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

2000 ◽  
Vol 346 (1) ◽  
pp. 223-232 ◽  
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.

scholarly journals Membrane insertion of the N-terminal α-helix of equinatoxin II, a sea anemone cytolytic toxin

2004 ◽  
Vol 384 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Ion GUTIÉRREZ-AGUIRRE ◽  
Ariana BARLIČ ◽  
Zdravko PODLESEK ◽  
Peter MAČEK ◽  
Gregor ANDERLUH ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Lipid Membrane ◽  
Model Systems ◽  
Tryptophan Residue ◽  
Sea Anemones ◽  
Fluorescence Measurements ◽  
Tryptophan Residues ◽  
Equinatoxin Ii ◽  
Α Helix ◽  
Pore Forming Proteins

Equinatoxin II (Eqt-II) is a member of the actinoporins, a unique family of cytotoxins comprising 20 kDa pore-forming proteins isolated from sea anemones. Actinoporins bind preferentially to lipid membranes containing sphingomyelin, and create cation-selective pores by oligomerization of three to four monomers. Previous studies have shown that regions of Eqt-II crucial for its cytolytic mechanism are an exposed aromatic cluster and the N-terminal region containing an amphipathic α-helix. In the present study, we have investigated the transfer of the N-terminal α-helix into the lipid membrane by the use of three mutants containing an additional tryptophan residue in different positions within the amphipathic α-helix (Ile18→Trp, Val22→Trp and Ala25→Trp). The interaction of the mutants with different model systems, such as lipid monolayers, erythrocytes and ghost membranes, was extensively characterized. Intrinsic fluorescence measurements and the use of vesicles containing brominated phospholipids indicated a deep localization of the N-terminal amphipathic helix in the lipid bilayer, except for the case of Val22→Trp. This mutant is stabilized in a state immediately prior to final pore formation. The introduction of additional tryptophan residues in the sequence of Eqt-II has proved to be a suitable approach to monitor the new environments that surround defined regions of the molecule upon membrane interaction.

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

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.

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

2008 ◽  
Vol 53 (2) ◽  
pp. 593-602 ◽  
Author(s):  
Adam A. Strömstedt ◽  
Mukesh Pasupuleti ◽  
Artur Schmidtchen ◽  
Martin Malmsten
Keyword(s):  
Lipid Membranes ◽  
Lipid Membrane ◽  
Antimicrobial Properties ◽  
Peptide Sequence ◽  
Proteolytic Degradation ◽  
Human Neutrophil Elastase ◽  
Helix Formation ◽  
Proteolytic Resistance ◽  
Model Lipid Membranes ◽  
Protease Cleavage Sites

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.

scholarly journals Mimicking the Mammalian Plasma Membrane: An Overview of Lipid Membrane Models for Biophysical Studies

Biomimetics ◽  
2020 ◽  
Vol 6 (1) ◽  
pp. 3
Author(s):  
Alessandra Luchini ◽  
Giuseppe Vitiello
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Cell Membranes ◽  
Lipid Membrane ◽  
Lipid Phase ◽  
Chemical Information ◽  
Lipid Species ◽  
Membrane Models ◽  
The Impact

Cell membranes are very complex biological systems including a large variety of lipids and proteins. Therefore, they are difficult to extract and directly investigate with biophysical methods. For many decades, the characterization of simpler biomimetic lipid membranes, which contain only a few lipid species, provided important physico-chemical information on the most abundant lipid species in cell membranes. These studies described physical and chemical properties that are most likely similar to those of real cell membranes. Indeed, biomimetic lipid membranes can be easily prepared in the lab and are compatible with multiple biophysical techniques. Lipid phase transitions, the bilayer structure, the impact of cholesterol on the structure and dynamics of lipid bilayers, and the selective recognition of target lipids by proteins, peptides, and drugs are all examples of the detailed information about cell membranes obtained by the investigation of biomimetic lipid membranes. This review focuses specifically on the advances that were achieved during the last decade in the field of biomimetic lipid membranes mimicking the mammalian plasma membrane. In particular, we provide a description of the most common types of lipid membrane models used for biophysical characterization, i.e., lipid membranes in solution and on surfaces, as well as recent examples of their applications for the investigation of protein-lipid and drug-lipid interactions. Altogether, promising directions for future developments of biomimetic lipid membranes are the further implementation of natural lipid mixtures for the development of more biologically relevant lipid membranes, as well as the development of sample preparation protocols that enable the incorporation of membrane proteins in the biomimetic lipid membranes.

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

2017 ◽  
Vol 6 (2) ◽  
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

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

1993 ◽  
Vol 131 (1) ◽  
pp. 11-22 ◽  
Author(s):  
Giovanna Belmonte ◽  
Cecilia Pederzolli ◽  
Peter Maček ◽  
Gianfranco Menestrina
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Lipid Membranes ◽  
Pore Formation ◽  
Sea Anemone ◽  
Model Lipid Membranes ◽  
Equinatoxin Ii

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

RSC Advances ◽  
2019 ◽  
Vol 9 (25) ◽  
pp. 13992-13997 ◽  
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.

High Pressure X-ray Studies of Lipid Membranes and Lipid Phase Transitions

2014 ◽  
Vol 228 (10-12) ◽  
Author(s):  
Nicholas J. Brooks ◽  
John M. Seddon
Keyword(s):  
High Pressure ◽  
Phase Transitions ◽  
Lipid Membranes ◽  
Soft Matter ◽  
Lipid Membrane ◽  
Lipid Phase ◽  
Pressure Jump ◽  
X Ray ◽  
Lipid Phase Transitions ◽  
X Ray Scattering

AbstractHydrostatic pressure has dramatic effects on biomembrane structure and stability and is a key thermodynamic parameter in the context of the biology of deep sea organisms. Furthermore, high-pressure and pressure-jump studies are very useful tools in biophysics and biotechnology, where they can be used to study the mechanism and kinetics of lipid phase transitions, biomolecular transformations, and protein folding/unfolding. Here, we first give an overview of the technology currently available for X-ray scattering studies of soft matter systems under pressure. We then illustrate the use of this technology to study a variety of lipid membrane systems.

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

Coatings ◽  
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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