Polypeptide Moiety Research Articles

One-pot functionalization of carbon dots with ecPis-4s antimicrobial peptide

The synthesis of nanobiostructures (CD@ecPis-4s) composed of carbon dots (CD) covalently bound to the antimicrobial peptide ecPis-4s is reported. Nanoparticle functionalization was achieved by pre-activating the oxygenated functional groups using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and ethyl (hydroxyimino)cyanoacetate in an aqueous medium. The obtained nanobiostructures were characterized by several techniques, including FTIR and 1H NMR spectroscopies. The functionalization does not compromise the optical properties of CD. The polypeptide moiety of the peptide-decorated nanoparticles retains the active α-helical conformation and lytic activity in the presence of anionic phospholipid vesicles as observed in the circular dichroism and calcein release experiments. Consequently, the CD@ecPis-4s nanobiostructures present similar activity against Gram-positive (S. aureus and S. agalactiae) and Gram-negative (E. coli and P. aeruginosa) bacteria strains when compared to the free peptide. To our knowledge, this is the first report on carbon dots containing a covalently bound antimicrobial peptide. Thereby, in an era of increasing antimicrobial resistance, the synthesis and investigations performed in this work led to materials with potential for monitoring, in loco, bacterial contamination via exposure to UV radiation.

A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell.

Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains, i.e. polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed peptidyl-tRNAenrichment using organic extraction and silica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using Escherichia coli, we identified ∼800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods to investigate nascent chains in the cell.

Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker.

Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.

Recognition of glycan and protein substrates by N-acetylglucosaminyltransferase-V

BackgroundN-Glycosylation is crucial for protein folding, trafficking, and functions. N-Glycans have a different number of N-acetylglucosamine (GlcNAc) branches in a protein-selective manner, and the β1,6-linked GlcNAc branch on specific proteins produced by N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) promotes cancer malignancy. However, little is known about how GnT-V acts on specific target proteins. MethodsBased on our structural model, we hypothesized that GnT-V interacts with the N-glycan core or polypeptide moiety as well as the accepter site of N-glycan. To explore this possibility, we selected four candidate residues involved in the interaction with the glycan core or surrounding amino acids, created point mutants of these residues, and examined the in vitro and in vivo activities of the mutants. ResultsOur in vitro enzyme assays using various types of substrates including oligosaccharides and glycoproteins revealed that the V354N mutant had dramatically reduced activity for all tested substrates with an altered substrate preference and that K361A had reduced activity for an oligosaccharide with asparagine (Asn), but not a shorter oligosaccharide without the reducing end of GlcNAc and Asn. These results suggest that V354 and K361 are involved in the recognition of N-glycan core and surrounding amino acids. We further performed rescue experiments using GnT-V knockout HeLa cells and confirmed the importance of these residues for modifications of glycoproteins in cells. ConclusionsWe identified several residues involved in the action of GnT-V toward N-glycan cores and surrounding amino acids. General significanceOur data provide new insights into how GnT-V recognizes glycoproteins.

Innovative gene therapy for lysosomal neuraminidase 1 (NEU1) deficiencies

N-Glycosylation is crucial for protein folding, trafficking, and functions. N-Glycans have a different number of N-acetylglucosamine (GlcNAc) branches in a protein-selective manner, and the β1,6-linked GlcNAc branch on specific proteins produced by N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) promotes cancer malignancy. However, little is known about how GnT-V acts on specific target proteins.Based on our structural model, we hypothesized that GnT-V interacts with the N-glycan core or polypeptide moiety as well as the accepter site of N-glycan. To explore this possibility, we selected four candidate residues involved in the interaction with the glycan core or surrounding amino acids, created point mutants of these residues, and examined the in vitro and in vivo activities of the mutants.Our in vitro enzyme assays using various types of substrates including oligosaccharides and glycoproteins revealed that the V354N mutant had dramatically reduced activity for all tested substrates with an altered substrate preference and that K361A had reduced activity for an oligosaccharide with asparagine (Asn), but not a shorter oligosaccharide without the reducing end of GlcNAc and Asn. These results suggest that V354 and K361 are involved in the recognition of N-glycan core and surrounding amino acids. We further performed rescue experiments using GnT-V knockout HeLa cells and confirmed the importance of these residues for modifications of glycoproteins in cells.We identified several residues involved in the action of GnT-V toward N-glycan cores and surrounding amino acids.Our data provide new insights into how GnT-V recognizes glycoproteins.

The Role of SUMOylation and Ubiquitination in Brain Ischaemia: Critical Concepts and Clinical Implications.

Brain ischaemia is a severe form of metabolic stress that activates a cascade of pathological events involving many signalling pathways. Modulation of these pathways is largely mediated by post-translational modifications (PTMs). Indeed, PTMs can rapidly modify pre-existing proteins by attaching chemical or polypeptide moieties to selected amino acid residues, altering their functions, stability, subcellular localizations, or interactions with other proteins. Subsequently, related signalling pathways can be substantially affected. Thus, PTMs are widely deployed by cells as an adaptive strategy at the front line to efficiently cope with internal and external stresses. Many types of PTMs have been identified, including phosphorylation, O-GlcNAcylation, small ubiquitin-like modifier (SUMO) modification (SUMOylation), and ubiquitination. All these PTMs have been studied in brain ischaemia to some extent. In particular, a large body of evidence has demonstrated that both global SUMOylation and ubiquitination are massively activated after brain ischaemia, and this activation may play a critical role in defining the fate and function of cells in the post-ischaemic brain. The goal of this review will be to summarize the current findings on SUMOylation and ubiquitination in brain ischaemia and discuss their clinical implications.

Glycan region of GPI anchored-protein is required for cytocidal oligomerization of an anticancer parasporin-2, Cry46Aa1 protein, from Bacillus thuringiensis strain A1547

Parasporin-2 (PS2), alternatively named Cry46Aa1, an anticancer protein derived from Bacillus thuringiensis strain A1547, causes specific cell damage via PS2 oligomerization in the cell membrane. Although PS2 requires glycosylphosphatidylinositol (GPI)-anchored proteins for its cytocidal action, their precise role is unknown. Here, we report that the glycan of GPI induces PS2 oligomerization, which causes cell death. Cytotoxicity, cell-binding and oligomerization of the toxin were not observed in GPI-anchored protein-deficient Chinese hamster ovary cells. Expression and protease-treatment analyses showed that the actions of the toxin were dependent on the glycan core, not the polypeptide moiety, of GPI-anchored proteins. However, surface expression of some GPI-anchored proteins is observed in PS2-insensitive cells. These data suggest that GPI-anchored proteins do not determine the target specificity, but instead function as a kind of coreceptor, in the cytocidal action of PS2.

Isolation and Screening of Protease Enzyme Producing Bacteria from Cheese at Dilla University, Ethiopia

Enzymes are biological catalysts that facilitate the conversion of substrates into products by providing favorable conditions that lower the activation energy of the reaction. An enzyme may be a protein or a glycoprotein and consists of at least one polypeptide moiety. Proteases are the most important industrial enzymes and comprise about 25% of commercial enzymes in the world. Two third of the industrially produced proteases are from microbial sources. The current study was focused on the screening of protease enzyme producing bacteria from dairy product of cheese at Dilla University. Cheese sample was collected from Dilla town market. The proteases enzyme production bacteria were screened on milk agar plate through spread plate method. As the result, certain bacteria were screened for protease enzyme production as well as were confirmed on milk agar medium. Almost all isolated (35 isolate) of bacteria from cheese food stuff were had a potential in the production of protease enzyme which help for various activities. Therefore, all isolate had a promising potential for production proteolytic enzyme which are used in industries and the other sectors.

Probing the interaction between heparan sulfate proteoglycan with biologically relevant molecules in mimetic models for cell membranes: A Langmuir film study

Investigating the role of proteoglycans associated to cell membranes is fundamental to comprehend biochemical process that occurs at the level of membrane surfaces. In this paper, we exploit syndecan-4, a heparan sulfate proteoglycan obtained from cell cultures, in lipid Langmuir monolayers at the air–water interface. The monolayer served as a model for half a membrane, and the molecular interactions involved could be evaluated with tensiometry and vibrational spectroscopy techniques. Polarization–modulation infrared reflection–absorption spectroscopy (PM-IRRAS) employed in a constant surface pressure regime showed that the main chemical groups for syndecan-4 were present at the air–water interface. Subsequent monolayer decompression and compression showed surface pressure-area isotherms with a large expansion for the lipid monolayers interacting with the cell culture reported to over-express syndecan-4, which was also an indication that the proteoglycan was inserted in the lipid monolayer. The introduction of biological molecules with affinity for syndecam-4, such as growth factors, which present a key role in biochemical process of cell signaling, changed the surface properties of the hybrid film, leading to a model, by which the growth factor binds to the sulfate groups present in the heparan sulfate chains. The polypeptide moiety of syndecan-4 responds to this interaction changing its conformation, which leads to lipid film relaxation and further monolayer condensation.

The interactions of calreticulin with immunoglobulin G and immunoglobulin Y

Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N-glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N-linked glycan, which is a ligand for calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of calreticulin. The experiments demonstrate the ability of calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY.

Synthesis of peptide-oligonucleotide conjugates using a heterobifunctional crosslinker.

Peptide-oligonucleotide conjugates (POCs) are molecular chimeras composed of a nucleic acid moiety covalently attached to a polypeptide moiety. POCs have been used in numerous applications from therapeutics to nanotechnology, and most recently as combinatorial agents in the assembly of bivalent protein affinity reagents. This unit describes the synthesis and purification of POC molecules using the heterobifunctional crosslinking reagent succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), which enables amine-modified oligonucleotides to become covalently linked to cysteine-modified polypeptides. This solution-based protocol consists of a two-step synthesis followed by a single purification step.

Human lactoferrin activates NF‐κB through the Toll‐like receptor 4 pathway while it interferes with the lipopolysaccharide‐stimulated TLR4 signaling

Lactoferrin (LF) has been implicated in innate immunity. Here we reveal the signal transduction pathway responsible for human LF (hLF)-triggered nuclear factor-kappaB (NF-kappaB) activation. Endotoxin-depleted hLF induces NF-kappaB activation at physiologically relevant concentrations in the human monocytic leukemia cell line, THP-1, and in mouse embryonic fibroblasts (MEFs). In MEFs, in which both tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 are deficient, hLF causes NF-kappaB activation at a level comparable to that seen in wild-type MEFs, whereas TRAF6-deficient MEFs show significantly impaired NF-kappaB activation in response to hLF. TRAF6 is known to be indispensable in leading to NF-kappaB activation in myeloid differentiating factor 88 (MyD88)-dependent signaling pathways, while the role of TRAF6 in the MyD88-independent signaling pathway has not been clarified extensively. When we examined the hLF-dependent NF-kappaB activation in MyD88-deficient MEFs, delayed, but remarkable, NF-kappaB activation occurred as a result of the treatment of cells with hLF, indicating that both MyD88-dependent and MyD88-independent pathways are involved. Indeed, hLF fails to activate NF-kappaB in MEFs lacking Toll-like receptor 4 (TLR4), a unique TLR group member that triggers both MyD88-depependent and MyD88-independent signalings. Importantly, the carbohydrate chains from hLF are shown to be responsible for TLR4 activation. Furthermore, we show that lipopolysaccharide-induced cytokine and chemokine production is attenuated by intact hLF but not by the carbohydrate chains from hLF. Thus, we present a novel model concerning the biological function of hLF: hLF induces moderate activation of TLR4-mediated innate immunity through its carbohydrate chains; however, hLF suppresses endotoxemia by interfering with lipopolysaccharide-dependent TLR4 activation, probably through its polypeptide moiety.

Facile and rapid access to linear and truncated microcystin analogues for the implementation of immunoassays

A series of simplified microcystin-LR analogues based on Adda [(2S,3S,8S,9S,4E,6E)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldecadienoic acid] or its corresponding aldol precursor linked to a polypeptide moiety have been synthesised and assessed for their binding affinity by the monoclonal antibody mAb MC159, an anti-microcystin-LR mAb recently selected by us for the detection of microcystins through various immunoassay formats. Some modifications have been brought to the enantiospecific synthesis of N-Boc-Adda developed by Pearson et al. (Org. Lett., 2000, 2, 2901) which enabled us to access in an economical and time-saving manner a small library of MC-LR linear analogues. Among which Adda was chosen to synthesise, as an illustrative example, a fluorescent probe derived from this beta-amino acid. This probe was subsequently solid-phase immobilised by means of oxime ligation in order to lead to biochips suitable for microcystin detection through the SPIT-FRI method.

Role of the polypeptide backbone and post-translational modifications in cross-reactivity of Art v 1, the major mugwort pollen allergen

Artemisia vulgaris (mugwort) is one of the main causes of late summer pollinosis in Europe, with >95% of patients sensitized to the glycoallergen Art v 1. Despite the importance of this allergen, little is known about its cross-reactive behavior. Here we investigated the occurrence of conserved Art v 1 antigenic determinants in sources known to display clinically relevant cross-reactivity with mugwort pollen. For this purpose, monoclonal antibodies specific for a cysteine-stabilized epitope of the Art v 1 defensin domain and for carbohydrates attached to the proline domain were produced by hybridoma and phage display technologies. Using polyclonal Art v 1-specific rabbit sera and antibodies against both the Art v 1 carbohydrate and polypeptide moieties, we could identify cross-reactive structures in pollen from botanically related Asteraceae weeds (Artemisia absinthium, Helianthus annuus and Ambrosia sp.). Homologous allergens were also recognized by IgE from mugwort-sensitized patients and the reactivity could be decreased by serum pre-incubation with natural and recombinant Art v 1. As no cross-reactive structures could be found in foods associated with mugwort pollinosis, we conclude that Art v 1 is poorly involved in mugwort cross-reactivity to food allergens.

Effect of GPI anchor moiety on the immunogenicity of DNA plasmids encoding the 19‐kDa C‐terminal portion of Plasmodium falciparum MSP‐1

The glycosylphosphatidylinositol (GPI)-anchored Plasmodium falciparum merozoite surface protein 1 (MSP-1) is a widely studied malaria vaccine candidate. The C-terminal 19-kDa portion of MSP-1 (MSP-1(19)) is of particular interest because this polypeptide moiety remains bound to the parasite even after erythrocyte invasion, while the remainder of MSP-1 is shed during invasion. Studies have shown that antibodies against MSP-1(19) inhibit merozoite invasion of erythrocytes efficiently, and that MSP-1(19) produces protective immunity in mice and monkeys. To investigate the efficacy of MSP-1(19 )DNA vaccine and role of GPI anchor moiety in the immunogenicity of MSP-1(19), we constructed expression vectors that produce MSP-1(19) as either secretory or GPI-anchored polypeptide. Both constructs efficiently expressed MSP-1(19) in transfected HEK-293 cells. While the recombinant plasmid lacking GPI anchor signal sequence expressed MSP-1(19) mainly as secreted polypeptide, that containing GPI anchor signal sequence produced GPI-anchored MSP-1(19 )on cell surface. In immunized mice, both constructs produced substantial levels of MSP-1(19)-specific IgG1, IgG2a, IgG2b, IgG3, IgA and IgM antibodies. In both cases, the IgG1 level was significantly higher than other isotypes. Interestingly, the plasmid containing GPI anchor signal sequence produced significantly higher levels of IgG2a and IgG2b than the plasmid that lacks GPI signal sequence.

Spectral Properties and DNA Targeting Features of a Thiazole Orange−Peptide Bioconjugate

The molecular recognition features of a DNA-sensitive fluorescent bioconjugate capable of targeting a specific DNA sequence with high efficiency are described. The bioconjugate combines a polypeptide from the Tc3 transposase DNA-binding domain with the dsDNA-sensitive fluorophore thiazole orange. Fluorescence spectroscopy and circular dichroism reveal that the polypeptide moiety determines the DNA sequence specificity as the intercalating dye makes nonspecific contributions to binding affinity. The conjugated thiazole orange is able to intercalate and fluoresce when the peptide binds at concentrations where little fluorescence is observed from either the bioconjugate alone or the bioconjugate mixed with DNA lacking the target sequence. Fluorescence studies indicate this molecular probe is sequence specific, binds the native Tc3 DNA target sequence with nanomolar affinity (KD approximately 15 nM), and is able to discriminate multiple point mutations in the cognate DNA site. The attachment of a sequence-specific binding peptide onto a functional probe provides a viable strategy for construction of synthetic enzymes and repressors, and facilitates dynamic studies of protein-DNA interactions.

Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries

Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and mu-calpain rapidly (k(2)/K(i) = 131,000 and 16,500 m(-1) s(-1), respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

Influence of the glycosylphosphatidylinositol anchor in the morphology and roughness of Langmuir–Blodgett films of phospholipids containing alkaline phosphatases

The morphology of phospholipid Langmuir–Blodgett films containing a glycosylphosphatidylinositol-anchored rat osseous plate alkaline phosphatase was studied by atomic force microscopy. Two forms of the enzyme were used in this study: an anchor-containing detergent-solubilized form, and a phospholipase-solubilized form, without the hydrophobic moiety. Direct measurements of catalytic activity in enzyme/phospholipid mixed films, and nanogravimetric estimations by quartz crystal microbalance confirmed the adsorption of both forms of alkaline phosphatase. Atomic force microscopy analysis showed that both enzyme forms originated aggregates when adsorbed on phospholipid Langmuir–Blodgett films. However, the phospholipase-solubilized form showed a higher roughness when compared to the detergent solubilized alkaline phosphatase. A model suggesting different alignments of the major axis of the ellipsoid polypeptide moiety relative to the film surface is proposed. This model, supported by surface density and catalytic activity data, might explain the difference of roughness observed for films containing each enzyme form adsorbed onto a phospholipid-modified solid support.

Hydration of a glycoprotein: relative water affinity of peptide and glycan moieties

Glycosylation, the most prevalent post-translational modification of proteins, affects a number of physical properties including the interactions with the surrounding aqueous solvent. Such glycan-water interactions have been discussed with respect to the increased solubility generally observed for glycoproteins, but experimental support of this correlation remains sparse. We have applied a two-channel calorimetric method to measure the free energy and enthalpy of hydration at 25 degrees C for the glycoprotein phytase (Phy) and a deglycosylated form (dgPhy) of the same protein. Comparisons of results for Phy and dgPhy show that the polypeptide moiety has a higher affinity for water than the glycans. In fact, at moderate hydration levels (approximately 0.3 g water/g macromolecule) the water uptake appears to be entirely governed by adsorption to the peptide groups. We conclude that strengthened interaction with the solvent is unlikely to be the mechanism underlying the increased solubility and lowered propensity of aggregation often reported to result from the glycosylation of proteins.

Incorporation conditions guiding the aggregation of a glycosylphosphatidyl inositol (GPI)-anchored protein in Langmuir monolayers

This work investigates the process of incorporation of a glycosylphosphatidyl inositol (GPI)-anchored alkaline phosphatase into Langmuir monolayers of dimyristoyl phosphatidic acid (DMPA). Three different methods of protein incorporation were assayed. When the protein solution was injected below the air–water interface after formation of the lipid monolayer a micro-heterogeneous distribution of alkaline phosphatase throughout the interface was observed. Adsorption kinetics studied by fluorescence microscopy, associated with surface pressure measurements, led to the proposition of a model in which the protein penetration is modulated by the surface packing of the monolayer and intermolecular interactions occurring between the phospholipid and the protein. At initial surface pressures higher than 20 mN m −1, the protein is quickly adsorbed on the interface and the lateral diffusion drives the alkyl chains to turn towards the air phase while the polypeptide moiety faces the aqueous subphase.