Evaluation of Strategies for Improving Proteolytic Resistance of Antimicrobial Peptides by Using Variants of EFK17, an Internal Segment of LL-37

ABSTRACT Methods for increasing the proteolytic stability of EFK17 (EFKRIVQRIKDFLRNLV), a new peptide sequence with antimicrobial properties derived from LL-37, were evaluated. EFK17 was modified by four d-enantiomer or tryptophan (W) substitutions at known protease cleavage sites as well as by terminal amidation and acetylation. The peptide variants were studied in terms of proteolytic resistance, antibacterial potency, and cytotoxicity but also in terms their adsorption at model lipid membranes, liposomal leakage generation, and secondary-structure behavior. The W substitutions resulted in a marked reduction in the proteolytic degradation caused by human neutrophil elastase, Staphylococcus aureus aureolysin, and V8 protease but not in the degradation caused by Pseudomonas aeruginosa elastase. For the former two endoproteases, amidation and acetylation of the terminals also reduced proteolytic degradation but only when used in combination with W substitutions. The d-enantiomer substitutions rendered the peptides indigestible by all four proteases; however, those peptides displayed little antimicrobial potency. The W- and end-modified peptides, on the other hand, showed an increased bactericidal potency compared to that of the native peptide sequence, coupled with a moderate cytotoxicity that was largely absent in serum. The bactericidal, cytotoxic, and liposome lytic properties correlated with each other as well as with the amount of peptide adsorbed at the lipid membrane and the extent of helix formation associated with the adsorption. The lytic properties of the W-substituted peptides were less impaired by increased ionic strength, presumably by a combination of W-mediated stabilization of the largely amphiphilic helix conformation and a nonelectrostatic W affinity for the bilayer interface. Overall, W substitutions constitute an interesting means to reduce the proteolytic susceptibility of EFK17 while also improving antimicrobial performance.

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Structural Study of the Hydration of Lipid Membranes Upon Interaction With Mesoporous Supports Prepared by Standard Methods and/or X‐Ray Irradiation

Frontiers in Materials ◽

10.3389/fmats.2021.686353 ◽

2021 ◽

Vol 8 ◽

Author(s):

Benedetta Marmiroli ◽

Barbara Sartori ◽

Adriana R. Kyvik ◽

Imma Ratera ◽

Heinz Amenitsch

Keyword(s):

Structural Study ◽

Lipid Membranes ◽

Lipid Membrane ◽

Subsequent Development ◽

Grazing Incidence ◽

Mesoporous Films ◽

X Ray ◽

Sensing Applications ◽

Model Lipid Membranes ◽

Biological Studies

Mesoporous materials feature ordered tailored structures with uniform pore sizes and highly accessible surface areas, making them an ideal host for functional organic molecules or nanoparticles for analytical and sensing applications. Moreover, as their porosity could be employed to deliver fluids, they could be suitable materials for nanofluidic devices. As a first step in this direction, we present a study of the hydration of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model lipid membranes on solid mesoporous support. POPC was selected as it changes the structure upon hydration at room temperature. Mesoporous films were prepared using two different templating agents, Pluronic P123 (PEO–PPO–PEO triblock copolymer where PEO is polyethylene oxide and PPO is polypropylene oxide) and Brij 58 (C16H33(EO)20OH where EO is ethylene oxide), both following the conventional route and by X-ray irradiation via deep X-ray lithography technique and subsequent development. The same samples were additionally functionalized with a self-assembly monolayer (SAM) of (3-aminopropyl)triethoxysilane. For every film, the contact angle was measured. A time resolved structural study was conducted using in situ grazing incidence small-angle X-ray scattering while increasing the external humidity (RH), from 15 to 75% in a specially designed chamber. The measurements evidenced that the lipid membrane hydration on mesoporous films occurs at a lower humidity value with respect to POPC deposited on silicon substrates, demonstrating the possibility of using porosity to convey water from below. A different level of hydration was reached by using the mesoporous thin film prepared with conventional methods or the irradiated ones, or by functionalizing the film using the SAM strategy, meaning that the hydration can be partially selectively tuned. Therefore, mesoporous films can be employed as “interactive” sample holders with specimens deposited on them. Moreover, thanks to the possibility of patterning the films using deep X-ray lithography, devices for biological studies of increasing complexity by selectively functionalizing the mesopores with biofunctional SAMs could be designed and fabricated.

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Impact of selected polyphenolics on the structural properties of model lipid membranes – a review

International Journal of Food Studies ◽

10.7455/ijfs.v6i2.391 ◽

2017 ◽

Vol 6 (2) ◽

Cited By ~ 1

Author(s):

Nataša P. Ulrih ◽

Ajda Ota ◽

Veronika Abram

Keyword(s):

Electron Paramagnetic Resonance ◽

Membrane Fluidity ◽

Phenolic Acids ◽

Lipid Membranes ◽

Lipid Membrane ◽

Biological Effects ◽

Resonance Spectroscopy ◽

Paramagnetic Resonance ◽

Model Lipid Membranes ◽

Electron Paramagnetic

This review is a presentation of data gathered on the interactions of several polyphenolics (i.e., phenolic acids, stilbenes, flavonoids) with lipid bilayers of different lipid compositions. These polyphenolics have been investigated through a combination of fluorescence spectroscopy, electron paramagnetic resonance spectroscopy, and differential scanning calorimetry, to detect changes in membrane fluidity. Among the investigated phenolic acids, the least polar phenolic acid, p-coumaric acid, has the greatest effect on lipid membrane structure. It appears to have a greater ability to cross membranes by passive transport than more polar phenolic acids. On the other hand, among the flavonoids that have been studied, the anthocyanins cyanidin 3-glucoside and its aglycone are inactive. All of the flavonols tested, except for epigallocatechin-3-gallate, promote small decreases in membrane fluidity. Computer simulation of electron paramagnetic resonance spectra for flavonoids indicated two or three regions in the phosphatidylcholine/ phosphatidylserine (2.4:1) membrane with different fluidity characteristics. The effects of the different flavonoids are correlated to their structural characteristics, whereby even the difference in one -OH group can be important, as can the number of H-bonds they form. The role of membrane composition and flavonoid structure in these interactions with lipid membranes are of great importance for bioavailability of these compounds and for their biological effects in an organism

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Anionic nanoparticle-lipid membrane interactions: the protonation of anionic ligands at the membrane surface reduces membrane disruption

RSC Advances ◽

10.1039/c9ra02462j ◽

2019 ◽

Vol 9 (25) ◽

pp. 13992-13997 ◽

Cited By ~ 4

Author(s):

Sebastian Salassi ◽

Ester Canepa ◽

Riccardo Ferrando ◽

Giulia Rossi

Keyword(s):

Lipid Membranes ◽

Au Nanoparticles ◽

Lipid Membrane ◽

Membrane Surface ◽

Membrane Disruption ◽

Membrane Interactions ◽

Carboxylate Groups ◽

Model Lipid Membranes ◽

Anionic Ligands

The interaction between anionic Au nanoparticles and model lipid membranes is facilitated by the spontaneous protonation of the NP ligand carboxylate groups, COO−˙ → COOH, in the lipid headgroup region.

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Structure, Formation, and Biological Interactions of Supported Lipid Bilayers (SLB) Incorporating Lipopolysaccharide

Coatings ◽

10.3390/coatings10100981 ◽

2020 ◽

Vol 10 (10) ◽

pp. 981

Author(s):

Palak Sondhi ◽

Dhanbir Lingden ◽

Keith J. Stine

Keyword(s):

Lipid Bilayers ◽

Lipid Membranes ◽

Lipid Membrane ◽

Biologically Active ◽

Supported Lipid Bilayers ◽

Biological Interactions ◽

Relevant Model ◽

Biologically Relevant ◽

Model Lipid Membranes ◽

Biomimetic Membrane

Biomimetic membrane systems play a crucial role in the field of biosensor engineering. Over the years, significant progress has been achieved creating artificial membranes by various strategies from vesicle fusion to Langmuir transfer approaches to meet an ever-growing demand for supported lipid bilayers on various substrates such as glass, mica, gold, polymer cushions, and many more. This paper reviews the diversity seen in the preparation of biologically relevant model lipid membranes which includes monolayers and bilayers of phospholipid and other crucial components such as proteins, characterization techniques, changes in the physical properties of the membranes during molecular interactions and the dynamics of the lipid membrane with biologically active molecules with special emphasis on lipopolysaccharides (LPS).

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Lipid Self-Assemblies under the Atomic Force Microscope

International Journal of Molecular Sciences ◽

10.3390/ijms221810085 ◽

2021 ◽

Vol 22 (18) ◽

pp. 10085

Author(s):

Aritz B. García-Arribas ◽

Félix M. Goñi ◽

Alicia Alonso

Keyword(s):

Lipid Membranes ◽

Lipid Membrane ◽

Domain Size ◽

Model Membranes ◽

Model Systems ◽

Systems Model ◽

Atomic Force ◽

Model Lipid Membranes ◽

The Impact

Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid–lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular membranes, that exhibit a wide variety of components, including lipids of different subfamilies (e.g., phospholipids, sphingolipids, sterols…), in addition to proteins and sugars. The capacity of lipids to segregate by themselves into different phases at the nanoscale (nanodomains) is an intriguing feature that is yet to be fully characterized in vivo due to the proposed transient nature of these domains in living systems. Model lipid membranes, instead, have the advantage of (usually) greater phase stability, together with the possibility of fully controlling the system lipid composition. Atomic force microscopy (AFM) is a powerful tool to detect the presence of meso- and nanodomains in a lipid membrane. It also allows the direct quantification of nanomechanical resistance in each phase present. In this review, we explore the main kinds of lipid assemblies used as model membranes and describe AFM experiments on model membranes. In addition, we discuss how these assemblies have extended our knowledge of membrane biophysics over the last two decades, particularly in issues related to the variability of different model membranes and the impact of supports/cytoskeleton on lipid behavior, such as segregated domain size or bilayer leaflet uncoupling.

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Structure–function studies of tryptophan mutants of equinatoxin II, a sea anemone pore-forming protein

Biochemical Journal ◽

10.1042/bj3460223 ◽

2000 ◽

Vol 346 (1) ◽

pp. 223-232 ◽

Cited By ~ 5

Author(s):

Petra MALOVRH ◽

Ariana BARLIĆ ◽

Zdravko PODLESEK ◽

Peter MAĆEK ◽

Gianfranco MENESTRINA ◽

...

Keyword(s):

Secondary Structure ◽

Lipid Membranes ◽

Lipid Membrane ◽

Expression System ◽

Random Coil ◽

Tryptophan Residue ◽

Lipid Phase ◽

Wild Type ◽

Model Lipid Membranes ◽

Equinatoxin Ii

Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes. It contains five tryptophan residues in three different regions of the molecule. In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp45, EqtII Trp116/117 and EqtII Trp149, were prepared in an Escherichia coli expression system [here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein]. They all possess a single intrinsic fluorescent centre. All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same. EqtII Trp116/117 resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties. Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane. The secondary structure of the other two mutants, EqtII Trp45 and EqtII Trp149, was altered to a certain extent. EqtII Trp149 was the most dissimilar from the wild-type, displaying a higher content of random-coil structure. It also retained the lowest number of nitrogen-bound protons after exchange with 2H2O, which might indicate a reduced compactness of the molecule. Tryptophans in EqtII Trp45 and EqtII Trp149 were more exposed to water, and also remained as such in the membrane-bound form.

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Electrochemical Properties of Lipid Membranes Self-Assembled from Bicelles

Membranes ◽

10.3390/membranes11010011 ◽

2020 ◽

Vol 11 (1) ◽

pp. 11

Author(s):

Damian Dziubak ◽

Kamil Strzelak ◽

Slawomir Sek

Keyword(s):

Electrochemical Properties ◽

Self Assembly ◽

Lipid Membranes ◽

Lipid Membrane ◽

Membrane Resistance ◽

Electrochemical Characteristics ◽

Freeze Thaw ◽

Lipid Films ◽

Supported Lipid Membranes

Supported lipid membranes are widely used platforms which serve as simplified models of cell membranes. Among numerous methods used for preparation of planar lipid films, self-assembly of bicelles appears to be promising strategy. Therefore, in this paper we have examined the mechanism of formation and the electrochemical properties of lipid films deposited onto thioglucose-modified gold electrodes from bicellar mixtures. It was found that adsorption of the bicelles occurs by replacement of interfacial water and it leads to formation of a double bilayer structure on the electrode surface. The resulting lipid assembly contains numerous defects and pinholes which affect the permeability of the membrane for ions and water. Significant improvement in morphology and electrochemical characteristics is achieved upon freeze–thaw treatment of the deposited membrane. The lipid assembly is rearranged to single bilayer configuration with locally occurring patches of the second bilayer, and the number of pinholes is substantially decreased. Electrochemical characterization of the lipid membrane after freeze–thaw treatment demonstrated that its permeability for ions and water is significantly reduced, which was manifested by the relatively high value of the membrane resistance.

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Interactions of Linear Analogues of Battacin with Negatively Charged Lipid Membranes

Membranes ◽

10.3390/membranes11030192 ◽

2021 ◽

Vol 11 (3) ◽

pp. 192

Author(s):

Kinga Burdach ◽

Dagmara Tymecka ◽

Aneta Urban ◽

Robert Lasek ◽

Dariusz Bartosik ◽

...

Keyword(s):

Lipid Membranes ◽

Liquid Crystalline ◽

Lipid Membrane ◽

Attenuated Total Reflection ◽

Gram Positive ◽

Insertion Depth ◽

Force Microscopy ◽

Acyl Chains ◽

Hydrophobic Portion ◽

Membrane Fluidization

The increasing resistance of bacteria to available antibiotics has stimulated the search for new antimicrobial compounds with less specific mechanisms of action. These include the ability to disrupt the structure of the cell membrane, which in turn leads to its damage. In this context, amphiphilic lipopeptides belong to the class of the compounds which may fulfill this requirement. In this paper, we describe two linear analogues of battacin with modified acyl chains to tune the balance between the hydrophilic and hydrophobic portion of lipopeptides. We demonstrate that both compounds display antimicrobial activity with the lowest values of minimum inhibitory concentrations found for Gram-positive pathogens. Therefore, their mechanism of action was evaluated on a molecular level using model lipid films mimicking the membrane of Gram-positive bacteria. The surface pressure measurements revealed that both lipopeptides show ability to bind and incorporate into the lipid monolayers, resulting in decreased ordering of lipids and membrane fluidization. Atomic force microscopy (AFM) imaging demonstrated that the exposure of the model bilayers to lipopeptides leads to a transition from the ordered gel phase to disordered liquid crystalline phase. This observation was confirmed by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) results, which revealed that lipopeptide action causes a substantial increase in the average tilt angle of lipid acyl chains with respect to the surface normal to compensate for lipopeptide insertion into the membrane. Moreover, the peptide moieties in both molecules do not adopt any well-defined secondary structure upon binding with the lipid membrane. It was also observed that a small difference in the structure of a lipophilic chain, altering the balance between hydrophobic and hydrophilic portion of the molecules, results in different insertion depth of the active compounds.

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An effect of antibiotic amphotericin B on ion transport across model lipid membranes and tonoplast membranes

Biochemical Pharmacology ◽

10.1016/j.bcp.2005.06.001 ◽

2005 ◽

Vol 70 (5) ◽

pp. 668-675 ◽

Cited By ~ 24

Author(s):

Monika Hereć ◽

Halina Dziubińska ◽

Kazimierz Trębacz ◽

Jacek W. Morzycki ◽

Wiesław I. Gruszecki

Keyword(s):

Ion Transport ◽

Amphotericin B ◽

Lipid Membranes ◽

Model Lipid Membranes

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Structure and Antimicrobial Properties of Monensin A and Its Derivatives: Summary of the Achievements

BioMed Research International ◽

10.1155/2013/742149 ◽

2013 ◽

Vol 2013 ◽

pp. 1-14 ◽

Cited By ~ 32

Author(s):

Daniel Łowicki ◽

Adam Huczyński

Keyword(s):

Antimicrobial Activity ◽

Lipid Membranes ◽

Antimicrobial Properties ◽

Biological Properties ◽

Sodium Cation ◽

Hydroxyl Groups ◽

Carboxyl Group ◽

Growth Promoter ◽

Monensin A

In this paper structural and microbiological studies on the ionophorous antibiotic monensin A and its derivatives have been collected. Monensin A is an ionophore which selectively complexes and transports sodium cation across lipid membranes, and therefore it shows a variety of biological properties. This antibiotic is commonly used as coccidiostat and nonhormonal growth promoter. The paper focuses on both the latest and earlier achievements concerning monensin A antimicrobial activity. The activities of monensin derivatives, including modifications of hydroxyl groups and carboxyl group, are also presented.

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