational properties of IMD, indicating the existence of nonlinear, nonplanar interconverting conformer populations. Such a structural flexibility may be important in the multiple, cooperative binding of IMD to sterically adjacent GAG sites.