Hydrophobic Peptide Research Articles

Immunoprecipitation methods impact the peptide repertoire in immunopeptidomics.

Mass spectrometry-based immunopeptidomics is the only unbiased method to identify naturally presented HLA ligands, which is an indispensable prerequisite for characterizing novel tumor antigens for immunotherapeutic approaches. In recent years, improvements based on devices and methodology have been made to optimize sensitivity and throughput in immunopeptidomics. However, developments in ligand isolation, mass spectrometric analysis, and subsequent data processing can have a marked impact on the quality and quantity of immunopeptidomics data. In this work, we compared the immunopeptidome composition in terms of peptide yields, spectra quality, hydrophobicity, retention time, and immunogenicity of two established immunoprecipitation methods (column-based and 96-well-based) using cell lines as well as primary solid and hematological tumor samples. Although, we identified comparable overall peptide yields, large proportions of method-exclusive peptides were detected with significantly higher hydrophobicity for the column-based method with potential implications for the identification of immunogenic tumor antigens. We showed that column preparation does not lose hydrophilic peptides in the hydrophilic washing step. In contrast, an additional 50% acetonitrile elution could partially regain lost hydrophobic peptides during 96-well preparation, suggesting a reduction of the bias towards the column-based method but not completely equalizing it. Together, this work showed how different immunoprecipitation methods and their adaptions can impact the peptide repertoire of immunopeptidomic analysis and therefore the identification of potential tumor-associated antigens.

A Frame-by-Frame Glance at Membrane Fusion Mechanisms: From Viral Infections to Fertilization.

Viral entry and fertilization are distinct biological processes that share a common mechanism: membrane fusion. In viral entry, enveloped viruses attach to the host cell membrane, triggering a series of conformational changes in the viral fusion proteins. This results in the exposure of a hydrophobic fusion peptide, which inserts into the host membrane and brings the viral and host membranes into close proximity. Subsequent structural rearrangements in opposing membranes lead to their fusion. Similarly, membrane fusion occurs when gametes merge during the fertilization process, though the exact mechanism remains unclear. Structural biology has played a pivotal role in elucidating the molecular mechanisms underlying membrane fusion. High-resolution structures of the viral and fertilization fusion-related proteins have provided valuable insights into the conformational changes that occur during this process. Understanding these mechanisms at a molecular level is essential for the development of antiviral therapeutics and tools to influence fertility. In this review, we will highlight the biological importance of membrane fusion and how protein structures have helped visualize both common elements and subtle divergences in the mechanisms behind fusion; in addition, we will examine the new tools that recent advances in structural biology provide researchers interested in a frame-by-frame understanding of membrane fusion.

Effect of helicity and hydrophobicity on cell-penetrating ability of arginine-rich peptides

Arginine (Arg)-rich peptides are one of the typical cell-penetrating peptides (CPPs), which can deliver membrane-impermeable compounds into intracellular compartments. Guanidino groups in Arg-rich peptides are critical for their high cell-penetrating ability, although it remains unclear whether peptide secondary structures contribute to this ability. In the current study, we designed four Arg-rich peptides containing α,α-disubstituted α-amino acids (dAAs), which prefer to adopt a helical structure. The four dAA-containing peptides adopted slightly different peptide secondary structures, from a random structure to a helical structure, with different hydrophobicities. In these peptides, dipropylglycine-containing peptide exhibited the highest helicity and hydrophobicity, and showed the best cell-penetrating ability. These findings suggested that the helicity and hydrophobicity of Arg-rich peptides contributes to their high cell-penetrating ability.

Changes in the volumetric properties of uracil in a buffered saline upon interaction with peptides: The effect of glycyl-L-tyrosine and glycyl-L-glutamic acid

Interactions between uracil (Ura) and peptides, glycyl-L-tyrosine (Gly-Tyr) and glycyl-L-glutamic acid (Gly-Glu), in a phosphate-buffered saline (pH = 7.4) were studied in temperature range of 288–313 K by a densimetry method. Using the obtained density data, the apparent molar volumes of Ura, standard apparent molar volumes at infinite dilution and their derivatives with respect to temperature have been calculated. The concentration dependences of the apparent molar volume of uracil have a maximum, the position of which characterizes the 1:1 stoichiometry in the resulting uracil:peptide complexes. The standard apparent molar volume of neutral uracil increases upon complexation with charged peptide particles, such as +GlyTyr− zwitterions and +GlyGlu2− anions. The effects of Ura concentration and temperature on the volume properties were discussed in terms of predominant types of molecular interactions (hydrophilic, hydrophobic, ionic and zwitterionic) on the basis of co-spheres overlap model. The positive values of the second derivative of the standard apparent molar volume of uracil with respect to temperature at constant pressure indicate an increase in the ordering of the solvent in the environment of uracil with the addition of peptides, which may be associated with the blocking of its polar groups due to hydrogen bonding with the peptides. The addition of the more hydrophobic peptide, glycyl-tyrosine, which takes the zwitterionic form in solution, has a stronger structuring effect compared to the more hydrophilic anions of glycyl-glutamic acid.

Effect of Marinating Temperature of Atlantic Herring on Meat Ripening, Peptide Fractions Proportion, and Antioxidant Activity of Meat and Brine

The temperature has a significant effect on cathepsin activity, but the effect of temperature on the ripening of marinades, and the formation of protein hydrolysis products, is less studied than other technological factors. The results of this study showed that herring marinated at 2 °C showed a higher mass yield, but lower non-protein nitrogen (NPN), peptides, and free amino acid fraction content, than after marinating at 7 and 12 °C. The higher temperature increased the free amino acid content the most, and decreased the hardness, as measured via sensory assessment, of the marinated meat. This was confirmed by the hardness measurement in the texture profile analysis. The highest activity of cathepsins D and B in the meat was found at 7 °C, while cathepsin L was found at 2 °C. Increasing the temperature by 10 °C increased the diffusion/loss of nitrogenous substances from the meat to the brine by 36%. The meat and brine showed high antioxidant activity, which depended on the marinating temperature, and originated mainly from the 5–10 or <5 kDa fraction. The meat had a higher ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate) activity than the brine, opposite to the DPPH (2,2-diphenyl-1-picrylhydrazyl radical) activity, while the FRAP (Ferric Reducing Antioxidant Power) capacity was similar for meat and brine. The fractionation of the meat and brine extracts increased the antioxidant potential of FRAP and ABTS only for the brine. The most hydrophobic peptides were released during marinating at 7 °C. The meat and brine were dominated by 2–4 kDa peptides, followed by 4–6 and 0.5–2 kDa. The higher temperature favored a higher proportion of <4 kDa than >4 kDa peptides in the brine.

Covalent Organic Framework Nanosheets for Fluorescence Quantification of Peptide.

Rapid and sensitive quantification of peptides plays an important role in clinical diagnosis. Fluorescence assay is one of the most promising peptide detection tools, but it relies on intrinsic fluorescence or additional derivatization, resulting in poor versatility. Covalent organic frameworks (COFs) have shown a good application prospect in the field of fluorescence detection, but their application scope is limited to heavy metal ions and some small polar organic molecules. Herein, we report the application of COFs nanosheet for fluorescence detection of peptides. Fluorescent sp2 acrylonitrile-linked COFs nanosheets (TTAN-CON) were prepared by water-assisted ultrasonic exfoliation which performed with excellent fluorescence properties with Stokes shifts of 146 nm and fluorescence quantum yield of up to 24.45%. Compared to the bulk fluorescent COFs, exfoliated CONs films performed with better stability of fluorescence signal in solution. We found the fluorescence of TTAN-CON can be effectively quenched by hydrophobic peptides at a very rapid rate (less than 5 min per sample). TTAN-CON presented good sensitivity and selectivity for hydrophobic peptides detection via the static and dynamic joint quenching mechanism. TTAN-CON was further used to detect NLLGLIEAK and ProGRP31-98, two target peptide fragments of lung cancer biomarker ProGRP. The fluorescence intensities of TTAN-CON were negative linearly correlated with the amounts of hydrophobic NLLGLIEAK over the range of 5-1000 ng/mL with the correlation coefficients over 0.99, and the limit of detection was 1.67 ng/mL, displaying higher sensitivity and convenience than traditional optical methods. What's more, the quantification of ProGRP31-98 was achieved by the quantification of hydrophobic peptides in its enzyme hydrolysis products. We anticipate COFs nanosheets to be a universal fluorescence detection work-box for peptides biomarkers with clinical significance.

Simultaneous Profiling of Palmitoylomics and Glycomics with Photo/pH Dual-Responsive Magnetic Nanocomposites.

Following an in-depth examination of a single type of protein posttranslational modification, the synergistic analysis of two or more modification types has gradually emerged as a focal point in proteomic research. Palmitoylation and glycosylation are both critical for protein, implicated in carcinogenesis and inflammation. In this study, novel dual-responsive magnetic nanocomposites that serve as an ideal platform for the sequential or simultaneous enrichment of palmitoyl and glycopeptides are reported. The nanocomposites denoted as magDVS-VBA are constructed by modifying magnetic nanoparticles with azobenzene and divinyl sulfone (DVS), and self-assembled with 4-vinylbenzeneboronic acid (VBA)-immobilized β-cyclodextrin, which responds to light. The incorporated DVS component possesses the ability to recognize palmitoyl or glycopeptides under different pH conditions, whereas the introduction of VBA enhances the affinity of the nanocomposite for glycopeptides. Notably, magDVS-VBA exhibits flexible photo-, pH-, and magnetic-responsive capabilities, enabling the simultaneous recognition of hydrophobic palmitoyl peptides and hydrophilic glycopeptides for the first time. The developed platform demonstrates high specificity for sensitive palmitoylomics and glycomics analysis of mouse liver tissue, providing an effective method for studying of their crosstalk, and potential implications in clinical applications.

The Key Features of Catechols and α,β Unsaturated Carbonyl Moieties: Interaction With α‐syn Hydrophobic peptide and Activation of Catecholamines Pathway in Cells

AbstractParkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide, and the treatment focuses on delivering L‐DOPA. In this work, we isolated and tested several compounds against α‐synuclein and the hydrophobic peptide 71VTGVTAVAQKTV82 including flavonols (kaempferol, quercetin, and isorhamnetin), isoflavone (genistein) and flavone (luteolin), and compounds with α, β unsaturated carbonyl moieties such as chlorogenic acid and the depsidone fumarprotocetraric acid. Most compounds inhibit both α‐synuclein and hydrophobic peptide fibrillization. Moreover, ITC experiments showed a Kd varying from 9 to 20 μM, and ΔH values vary from −1.94 to −10.5 among the compounds. Docking experiments showed the intermolecular interactions within the sites 2, 9, and 3/13 of α‐synuclein, and with the hydrophobic peptide. In cultured cells, the presence of the compounds showed that most of them can promote cell proliferation and differentiation. Considering that treatments for neurodegenerative disorders, including PD, is only palliative the evaluation of these compounds that can prevent the fibrillization of α‐synuclein and stimulate the catecholamines pathway is promising.

Recognition Receptor for Methylated Arginine at the Single Molecular Level.

Among the various types of post-translational modifications (PTMs), methylation is the simple functionalized one that regulates the functions of proteins and affects interactions of protein-protein and protein-DNA/RNA, which will further influence diverse cellular processes. The methylation modification has only a slight effect on the size and hydrophobicity of proteins or peptides, and it cannot change their net charges at all, so the methods for recognizing methylated protein are still limited. Here, we designed a recognition receptor consisting of a α-hemolysin (α-HL) nanopore and polyamine decorated γ-cyclodextrin (am8γ-CD) to differentiate the methylation of peptide derived from a heterogeneous nuclear ribonucleoprotein at the single molecule level. The results indicate that the modification of a methyl group enhances the interaction between the peptide and the recognition receptor. The results of molecular simulations were consistent with the experiments; the methylated peptide interacts with the receptor strongly due to the more formation of hydrogen bonds. This proposed strategy also can be used to detect PTM in real biological samples and possesses the advantages of low-cost and high sensitivity and is label-free. Furthermore, the success in the construction of this recognition receptor will greatly facilitate the investigation of pathogenesis related to methylated arginine.

Moisturizing at a molecular level - The basis of Corneocare.

This review covers the last 20 years of research we and our collaborators have conducted on ethnic differences in facial skin moisturization placed in historical context with previous research. We have focussed particularly on the biochemical and cellular gradients of the stratum corneum (SC) with the aim of discovering new skin moisturization and SC maturation mechanisms, identifying new technologies and/or providing conceptual innovations for ingredients that will improve our understanding and treatment of dry skin. Specifically, we discuss gradients for corneodesmosomes and proteases, corneocyte phenotype-inducing enzymes, filaggrin and natural moisturizing factor (NMF), and barrier lipids. These gradients are interdependent and influence greatly corneocyte maturation. The interrelationship between corneodesmolysis and the covalent attachment of ω-hydroxy ceramides and ω-hydroxy fatty acids to the corneocyte protein envelope forming the corneocyte lipid envelope is especially relevant in our new understanding of mechanisms leading to dry skin. This process is initiated by a linoleoyl-ω-acyl ceramide transforming enzyme cascade including 12R lipoxygenase (12R-LOX), epidermal lipoxygenase-3 (eLOX3), epoxide hydrolase 3 (EPHX3), short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7), ceramidase and transglutaminase 1. Our research has opened the opportunity of using novel treatment systems for dry skin based on lipids, humectants, niacinamide and inhibitors of the plasminogen system. It is clear that skin moisturization is a more complex mechanism than simple skin hydration.

Fine‐tuning the antimicrobial activity of β‐hairpin peptides with fluorinated amino acids

AbstractAntimicrobial peptides (AMPs) possess bactericidal activity against a variety of pathogens depending on an overall balance of positively charged and hydrophobic residues. Selective fluorination of peptides serves to fine‐tune the intrinsic hydrophobicity and that could improve AMP bioactivity without affecting the sequence length. Only a few studies have focused on the impact of this unique element on antimicrobial potency and came to somewhat contractionary results. Moreover, the influence of fluorinated amino acids on peptide proteolysis is yet not fully understood. In this work, we tackle the link between side chain fluorination and both antimicrobial activity and proteolytic stability for two series of amphiphilic β‐hairpin peptides. In particular, a synergy between antimicrobial activity and peptide hydrophobicity was determined. All peptides were found to be barely hemolytic and non‐toxic. Most surprisingly, the fluorinated peptides were susceptible to enzymatic degradation. Hence, the distinctive properties of these polyfluorinated AMPs will serve for the future design of peptide‐based drugs.

A point mutation in GPI-attachment signal peptide accelerates the development of prion disease.

A missense variant from methionine to arginine at codon 232 (M232R) of the prion protein gene accounts for ~ 15% of Japanese patients with genetic prion diseases. However, pathogenic roles of the M232R substitution for the induction of prion disease have remained elusive because family history is usually absent in patients with M232R. In addition, the clinicopathologic phenotypes of patients with M232R are indistinguishable from those of sporadic Creutzfeldt-Jakob disease patients. Furthermore, the M232R substitution is located in the glycosylphosphatidylinositol (GPI)-attachment signal peptide that is cleaved off during the maturation of prion proteins. Therefore, there has been an argument that the M232R substitution might be an uncommon polymorphism rather than a pathogenic mutation. To unveil the role of the M232R substitution in the GPI-attachment signal peptide of prion protein in the pathogenesis of prion disease, here we generated a mouse model expressing human prion proteins with M232R and investigated the susceptibility to prion disease. The M232R substitution accelerates the development of prion disease in a prion strain-dependent manner, without affecting prion strain-specific histopathologic and biochemical features. The M232R substitution did not alter the attachment of GPI nor GPI-attachment site. Instead, the substitution altered endoplasmic reticulum translocation pathway of prion proteins by reducing the hydrophobicity of the GPI-attachment signal peptide, resulting in the reduction of N-linked glycosylation and GPI glycosylation of prion proteins. To the best of our knowledge, this is the first time to show a direct relationship between a point mutation in the GPI-attachment signal peptide and the development of disease.

Preparation of whey protein hydrolysates with ACE‐inhibitory activity using cysteine peptidases from Bromelia hieronymi Mez. (Bromeliaceae)

A proteolytic extract from fruits of Bromelia hieronymi was used to hydrolyse bovine whey proteins. The peptide profile obtained exhibited a gradual fading of the main whey proteins and a decrease in the content of hydrophobic peptides. The 180‐min hydrolysate showed a hydrolysis degree of 15.2% and inhibited the angiotensin‐converting enzyme (ACE), showing an IC50 of 0.17 mg/mL. By bioinformatics analysis, several theoretical sequences of possible ACE‐inhibitory peptides were deduced. These results showed that proteolytic extracts from B. hieronymi can be used to prepare whey hydrolysates that would serve as a potential bioactive ingredient of functional foods.

A Novel Surfactant with Short Hydrophobic Head and Long Hydrophilic Tail Generates Vesicles with Unique Structural Feature.

Surfactant molecules typically have a long hydrophobic tail and a short hydrophilic head group. It remains unexplored if surfactants can have a short hydrophobic head group and a long hydrophilic tail. Designing such surfactants is a challenge as a lengthy hydrophilic tail would completely solubilize the molecules. In this context, herein, the Fmoc-functionalized Gly-Pro-Hyp (GPO) tripeptide repeat-based molecule (Fm-GPO) with fluorenyl moiety as a short hydrophobic head and peptide as a long hydrophilic tail is demonstrated as a reverse surfactant at physiological pH, for the first time. π-π stacking of the fluorenyl moieties and intermolecular hydrogen bonding between the peptide chains with extended polyproline-II structure promoted the self-assembly into spherical vesicles with a unique feature of a large hydrophilic area in the interior and exterior of the bilayer. The current Fm-GPO system offers a new class of surfactants with unique features that can aid in the design of drug-loaded vehicles, which can be target-specific as the peptide chain can be manipulated with different functional ultra-short peptide sequences.

Identification of ACE I-Inhibitory Peptides Released by the Hydrolysis of Tub Gurnard (Chelidonichthys lucerna) Skin Proteins and the Impact of Their In Silico Gastrointestinal Digestion.

Tub gurnard is a highly abundant fishery species caught as a discard in the Mediterranean Sea. This work proposes its valorisation through the release of potential antihypertensive peptides and glycosaminoglycans (GAGs) through the controlled hydrolysis of tub gurnard skin proteins. Four proteases (Esperase, Alcalase, Trypsin and Pronase E) were used to obtain potent angiotensin converting enzyme I (ACE)-inhibitory hydrolysates. Peptides and GAGs were separated and evaluated for their antihypertensive potential by fluorometry. The peptide-rich fractions derived from the Esperase and Alcalase hydrolysates showed very low IC50 values (47 and 68 μg/mL, respectively). Only the GAGs from the Trypsin and Esperase hydrolysates were relevant ACE inhibitors (63 and 52% at 1 mg/mL, respectively). The peptide composition of the most potent ACE-inhibitory fractions derived from the Esperase and Alcalase hydrolysates (IC50 values of 33 and 29 μg/mL, respectively) was analysed by RP-LC-ESI-MS/MS. The analysis suggests that the ACE-inhibitory activity is related to the peptide hydrophobicity, as well as to the presence of specific residues at any of the last four C-terminal positions. The in silico gastrointestinal digestion of these fractions yielded small peptides with antihypertensive potential.

Controlling Amyloid Beta Peptide Aggregation and Toxicity by Protease-Stable Ligands.

Polymerization of soluble amyloid beta (Aβ) peptide into protease-stable insoluble fibrillary aggregates is a critical step in the pathogenesis of Alzheimer's disease (AD). The N-terminal (NT) hydrophobic central domain fragment 16KLVFF20 plays an important role in the formation and stabilization of β-sheets by self-recognition of the parent Aβ peptide, followed by aggregation of Aβ in the AD brain. Here, we analyze the effect of the NT region inducing β-sheet formation in the Aβ peptide by a single amino acid mutation in the native Aβ peptide fragment. We designed 14 hydrophobic peptides (NT-01 to NT-14) by a single mutation at 18Val by using hydrophobic residues leucine and proline in the natural Aβ peptide fragment (KLVFFAE) and analyzed its effect on the formation of Aβ aggregates. Among all these peptides, NT-02, NT-03, and NT-13 significantly affected the Aβ aggregate formation. When the NT peptides were coincubated with the Aβ peptide, a significant reduction in β-sheet formation and increment in random coil content of Aβ was seen, confirmed by circular dichroism spectroscopy and Fourier transform infrared spectroscopy, followed by the reduction of fibril formation measured by the thioflavin-T (ThT) binding assay. The aggregation inhibition was monitored by Congo red and ThT staining and electron microscopic examination. Moreover, the NT peptides protect the PC-12 differentiated neurons from Aβ-induced toxicity and apoptosis in vitro. Thus, manipulation of the Aβ secondary structure with protease-stable ligands that promote the random coil conformation may provide a tool to control the Aβ aggregates observed in AD patients.

Two transporters cooperate to secrete amphipathic peptides from the cytoplasmic and membranous milieus

Diverse organisms secrete amphipathic biomolecules for competitive gains. However, how cells cope with producing these membrane-permeabilizing molecules is unclear. We focused on the PSM family of secreted amphipathic peptides in the pathogen Staphylococcus aureus that uses two ABC transporters, PmtCD and AbcA, to export peptides across the bacterial cell membrane. We found that increased peptide hydrophobicity favors PSM secretion through PmtCD over AbcA and that only PmtCD protected cells against amphipathic peptides. We propose a two-system model in which PmtCD and AbcA independently export PSMs from either membrane or cytosolic environments, respectively. Our model provides a rationale for the encoding of multiple transport systems on diverse biosynthetic gene clusters used to produce distinct amphipathic molecules. In addition, our data serve as a guide for selectively blocking PSM secretion to achieve antimicrobial or antivirulence approaches and to disrupt established roles of PSM-mediated virulence.

Tuning the size and solvent-exposed hydrophobicity of a pore-forming peptide eliminates its cytotoxicity using a natural product.

Pore-forming toxins (PFTs) are a diverse group of peptides and proteins that interact with the cell membrane to form transmembrane pores which lead to to ion dyshomeostasis and cell death. Our previous work showed that claramine, an aminosterol derivative of cholesterol, protects brain and kidney cells from prototypical PFTs. This effect was not induced by a change in protein structure, but instead by incorporating into and changing the physico-chemical properties of the plasma membrane to prevent the binding of these toxins. In the search for compounds that can target the structures of PFTs directly to eliminate their toxicity, we found that the polyphenol epigallocatechin gallate (EGCG) directly interacts with the monomeric PFT melittin, the active component of honeybee venom, resulting in the elimination of its binding to the cell membrane and neutralization of its cytotoxicity. EGCG achieved this effect by lowering the solvent-exposed hydrophobicity of melittin and promoting its oligomerization into larger aggregates. These biophysical properties have also shown to play an important role in the membrane binding and associated cytotoxicity of misfolded protein oligomers implicit in neurodegenerative diseases. Our results show that this relationship translates to the attenuation of melittin cytotoxicity by EGCG. These findings emphasize the importance of the physico-chemical properties of PFTs in controlling their binding to cell membranes and suggest a potential mechanism for therapeutic development against PFTs.

Post‐translational modification and phenotype

Post‐translational modification and phenotype

Mechanism of action of beta-stranded antimicrobial (KL)n model peptides.

Here, we investigate model amphipathic β-stranded peptides, consisting of alternating lysine and leucine residues. These so called KLn peptides ([KL]n/2-NH2) show high antimicrobial activity against various bacterial strains. At the same time, they tend to aggregate into β-pleated amyloid-like fibrils. We systematically investigated their membrane affinity and their structural behaviour in the presence of lipid membranes as a function of peptide length, charge and hydrophobicity, using peptides with 6-26 amino acids. Different biophysical techniques (circular dichroism, solid-state NMR and fluorescence assays) were used and the results correlated with results of biological assays (antimicrobial and hemolytic assays), to identify possible mechanisms of action of KLn peptides. We find that the very short KL peptides are inactive, but longer peptides are highly hemolytic and aggregates strongly, especially in the presence of phosphate [1]. There is an optimal length of around 10 amino acids where the KL peptides have a high antimicrobial effect and low hemolysis. At this length, peptides are long enough to form stable β-stranded structure but flexible enough to not aggregate very fast in solution. A similar length behaviour has been observed for other similar peptides made from alternating hydrophobic and charged residues [2]. Vesicle leakage is length-dependent and peptides need at least 12 amino acids to induce high leakage. This threshold length does not depend on membrane thickness. This indicates that pores are probably not formed by these peptides, and this was further confirmed by solid-state 15N- and 19F-NMR experiments, whose results were incompatible with pore models [3]. Thus, it seems that the peptides are bound to the membrane surface and destabilize the membrane by some kind of carpet mechanism.