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The odorant binding protein, OBP44a is one of the most abundant proteins expressed in the brain of the developing fruit fly Drosophila melanogaster. Its cellular function has not yet been determined. The OBP family of proteins is well established to recognize hydrophobic molecules. In this study, NMR is employed to structurally characterize OBP44a. NMR chemical shift perturbation measurements confirm that OBP44a binds to fatty acids. Complete assignments of the backbone chemical shifts and secondary chemical shift analysis demonstrate that the apo state of OBP44a is comprised of six α-helices. Upon binding 8(Z)-eicosenoic acid (8(Z)-C20:1), the OBP44a C-terminal region undergoes a conformational change, from unstructured to α-helical. In addition to C-terminal restructuring upon ligand binding, some hydrophobic residues show dramatic chemical shift changes. Surprisingly, several charged residues are also strongly affected by lipid binding. Some of these residues could represent key structural features that OBP44a relies on to perform its cellular function. The NMR chemical shift assignment is the first step towards characterizing the structure of OBP44a and how specific residues might play a role in lipid binding and release. This information will be important in deciphering the biological function of OBP44a during fly brain development. [ABSTRACT FROM AUTHOR]

Lipids are the primary form of energy storage and a major component of plasma membranes, which form the interface between the cell and the extracellular environment. Several lipids — including phosphoinositide, phosphatidic acid, sphingolipids, lysophospholipids, oxylipins, and free fatty acids — also serve as substrates for the generation of signalling molecules. Abiotic stresses, such as drought and temperature stress, are known to affect plant growth. In addition, abiotic stresses can activate certain lipid‐dependent signalling pathways that control the expression of stress‐responsive genes and contribute to plant stress adaptation. Many studies have focused either on the enzymatic production and metabolism of lipids, or on the mechanisms of abiotic stress response. However, there is little information regarding the roles of plant lipids in plant responses to abiotic stress. In this review, we describe the metabolism of plant lipids and discuss their involvement in plant responses to abiotic stress. As such, this review provides crucial background for further research on the interactions between plant lipids and abiotic stress. [ABSTRACT FROM AUTHOR]

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid‐binding features of αS through the design of site‐directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane‐bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death. Brief statement outlining significance: Modulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane‐bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS. [ABSTRACT FROM AUTHOR]

With advancements of modern biophysical tools and superresolution imaging, cell biology is entering a new phase of research with technological power fitting for membrane dynamics analyses. However, our current knowledge base of cellular signaling events is mostly built on a network of protein interactions, which is incompatible with the essential roles of membrane activities in those events. The lack of a theoretical platform is rendering biophysical analyses of membrane biology supplementary to the protein-centric paradigm. We hypothesize a framework of signaling events mediated by lipid dynamics and argue that this is the evolutionarily obligatory developmental path of cellular complexity buildup. In this framework, receptors are the late comers, integrating into the pre-existing membrane based signaling events using their lipid interface as the point of entry. We further suggest that the reason for cell surface receptors to remain silent at the resting state is via the suppression effects of their surrounding lipids. The avoidance of such a suppression, via ligand binding or lipid domain disruption, enables the receptors to autonomously integrate themselves into the preexisting networks of signaling cascades.

With advancements of modern biophysical tools and superresolution imaging, cell biology is entering a new phase of research with technological power fitting for membrane dynamics analyses. However, our current knowledge base of cellular signaling events is mostly built on a network of protein interactions, which is incompatible with the essential roles of membrane activities in those events. The lack of a theoretical platform is rendering biophysical analyses of membrane biology supplementary to the protein-centric paradigm. We hypothesize a framework of signaling events mediated by lipid dynamics and argue that this is the evolutionarily obligatory developmental path of cellular complexity buildup. In this framework, receptors are the late comers, integrating into the pre-existing membrane based signaling events using their lipid interface as the point of entry. We further suggest that the reason for cell surface receptors to remain silent at the resting state is via the suppression effects of their surrounding lipids. The avoidance of such a suppression, via ligand binding or lipid domain disruption, enables the receptors to autonomously integrate themselves into the preexisting networks of signaling cascades. [ABSTRACT FROM AUTHOR]

The use of silver nanoparticles (AgNPs) with their novel and distinct physical, chemical, and biological properties, has proven to be an alternative for the development of new antibacterial agents. In particular, the possibility to generate AgNPs coated with novel capping agents, such as phytomolecules obtained via a green synthesis (G-AgNPs), is attracting great attention in scientific research. Recently, we showed that membrane interactions seem to be involved in the antibacterial activity of AgNPs obtained via a green chemical synthesis using the aqueous leaf extract of chicory (Cichorium intybus L.). Furthermore, we observed that these G-AgNPs exhibited higher antibacterial activity than those obtained by chemical synthesis. In order to achieve the green AgNPs mode of action as well as their cellular target, we aimed to study the antibacterial activity of this novel green AgNPs against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The effect of the G-AgNPs on the bacterial surface was first evaluated by zeta potential measurements and correlated with direct plate count agar method. Afterwards, atomic force microscopy was applied to directly unravel the effects of these G-AgNPs on bacterial envelopes. Overall, the data obtained in this study seems correlate with a multi-step mechanism by which G-AgNPs-lipid membrane interactions is the first step prior to membrane disruption, resulting in antibacterial activity. Unlabelled Image • Green AgNPs-bacteria interaction was studied by zeta potential and AFM imaging. • G-AgNPs are able to induce several damages on the bacterial envelope. • G-AgNPs promote no lipid packing disruption on a bacterial model membrane. • G-AgNPs can lead to bacterial death via a multifaceted mechanism. • Green synthesis allows producing AgNPs with enhanced antibacterial activity. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Green AgNPs (G-AgNPs) can interact with lipid layers used as membrane-mimetic models. • Biophysical characterization proves an interfacial AgNPs-lipid membrane interaction. • Aromatic/hydrophobic moieties in capping agents favors the G-AgNPs insertion into lipid layers. • Membrane affinity should be an essential step in the antibacterial activity of G-AgNPs. Abstract Silver nanoparticles (AgNPs) constitute a very promising approach for overcoming the emergence of antibiotic resistance bacteria. Although their mode of action could be related with membrane damage, the AgNPs-lipid membrane interaction is still unclear. In this sense, the present work investigated the interaction of model lipid membranes with AgNPs coated with different capping agents such as citrate (C-AgNPs) and phytomolecules (G-AgNPs) obtained via a green synthesis. The AgNPs-membrane interactions were evaluated studying i) the surface pressure changes on both zwitterionic (DMPC) and negatively charged (DMPC:DMPG) lipid monolayers, ii) the zeta potential and DLS of DMPC:DMPG liposomes and iii) Zeta potential on Escherichia coli membranes, incubated with this nanomaterials. The results showed that both negatively charged-AgNPs can interact with these lipid monolayers inducing an increase in the surface pressure but G-AgNPs presented a significantly higher affinity toward both monolayers in comparison with C-AgNPs. Zeta potential data confirmed again the interaction event showing that both DMPC:DMPG liposomes and E. coli bacteria became more negative with the addition of G-AgNPs. This increased net negative charge of the liposomes and E. coli allows to indicate an interfacial interaction where the green nanometal should keep adsorbed to the membrane via the insertion of aromatic/hydrophobic moieties of capping agents on the surface of AgNPs into the lipid bilayer. Summarizing, the AgNPs-membrane interaction should be an essential step in the antibacterial activity either because the membrane is the main target or by increasing the local concentration of silver from G-AgNPs accumulation which could cause the bactericidal effect. [ABSTRACT FROM AUTHOR]

The cleavage of the amyloid precursor protein by β- and γ-secretases is a key event in Alzheimer's disease. A fusion protein was constructed to investigate the cleavage rate and aggregation kinetics of amyloid-beta (1–40) (Aβ(1–40)) peptides. The peptide was expressed with a Small Ubiquitin-Like Modifier (SUMO) on the N-terminus and cleaved by a SUMO protease Ulp1. The time course of the cleavage reaction was monitored by SDS-PAGE gel with 100:1 or 1000:1 SUMO-Aβ(1–40) to Ulp1 molar ratio and in the presence of brain total lipid extract unilamellar vesicles. Similarly, the aggregation of Aβ(1–40) peptides upon cleavage was monitored by thioflavin T fluorescence assays and by circular dichroism. The cleavage reaction was modulated by the concentration of Ulp1, with fast release of Aβ(1–40) peptides producing shorter lag time before fibril formation, but with similar elongation rate. The presence of lipids significantly reduced the cleavage completion at 1000:1, but reduced the lag time before fibril formation, while at 100:1 similar cleavage and aggregation kinetics were observed compared to the lipid-free condition. Overall, the results showed that the fusion protein SUMO-Aβ(1–40) is a means to study the cleavage and aggregation of amyloid peptides and that the presence of lipids and the fast release rate accelerated the aggregation of Aβ(1–40) peptides. [ABSTRACT FROM AUTHOR]

Merozoite surface protein 2 ( MSP2) is a highly abundant, GPI-anchored antigen on the malaria parasite Plasmodium falciparum. MSP2 induces an immune response in the context of natural infections and vaccine trials, and these responses are associated with protection from parasite infection. Recombinant MSP2 is highly disordered in solution but antigenic analyses suggest that it is more ordered on the merozoite surface. We have shown previously that the interaction of recombinant full-length MSP2 with lipid surfaces induces a conformational change in the conserved N-terminal region of MSP2, which contributes to epitope masking in this region. To explore the impacts of lipid interactions on the conformation and antigenicity of the conserved C-terminal region of MSP2, a construct corresponding to this domain, MSP2172-221, was designed. NMR studies indicate that many residues in MSP2172-221 interact with DPC micelles, including some in epitopes recognised by C-terminal-specific monoclonal antibodies, but, in contrast to the MSP2 N-terminus, there is no indication of stable helical conformation. The binding affinities of a panel of monoclonal antibodies indicate that MSP2172-221 is antigenically similar to full-length MSP2 and show that liposome conjugation alters the antigenicity in a manner that may mimic native MSP2 on the merozoite surface. These findings highlight the impact of lipid interactions on the conformation and antigenicity of MSP2172-221 and will assist in the design of recombinant MSP2 immunogens for use as malaria vaccine candidates. Databases Resonance assignments are available in the BioMagResBank (BMRB) database under the accession number 27134. [ABSTRACT FROM AUTHOR]

Structures can now be predicted for any protein using programs like AlphaFold and Rosetta, which rely on a foundation of experimentally determined structures of architecturally diverse proteins. The accuracy of such artificial intelligence and machine learning (AI/ML) approaches benefits from the specification of restraints which assist in navigating the universe of folds to converge on models most representative of a given protein's physiological structure. This is especially pertinent for membrane proteins, with structures and functions that depend on their presence in lipid bilayers. Structures of proteins in their membrane environments could conceivably be predicted from AI/ML approaches with user-specificized parameters that describe each element of the architecture of a membrane protein accompanied by its lipid environment. We propose the Classification Of Membrane Proteins based On Structures Engaging Lipids (COMPOSEL), which builds on existing nomenclature types for monotopic, bitopic, polytopic and peripheral membrane proteins as well as lipids. Functional and regulatory elements are also defined in the scripts, as shown with membrane fusing synaptotagmins, multidomain PDZD8 and Protrudin proteins that recognize phosphoinositide (PI) lipids, the intrinsically disordered MARCKS protein, caveolins, the β barrel assembly machine (BAM), an adhesion G-protein coupled receptor (aGPCR) and two lipid modifying enzymes - diacylglycerol kinase DGKε and fatty aldehyde dehydrogenase FALDH. This demonstrates how COMPOSEL communicates lipid interactivity as well as signaling mechanisms and binding of metabolites, drug molecules, polypeptides or nucleic acids to describe the operations of any protein. Moreover COMPOSEL can be scaled to express how genomes encode membrane structures and how our organs are infiltrated by pathogens such as SARS-CoV-2. [Display omitted] [ABSTRACT FROM AUTHOR]

Cell membranes are composed of a lipid bilayer containing proteins that cross and/or interact with lipids on either side of the two leaflets. The basic structure of cell membranes is this bilayer, composed of two opposing lipid monolayers with fascinating properties designed to perform all the functions the cell requires. To coordinate these functions, lipid composition of cellular membranes is tailored to suit their specialized tasks. In this review, we describe the general mechanisms of membrane–protein interactions and relate them to some of the molecular strategies organisms use to adjust the membrane lipid composition in response to a decrease in environmental temperature. While the activities of all biomolecules are altered as a function of temperature, the thermosensors we focus on here are molecules whose temperature sensitivity appears to be linked to changes in the biophysical properties of membrane lipids. This article is part of a Special Issue entitled: Lipid–protein interactions. [ABSTRACT FROM AUTHOR]

Composition and phase dependence of the mixing of 1,2-Dipalmitoyl- sn -glycero-3-phosphocholine (DPPC), and 1,2-Dioleoyl- sn -glycero-3-phosphocholine (DOPC), with the oxidized phospholipid, 1-palmitoyl-2-glutaryl- sn -glycero-3-phosphocholine (PGPC) were investigated by characterizing the aggregation states of DPPC/PGPC and DOPC/PGPC using a fluorescence quenching assay, dynamic light scattering, and time-resolved fluorescence quenching in the temperature range 5–60 °C. PGPC forms 3.5 nm radii micelles of aggregation number 33. In the gel phase, DPPC and PGPC fuse to form mixed vesicles for PGPC molar fraction, X PGPC ≤ 0.3 and coexisting vesicles and micelles at higher X PGPC . Data suggest that liquid phase DPPC at 50 °C forms mixed vesicles with segregated or hemi fused DPPC and PGPC for X PGPC ≤ 0.3. At 60 °C, DPPC and PGPC do not mix, but form coexisting vesicles and micelles. DOPC and PGPC do not mix in any proportion in the liquid phase. Two dissimilar aggregates of the sizes of vesicles and PGPC micelles were observed for all X PGPC for T ≥ 22 °C. DOPC–PGPC and DPPC–PGPC mixing is non-ideal for X PGPC > 0.3 in both gel and fluid phases resulting in exclusion of PGPC from the bilayer. Formation of mixed vesicles is favored in the gel phase but not in the liquid phase for X PGPC ≤ 0.3. For X PGPC ≤ 0.3, aggregation states change progressively from mixed vesicles in the gel phase to component segregated mixed vesicles in the liquid phase close to the chain melting transition temperature to separated coexisting vesicles and micelles at higher temperatures. [ABSTRACT FROM AUTHOR]

Phenotypical analysis of the lipid interacting residues in the closed state of the mechanosensitive channel of small conductance (MscS) from Escherichia coli ( E. coli ) has previously shown that these residues are critical for channel function. In the closed state, mutation of individual hydrophobic lipid lining residues to alanine, thus reducing the hydrophobicity, resulted in phenotypic changes that were observable using in vivo assays. Here, in an analogous set of experiments, we identify eleven residues in the first transmembrane domain of the open state of MscS that interact with the lipid bilayer. Each of these residues was mutated to alanine and leucine to modulate their hydrophobic interaction with the lipid tail-groups in the open state. The effects of these changes on channel function were analyzed using in vivo bacterial assays and patch clamp electrophysiology. Mutant channels were found to be functionally indistinguishable from wildtype MscS. Thus, mutation of open-state lipid interacting residues does not differentially stabilize or destabilize the open, closed, intermediate, or transition states of MscS. Based on these results and other data from the literature, we propose a new gating paradigm for MscS where MscS acts as a “Jack-In-The-Box” with the intrinsic bilayer lateral pressure holding the channel in the closed state. In this model, upon application of extrinsic tension the channel springs into the open state due to relief of the intrinsic lipid bilayer pressure. [ABSTRACT FROM AUTHOR]

Human apolipoprotein E (apoE) isoforms exhibit different conformational stabilities and lipid-binding properties that give rise to altered cholesterol metabolism among the isoforms. Using Trp-substituted mutations and site-directed fluorescence labeling, we made a comprehensive comparison of the conformational organization of the N- and C-terminal domains and lipid interactions between the apoE3 and apoE4 isoforms. Trp fluorescence measurements for selectively Trp-substituted variants of apoE isoforms demonstrated that apoE4 adopts less stable conformations in both the N- and C-terminal domains compared to apoE3. Consistent with this, the conformational reorganization of the N-terminal helix bundle occurs at lower guanidine hydrochloride concentration in apoE4 than in apoE3 as monitored by fluorescence resonance energy transfer (FRET) from Trp residues to acrylodan attached at the N-terminal helix. Upon binding of apoE3 and apoE4 variants to egg phosphatidylcholine small unilamellar vesicles, similar changes in Trp fluorescence or FRET efficiency were observed for the isoforms, indicating that the opening of the N-terminal helix bundle occurs similarly in apoE3 and apoE4. Introduction of mutations into the C-terminal domain of the apoE isoforms to prevent self-association and maintain the monomeric state resulted in great increase in the rate of binding of the C-terminal helices to a lipid surface. Overall, our results demonstrate that the different conformational organizations of the N- and C-terminal domains have a minor effect on the steady-state lipid-binding behavior of apoE3 and apoE4: rather, self-association property is a critical determinant in the kinetics of lipid binding through the C-terminal helices of apoE isoforms. [ABSTRACT FROM AUTHOR]

Abstract: In this study we have prepared ceramide phosphoserine (CerPS) and examined its sterol-interacting properties. CerPS is a hydrogen-bonding sphingolipid, but its head group differs from that found in sphingomyelin (SM). Based on diphenylhexatriene steady-state anisotropy measurements, we observed that fully hydrated N-palmitoyl CerPS had a gel-to-liquid crystalline phase transition temperature of about 51°C in 50mM sodium phosphate buffer (pH 7.4). This was close to the Tm measured for 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) bilayers (Tm 50.5°C). Based on cholestatrienol (CTL) quenching experiments in liquid disordered ternary bilayers (containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphcholine; POPC), cholesterol/CTL formed sterol-enriched ordered domains with CerPS. These had similar thermostability as the sterol domains formed with N-palmitoyl SM. Cholesterol failed to form sterol-enriched ordered domains with DPPS under comparable conditions. Based on the equilibrium partitioning of CTL, we observed that the affinity of sterol for bilayers containing POPC/CerPS/cholesterol (6:3:1 by mol) was much higher than the affinity measured for control fluid POPC/cholesterol (9:1 by mol) bilayers, but slightly less than seen for comparable PSM-containing bilayers. We conclude that the phosphoserine head group was less efficient than the phosphocholine head group in stabilizing sterol/sphingolipid interaction. However, hydrogen bonding apparently can overcome some of the negative effects of the phosphoserine head group, since CerPS interacted more favorably with cholesterol compared to DPPS. [Copyright &y& Elsevier]

Abstract: Sphingolipids have been found to have single methyl branchings both in their long-chain base and in their N-linked acyl chains. In this study we determined how methyl-branching in the N-linked acyl chain of sphingomyelin (SM) affected their membrane properties. SM analogs with a single methyl-branching at carbon 15 (of a 17:0 acyl chain; anteiso) had a lower gel–liquid transition temperature as compared to an iso-branched SM analog. Phytanoyl SM (methyls at carbons 3, 7, 11 and 15) as well as a SM analog with a methyl on carbon 10 in a hexadecanoyl chain failed to show a gel–liquid transition above 10°C. Only the two distally branched SM analogs (iso and anteiso) formed ordered domains with cholesterol in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer. However, domains formed by the branched SM analogs appeared to contain less sterol when compared to palmitoyl SM (PSM) as the saturated phospholipid. Sterol-enriched domains formed by the anteiso SM analog were also less stable against temperature than domains formed by PSM. Both the 10-methyl and phytanoyl SM analogs failed to form sterol-enriched domains in the POPC bilayer. Acyl chain branching weakened SM/sterol interactions markedly when compared to PSM, as also evidenced from the decreased affinity of cholestatrienol to bilayers containing branched SM analogs. Our results show that methyl-branching weakened intermolecular interactions in a position-dependent manner. [ABSTRACT FROM AUTHOR]

Abstract: Soluble oligomeric aggregates of α-synuclein have been implicated to play a central role in the pathogenesis of Parkinson''s disease. Disruption and permeabilization of lipid bilayers by α-synuclein oligomers is postulated as a toxic mechanism, but the molecular details controlling the oligomer–membrane interaction are still unknown. Here we show that membrane disruption strongly depends on the accessibility of the hydrophobic membrane core and that charge interactions play an important but complex role. We systematically studied the influence of the physical membrane properties and solution conditions on lipid bilayer disruption by oligomers using a dye release assay. Varying the lipid headgroup composition revealed that membrane disruption only occurs for negatively charged bilayers. Furthermore, the electrostatic repulsion between the negatively charged α-synuclein and the negative surface charge of the bilayer inhibits vesicle disruption at low ionic strength. The disruption of negatively charged vesicles further depends on lipid packing parameters. Bilayer composition changes that result in an increased lipid headgroup spacing make vesicles more prone to disruption, suggesting that the accessibility of the bilayer hydrocarbon core modulates oligomer–membrane interaction. These data shed important new insights into the driving forces governing the highly debated process of oligomer–membrane interactions. [Copyright &y& Elsevier]

Chitosan is a cationic biopolymer that has been used extensively in dietary supplements to reduce fat absorption in the fight against obesity. The mechanism of fat binding of chitosan is still not fully understood and has been the subject of controversy. This study was designed to improve the understanding of the underlying mechanism by investigating the interaction of chitosan with oil-in-water emulsion drop- lets. Our results indicated that (0.1% w/w) chitosan adsorbed to a 20% w/w phosphatidyicholine-stabilized anionic primary emulsion to form a secondary cationic emulsion by electrostatic attraction forces under conditions resembling the stomach (pH 2). Bile salts (6 mM) were added to simulate secretion in the small intestine and pH increase. Bile salts adsorbed to the chitosan secondary emulsion, which resulted in aggregation of oil droplets followed by coalescence due to close packing of droplets. Increased viscosity (267-2531 cp) and increased degree of deacetylation (40-92% DDA) of chitosan enhanced emulsion breakdown. Increasing the pH to 7.5 without addition of bile salts yielded little aggregation. Pronounced aggregation is thought to decrease the accessibility of lipase to the oil resulting in lower bio- availability and reduced caloric intake. Understanding how chitosan interacts with oil droplets in the digestive tract is vital to developing a comprehensive model of the influence of chitosan on the bioavailability of dietary lipids. The information gained in this study may be useful for the interpretation and experimental design of animal and human feeding studies and for the rational design of chitosan-based functional foods for fat reduction. [ABSTRACT FROM AUTHOR]

Abstract: Maurocalcine (MCa) is a 33-amino acid residue peptide that was initially identified in the Tunisian scorpion Scorpio maurus palmatus. This peptide triggers interest for three main reasons. First, it helps unravelling the mechanistic basis of Ca2+ mobilization from the sarcoplasmic reticulum because of its sequence homology with a calcium channel domain involved in excitation–contraction coupling. Second, it shows potent pharmacological properties because of its ability to activate the ryanodine receptor. Finally, it is of technological value because of its ability to carry cell-impermeable compounds across the plasma membrane. Herein, we characterized the molecular determinants that underlie the pharmacological and cell-penetrating properties of maurocalcine. We identify several key amino acid residues of the peptide that will help the design of cell-penetrating analogues devoid of pharmacological activity and cell toxicity. Close examination of the determinants underlying cell penetration of maurocalcine reveals that basic amino acid residues are required for an interaction with negatively charged lipids of the plasma membrane. Maurocalcine analogues that penetrate better have also stronger interaction with negatively charged lipids. Conversely, less effective analogues present a diminished ability to interact with these lipids. These findings will also help the design of still more potent cell penetrating analogues of maurocalcine. [Copyright &y& Elsevier]

Abstract: Amoebapores, the pore-forming proteins of Entamoeba histolytica, are major pathogenicity factors of the parasite. Upon a comprehensive survey in the recently completed genome data sets for the protozoon, we identified in addition to the three amoebapore genes, 16 genes which are constitutively expressed and code for structurally similar proteins, all belonging to the family of saposin-like proteins. Here, we recombinantly expressed in bacteria a defined single entity of this expansive amoebic protein family, namely SAPLIP 3. The protein consists of the saposin-like domain only, comparable to amoebapores, and we characterized its interactions with membranes using different assays. In contrast to amoebapores, SAPLIP 3 neither forms pores in liposomes nor permeabilizes bacterial membranes. However, SAPLIP 3 induces leaky fusion of lipid vesicles as evidenced by fluorescence microscopic analysis and by using a fusion assay that monitors the dequenching of a lipophilic dye. The membrane-fusogenic activity of SAPLIP 3 which is dependent on the presence of negatively charged lipids and on acidic pH resembles in combination with the negative surface charge of the protein characteristics of human saposin C. Beside its function as a cofactor of sphingolipid hydrolysing enzymes, the human protein is considered to be involved in the reorganization of lysosomal compartments due to its fusogenic activity. We hypothesize that in the amoeba, SAPLIP 3 fulfils a similar function in the multifarious endo- and exocytotic transport processes. [Copyright &y& Elsevier]

Abstract: The barrier function of skin resides in the lipid components of the stratum corneum, particularly their spatial organisation. FTIR spectroscopy has already been used as a relevant tool to study this lipid organisation: IR vibration band shifts have been attributed to the variations in lipid organisation induced by temperature. Our study included a stratum corneum model, composed of the three main lipids: palmitic acid as an example of fatty acids, cholesterol and ceramide III as an example of ceramide. Different films with various ratios of these lipids were studied. In our analytical strategy, the interest of using a chemometric analysis of global data obtained from ATR-FTIR spectra to highlight the main interactions involved in the molecular organisation of lipids has been demonstrated. Two kinds of interaction between the three main lipids have been shown: a non polar interaction between the long hydrocarbon chains and a polar interaction as the hydrogen bonding between polar functional groups. By varying the lipid ratio, we have shown first that the relative importance of each interaction was modified, second, that the induced modification of organisation can be detected by chemometric analysis of the ATR-FTIR spectra. The role of each kind of lipid in the organisation has been discussed. In conclusion, associating the ATR-FTIR with chemometric treatment is a promising tool: firstly, to understand the consequence of lipid relative compositions on the structural organisation of the stratum corneum, secondly, to show the relationship between lipid organisation and percutaneous penetration data. Indeed, this methodology will be transposed to in vivo studies with IR measurements through a probe. [Copyright &y& Elsevier]

Abstract α-Synuclein co-exists with lipids in the Lewy bodies, a pathological hallmark of Parkinson's disease. Molecular interaction between α-synuclein and lipids has been examined by observing lipid-induced protein self-oligomerization in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. Lipids such as phosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and even arachidonic acid induced the self-oligomerization whereas phosphatidylcholine did not affect the protein. Because the oligomerizations occurred from critical micelle concentrations of the lipids, the self interaction of α-synuclein was shown to be a lipid-surface dependent phenomenon with head group specificity. By employing β-synuclein and a C-terminally truncated α-synuclein (α-syn97), the head-group dependent self-oligomerization was demonstrated to occur preferentially at the N-terminal region while the fatty acid interaction leading to the protein self-association required the presence of the acidic C-terminus of α-synuclein. In the presence of Cu[sup 2+] and H[sub 2]O[sub 2], phosphatidylinositol (PI), along with other acidic lipids, actually enhanced the metal-catalyzed oxidative self-oligomerization of α-synuclein. The dityrosine crosslink formation responsible for the PI-enhanced covalent self-oligomerization was more sensitive to variation of copper concentrations than that of H[sub 2]O[sub 2] during the metal-catalyzed oxidation. The enhancement by PI was shown to be due to facilitation of copper localization to the protein because actual binding affinity between copper and α-synuclein increased from K[sub d] of 44.7 μ m to 5.9 μ m in the presence of the lipid. Taken together, PI not only affects α-synuclein to be more self-interactive by providing the lipid surface, but also enhances the metal-catalyzed oxidative protein self-oligomerization by facilitating copper localization to the protein when the metal and H[sub 2]O[sub 2] are provided. This observation therefore could be implicated in the formation of Lewy bodies as lipids and metal-catalyzed oxidative stress have been considered to be a part of pathological causes leading to the neurodegeneration. [ABSTRACT FROM AUTHOR]

Sphingomyelin and phosphatidylcholine are important components in the external leaflet of cellular plasma membranes. In this review we compare the structure of these lipid molecules, with emphasis on the differences in hydrogen bonding capacity and membrane properties that arise from the small but significant differences in molecular structure. The membrane properties of sphingomyelins and the implications that these have, or might have, in biological membranes and for raft function are further discussed. [Copyright &y& Elsevier]

Phospholipids, when suspended in water, may self-aggregate into vesicle-like structures with an internal aqueous compartment. Such structures, termed liposomes (Bangham ct al. 1965), share many properties with biologic membranes (Lenaz 1977). Liposomes of various composition, including those prepared by incorporating cholesterol and specific-viral receptors, serve as models of natural membranes for examining the attachment mode of animal viruses. For instance, the interaction of enveloped Sindbis virus with liposomes is similar to that with the intact erythrocytes (Mooney et al. 1975). Similarly, after attaching, Sendai virus (a paramyxovirus) membrane fuses with liposomes and the viral nucleoprotein is then ingested by phagocytosis (Haywood 1978); this process resembles virus interaction with plasma membrane and subsequent entry into the host cell. Nonenveloped poliovirus also interacts strongly with liposomes (Taber et al. 1978). There is little information, however, concerning interaction between plant viruses and lipids. Kiho and Abe (1980) reported that tobacco mosaic virus (TMV) is destabilized when treated with lecithin and polyornithine. Liposome-encapsulated TMV RNA (Fraley ct al. 1982, Watanabe et al. 1982) and turnip rosette virus RNA (Roixo and Hull 1982) were used recently for infecting plant protoplasts. Southern bean mosaic virus (SBMV) is a RNA-containing plant virus for which considerable information is available on the nature of stabilizing interactions (Hsu ct al. 1977, Sehgal 1981) and capsid topography (Hermodson et al. 1982). There is also evidence that SBMV may infect via uninjured leaf cells (Schneider and Worley 1959). Lipid-containing cellular structures or hydrophobic environment of a cell membrane have been postulated (Caspar 1963, Durham 1978) as the disassembly sites of viruses. It was of interest therefore, to determine if SBMV will interact with lipids and if so, can any alterations be detected in its stability. We report that SBMV... [ABSTRACT FROM AUTHOR]

Surface plasmon resonance (SPR)/BIAcore technology enables the characterization of molecular interactions, including determination of affinities and kinetics. In BIAcore, one of the interaction partners (the ligand) is immobilized on a chip and the other (the analyte) is provided in solution. BIAcore allows to study association and dissociation rates in real time without the use of labeling. BIAcore can be applied to molecular interactions involving small compounds and biological macromolecules such as proteins, lipids, nucleic acids, or carbohydrates. Here we describe protocols for the measurements of PDZ domain-peptide (oriented biotinylated peptides), PDZ domain-liposomes (lipid membranes), and PDZ-lipid-peptide tripartite interactions.

Fӧrster-type resonance energy transfer (FRET) with fluorescence cross-correlation spectroscopy (FCCS) is a powerful combination for observing intramolecular conformational dynamics on the micro- to millisecond timescale. Owing to its sensitivity to various physical parameters, FRET-FCCS has also been used to detect the reagent effects on proteins dynamics. However, FRET-FCCS alone cannot acquire the exact measurements of rate constants. Moreover, this technique is highly model dependent and can be unreliable when determining too many parameters at once. On the contrary, single-molecular FRET (smFRET) can measure the conformational states and their populations directly, although it is extremely challenging for probing fast dynamics under 1 ms. In this chapter, we describe how to realize sub-millisecond conformational dynamics measurements of a SNARE protein Ykt6 under lipid environments by smFRET and FRET-FCCS. This protocol includes sample preparation, microscope designs, data acquisition, and analysis methodology.