Structural investigation of interactions between halogenated flavonoids and the lipid membrane along with their role as cytotoxic agents.

This study focuses on understanding the structural and molecular changes in lipid membranes under the influence of six halogenated flavonoid derivatives differing in the number and position of substitution of chlorine and bromine atoms (D1-D6). Utilizing various analytical techniques, including fluorometric methods, dynamic light scattering (DLS), attenuated Fourier transform infrared spectroscopy (ATR- FTIR), and FT-Raman spectroscopy, the research aims to elucidate the mechanisms underlying the interaction of flavonoids with cell membranes. Additionally, the study includes in silico analyses to explore the physicochemical properties of these compounds and their potential pharmaceutical applications, along with toxicity studies to assess their effects on cancer, normal, and red blood cells. Our study showed the ability of halogenated derivatives to interact mostly with the outer part of the membrane, especially in the lipid heads region however, some of them were able to penetrate deeper into the membrane and affect the fluidity of hydrocarbon chains. The potential to reduce cancer cell viability, the lack of toxicity towards erythrocytes, and the favourable physicochemical and pharmacokinetic properties suggest these halogenated flavonoids potential candidates for exploring their potential for medical use.

Sum-frequency vibrational spectroscopy, a tutorial: Applications for the study of lipid membrane structure and dynamics.

Planar supported lipid bilayers (PSLBs) are an ideal model for the study of lipid membrane structures and dynamics when using sum-frequency vibrational spectroscopy (SFVS). In this paper, we describe the construction of asymmetric PSLBs and the basic SFVS theory needed to understand and make measurements on these membranes. Several examples are presented, including the determination of phospholipid orientation and measuring phospholipid transmembrane translocation (flip-flop).

Pixelated High-Q Metasurfaces for in Situ Biospectroscopy and Artificial Intelligence-Enabled Classification of Lipid Membrane Photoswitching Dynamics.

Nanophotonic devices excel at confining light into intense hot spots of electromagnetic near fields, creating exceptional opportunities for light-matter coupling and surface-enhanced sensing. Recently, all-dielectric metasurfaces with ultrasharp resonances enabled by photonic bound states in the continuum (BICs) have unlocked additional functionalities for surface-enhanced biospectroscopy by precisely targeting and reading out the molecular absorption signatures of diverse molecular systems. However, BIC-driven molecular spectroscopy has so far focused on end point measurements in dry conditions, neglecting the crucial interaction dynamics of biological systems. Here, we combine the advantages of pixelated all-dielectric metasurfaces with deep learning-enabled feature extraction and prediction to realize an integrated optofluidic platform for time-resolved in situ biospectroscopy. Our approach harnesses high-Q metasurfaces specifically designed for operation in a lossy aqueous environment together with advanced spectral sampling techniques to temporally resolve the dynamic behavior of photoswitchable lipid membranes. Enabled by a software convolutional neural network, we further demonstrate the real-time classification of the characteristic cis and trans membrane conformations with 98% accuracy. Our synergistic sensing platform incorporating metasurfaces, optofluidics, and deep learning reveals exciting possibilities for studying multimolecular biological systems, ranging from the behavior of transmembrane proteins to the dynamic processes associated with cellular communication.

Evaluation of Lipids for the Study of Photosynthetic Membranes.

The biological role of lipids goes far beyond the formation of a structural membrane bilayer platform for membrane proteins and controlling fluxes across the membranes. For example, in photosynthetic thylakoid membranes, lipids occupy well-defined binding niches within protein complexes and determine the structural organization of membrane proteins and their function by controlling generic physicochemical membrane properties. In this chapter, two-dimensional thin-layer chromatography (2D TLC) and gas chromatography (GC) techniques are presented for quantitative analysis of lipid classes and fatty acids in thylakoid membranes. In addition, lipid extraction methods from isolated thylakoid membranes and leaves are described together with a procedure for the derivatization of fatty acids to fatty acid methyl esters (FAME) that is required for GC analysis.

The antimicrobial fibupeptide lugdunin forms water-filled channel structures in lipid membranes.

Recently, a novel cyclo-heptapeptide composed of alternating D,L-amino acids and a unique thiazolidine heterocycle, called lugdunin, was discovered, which is produced by the nasal and skin commensal Staphylococcus lugdunensis. Lugdunin displays potent antimicrobial activity against a broad spectrum of Gram-positive bacteria, including challenging-to-treat methicillin-resistant Staphylococcus aureus (MRSA). Lugdunin specifically inhibits target bacteria by dissipating their membrane potential. However, the precise mode of action of this new class of fibupeptides remains largely elusive. Here, we disclose the mechanism by which lugdunin rapidly destabilizes the bacterial membrane potential using an in vitro approach. The peptide strongly partitions into lipid compositions resembling Gram-positive bacterial membranes but less in those harboring the eukaryotic membrane component cholesterol. Upon insertion, lugdunin forms hydrogen-bonded antiparallel ß-sheets by the formation of peptide nanotubes, as demonstrated by ATR-FTIR spectroscopy and molecular dynamics simulations. These hydrophilic nanotubes filled with a water wire facilitate not only the translocation of protons but also of monovalent cations as demonstrated by voltage-clamp experiments on black lipid membranes. Collectively, our results provide evidence that the natural fibupeptide lugdunin acts as a peptidic channel that is spontaneously formed by an intricate stacking mechanism, leading to the dissipation of a bacterial cell's membrane potential.

Influence of pH and lipid membrane on the liquid-liquid phase separation of wheat γ-gliadin in aqueous conditions.

Protein body (PB) formation in wheat seeds is a critical process influencing seed content and nutritional quality. In this study, we investigate the potential mechanisms governing PB formation through an in vitro approach, focusing on γ-gliadin, a key wheat storage protein. We used a microfluidic technique to encapsulate γ-gliadin within giant unilamellar vesicles (GUVs) and tune the physicochemical conditions in a controlled and rapid way. We examined the influence of pH and protein concentration on LLPS and protein-membrane interactions using various microscopy and spectroscopy techniques. We showed that γ-gliadin encapsulated in GUVs can undergo a pH-triggered liquid-liquid phase separation (LLPS) by two distinct mechanisms depending on the γ-gliadin concentration. At low protein concentrations, γ-gliadins phase separate by a nucleation and growth-like process, while, at higher protein concentration and pH above 6.0, γ-gliadin formed a bi-continuous phase suggesting a spinodal decomposition-like mechanism. Fluorescence and microscopy data suggested that γ-gliadin dense phase exhibited affinity for the GUV membrane, forming a layer at the interface and affecting the reversibility of the phase separation.

The Chemical Reactivity of Membrane Lipids.

It is well-known that aqueous dispersions of phospholipids spontaneously assemble into bilayer structures. These structures have numerous applications across chemistry and materials science and form the fundamental structural unit of the biological membrane. The particular environment of the lipid bilayer, with a water-poor low dielectric core surrounded by a more polar and better hydrated interfacial region, gives the membrane particular biophysical and physicochemical properties and presents a unique environment for chemical reactions to occur. Many different types of molecule spanning a range of sizes, from dissolved gases through small organics to proteins, are able to interact with membranes and promote chemical changes to lipids that subsequently affect the physicochemical properties of the bilayer. This Review describes the chemical reactivity exhibited by lipids in their membrane form, with an emphasis on conditions where the lipids are well hydrated in the form of bilayers. Key topics include the following: lytic reactions of glyceryl esters, including hydrolysis, aminolysis, and transesterification; oxidation reactions of alkenes in unsaturated fatty acids and sterols, including autoxidation and oxidation by singlet oxygen; reactivity of headgroups, particularly with reactive carbonyl species; and E/Z isomerization of alkenes. The consequences of reactivity for biological activity and biophysical properties are also discussed.

Structure and assembly of a bacterial gasdermin pore.

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning ß-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

Explorative characterization and taxonomy-aligned comparison of alterations in lipids and other biomolecules in Antarctic bacteria grown at different temperatures.

Temperature significantly impacts bacterial physiology, metabolism and cell chemistry. In this study, we analysed lipids and the total cellular biochemical profile of 74 fast-growing Antarctic bacteria grown at different temperatures. Fatty acid diversity and temperature-induced alterations aligned with bacterial classification-Gram-groups, phylum, genus and species. Total lipid content, varied from 4% to 19% of cell dry weight, was genus- and species-specific. Most bacteria increased lipid content at lower temperatures. The effect of temperature on the profile was complex and more species-specific, while some common for all bacteria responses were recorded. Gram-negative bacteria adjusted unsaturation and acyl chain length. Gram-positive bacteria adjusted methyl branching (anteiso-/iso-), chain length and unsaturation. Fourier transform infrared spectroscopy analysis revealed Gram-, genus- and species-specific changes in the total cellular biochemical profile triggered by temperature fluctuations. The most significant temperature-related alterations detected on all taxonomy levels were recorded for mixed region 1500-900 cm-1 , specifically the band at 1083 cm-1 related to phosphodiester groups mainly from phospholipids (for Gram-negative bacteria) and teichoic/lipoteichoic acids (for Gram-positive bacteria). Some changes in protein region were detected for a few genera, while the lipid region remained relatively stable despite the temperature fluctuations.

Carlos Gutiérrez-Merino: Synergy of Theory and Experimentation in Biological Membrane Research.

Professor Carlos Gutiérrez-Merino, a prominent scientist working in the complex realm of biological membranes, has made significant theoretical and experimental contributions to the field. Contemporaneous with the development of the fluid-mosaic model of Singer and Nicolson, the Förster resonance energy transfer (FRET) approach has become an invaluable tool for studying molecular interactions in membranes, providing structural insights on a scale of 1-10 nm and remaining important alongside evolving perspectives on membrane structures. In the last few decades, Gutiérrez-Merino's work has covered multiple facets in the field of FRET, with his contributions producing significant advances in quantitative membrane biology. His more recent experimental work expanded the ground concepts of FRET to high-resolution cell imaging. Commencing in the late 1980s, a series of collaborations between Gutiérrez-Merino and the authors involved research visits and joint investigations focused on the nicotinic acetylcholine receptor and its relation to membrane lipids, fostering a lasting friendship.

Charge of a transmembrane peptide alters its interaction with lipid membranes.

Transmembrane (TM) proteins interact closely with the surrounding membrane lipids. Lipids in the vicinity of TM proteins were reported to have hindered mobility, which has been associated with lipids being caught up in the rough surface of the TM domains. These reports, however, neglect one important factor that largely influences the membrane behavior - electrostatics of the TM peptides that are usually positively charged at their cytosolic end. Here, we study on the example of a neutral and a positively charged WALP peptide, how the charge of a TM peptide influences the membrane. We investigate both its dynamics and mechanics by: (i) time dependent fluorescent shift in combination with classical and FRET generalized polarization to evaluate the mobility of lipids at short and long-range distance from the peptide, (ii) atomic force microscopy to observe the mechanical stability of the peptide-containing membranes, and (iii) molecular dynamics simulations to analyze the peptide-lipid interactions. We show that both TM peptides lower lipid mobility in their closest surroundings. The peptides cause lateral heterogeneity in lipid mobility, which in turn prevents free lipid rearrangement and lowers the membrane ability to seal ruptures after mechanical indentations. Introduction of a positive charge to the peptide largely enhances these effects, affecting the whole membrane. We thus highlight that unspecific peptide-lipid interactions, especially the electrostatics, should not be overlooked as they have a great impact on the mechanics and dynamics of the whole membrane.

Conformational ensemble-dependent lipid recognition and segregation by prenylated intrinsically disordered regions in small GTPases.

We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. Here we show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we find that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.

Bacterial susceptibility and resistance to modelin-5.

Modelin-5 (M5-NH2) killed Pseudomonas aeruginosa with a minimum lethal concentration (MLC) of 5.86 µM and strongly bound its cytoplasmic membrane (CM) with a Kd of 23.5 µM. The peptide adopted high levels of amphiphilic α-helical structure (75.0%) and penetrated the CM hydrophobic core (8.0 mN m-1). This insertion destabilised CM structure via increased lipid packing and decreased fluidity (ΔGmix < 0), which promoted high levels of lysis (84.1%) and P. aeruginosa cell death. M5-NH2 showed a very strong affinity (Kd = 3.5 µM) and very high levels of amphiphilic α-helical structure with cardiolipin membranes (96.0%,) which primarily drove the peptide's membranolytic action against P. aeruginosa. In contrast, M5-NH2 killed Staphylococcus aureus with an MLC of 147.6 µM and weakly bound its CM with a Kd of 117.6 µM, The peptide adopted low levels of amphiphilic α-helical structure (35.0%) and only penetrated the upper regions of the CM (3.3 mN m-1). This insertion stabilised CM structure via decreased lipid packing and increased fluidity (ΔGmix > 0) and promoted only low levels of lysis (24.3%). The insertion and lysis of the S. aureus CM by M5-NH2 showed a strong negative correlation with its lysyl phosphatidylglycerol (Lys-PG) content (R2 > 0.98). In combination, these data suggested that Lys-PG mediated mechanisms inhibited the membranolytic action of M5-NH2 against S. aureus, thereby rendering the organism resistant to the peptide. These results are discussed in relation to structure/function relationships of M5-NH2 and CM lipids that underpin bacterial susceptibility and resistance to the peptide.

The asymmetric plasma membrane-A composite material combining different functionalities?: Balancing Barrier Function and Fluidity for Effective Signaling.

One persistent puzzle in the life sciences is the asymmetric lipid composition of the cellular plasma membrane: while the exoplasmic leaflet is enriched in lipids carrying predominantly saturated fatty acids, the cytoplasmic leaflet hosts preferentially lipids with (poly-)unsaturated fatty acids. Given the high energy requirements necessary for cells to maintain this asymmetry, the question naturally arises regarding its inherent benefits. In this paper, we propose asymmetry to represent a potential solution for harmonizing two conflicting requirements for the plasma membrane: first, the need to build a barrier for the uncontrolled influx or efflux of substances; and second, the need to form a fluid and dynamic two-dimensional substrate for signaling processes. We hence view here the plasma membrane as a composite material, where the exoplasmic leaflet is mainly responsible for the functional integrity of the barrier and the cytoplasmic leaflet for fluidity. We reinforce the validity of the proposed mechanism by presenting quantitative data from the literature, along with multiple examples that bolster our model.

A Survey of Models of Cell Membranes: Toward a New Understanding of Membrane Organization.

The cell membrane, the boundary that separates living cells from their environment, has been the subject of study for over a century. The fluid-mosaic model of Singer and Nicolson in 1972 proposed the plasma membrane as a two-dimensional fluid composed of lipids and proteins. Fifty years hence, advances in biophysical and biochemical tools, particularly optical imaging techniques, have allowed for a better understanding of the physical nature, organization, and composition of cell membranes. This has been made possible by visualizing membrane heterogeneities and their dynamics and appreciating the asymmetrical arrangement of lipids in living cell membranes. Despite these advances, mechanisms underlying the local spatiotemporal organization of membrane components remain unclear. This review surveys various models of membrane organization, culminating in a new model that incorporates nonequilibrium processes and forces exerted by interactions with extramembrane elements such as the actin cytoskeleton. The proposed model provides a comprehensive understanding of membrane organization, taking into account the dynamic nature of the cell membrane and its interactions with its immediate environment.

Studying Membrane Protein-Lipid Specificity through Direct Native Mass Spectrometric Analysis from Tunable Proteoliposomes.

Native mass spectrometry (nMS) has emerged as a key analytical tool to study the organizational states of proteins and their complexes with both endogenous and exogenous ligands. Specifically, for membrane proteins, it provides a key analytical dimension to determine the identity of bound lipids and to decipher their effects on the observed structural assembly. We recently developed an approach to study membrane proteins directly from intact and tunable lipid membranes where both the biophysical properties of the membrane and its lipid compositions can be customized. Extending this, we use our liposome-nMS platform to decipher the lipid specificity of membrane proteins through their multiorganelle trafficking pathways. To demonstrate this, we used VAMP2 and reconstituted it in the endoplasmic reticulum (ER), Golgi, synaptic vesicle (SV), and plasma membrane (PM) mimicking liposomes. By directly studying VAMP2 from these customized liposomes, we show how the same transmembrane protein can bind to different sets of lipids in different organellar-mimicking membranes. Considering that the cellular trafficking pathway of most eukaryotic integral membrane proteins involves residence in multiple organellar membranes, this study highlights how the lipid-specificity of the same integral membrane protein may change depending on the membrane context. Further, leveraging the capability of the platform to study membrane proteins from liposomes with curated biophysical properties, we show how we can disentangle chemical versus biophysical properties, of individual lipids in regulating membrane protein assembly.

Photoinduced bidirectional switching in lipid membranes containing azobenzene glycolipids.

Following the reaction of biological membranes to external stimuli reveals fundamental insights into cellular function. Here, self-assembled lipid monolayers act as model membranes containing photoswitchable azobenzene glycolipids for investigating structural response during isomerization by combining Langmuir isotherms with X-ray scattering. Controlled in-situ trans/cis photoswitching of the azobenzene N = N double bond alters the DPPC monolayer structure, causing reproducible changes in surface pressure and layer thickness, indicating monolayer reorientation. Interestingly, for monolayers containing azobenzene glycolipids, along with the expected DPPC phase transitions an additional discontinuity is observed. The associated reorintation represents a crossover point, with the surface pressure and layer thickness changing in opposite directions above and below. This is evidence that the azobenzene glycolipids themselves change orientation within the monolayer. Such behaviour suggests that azobenzene glycolipids can act as a bidirectional switch in DPPC monolayers providing a tool to investigate membrane structure-function relationships in depth.

Beyond Fluidity: The Role of Lipid Unsaturation in Membrane Function.

The number of double bonds in the acyl chains of membrane lipids varies tremendously at all scales of life, from the organism level to the subcellular level, where differences in lipid unsaturation can be observed between two membrane leaflets or between two continuous regions of the same organelle. Here, we review different approaches that have been used to understand the variability in the acyl chain composition of lipid membranes. We suggest that a full understanding of lipid unsaturation is limited not only by technical difficulties but also because some properties afforded by unsaturated lipids in membrane lipids are likely to be subtler than a mere effect on 2D fluidity, notably, the way the position of double bonds in the acyl chains affect the motion of transmembrane proteins, the adsorption of peripheral proteins, or some mechanical properties of the membrane.

Membrane plasticity induced by myo-inositol derived archaeal lipids: chemical synthesis and biophysical characterization.

Archaeal membrane lipids have specific structures that allow Archaea to withstand extreme conditions of temperature and pressure. In order to understand the molecular parameters that govern such resistance, the synthesis of 1,2-di-O-phytanyl-sn-glycero-3-phosphoinositol (DoPhPI), an archaeal lipid derived from myo-inositol, is reported. Benzyl protected myo-inositol was first prepared and then transformed to phosphodiester derivatives using a phosphoramidite based-coupling reaction with archaeol. Aqueous dispersions of DoPhPI alone or mixed with DoPhPC can be extruded and form small unilamellar vesicles, as detected by DLS. Neutron, SAXS, and solid-state NMR demonstrated that the water dispersions could form a lamellar phase at room temperature that then evolves into cubic and hexagonal phases with increasing temperature. Phytanyl chains were also found to impart remarkable and nearly constant dynamics to the bilayer over wide temperature ranges. All these new properties of archaeal lipids are proposed as providers of plasticity and thus means for the archaeal membrane to resist extreme conditions.

Labyrinthopeptin A2 disrupts raft domains.

Labyrinthopeptins constitute a class of ribosomal synthesized peptides belonging to the type III family of lantibiotics. They exist in different variants and display broad antiviral activities as well as show antiallodynic activity. Although their mechanism of action is not understood, it has been described that Labyrinthopeptins interact with membrane phospholipids modulating its biophysical properties and point out to membrane destabilization as its main point of action. We have used all-atom molecular dynamics to study the location of labyrinthopeptin A2 in a complex membrane as well as the existence of specific interactions with membrane lipids. Our results indicate that labyrinthopeptin A2, maintaining its globular structure, tends to be placed at the membrane interface, mainly between the phosphate atoms of the phospholipids and the oxygen atom of cholesterol modulating the biophysical properties of the membrane lipids. Outstandingly, we have found that labyrinthopeptin A2 tends to be preferentially surrounded by sphingomyelin while excluding cholesterol. The bioactive properties of labyrinthopeptin A2 could be attributed to the specific disorganization of raft domains in the membrane and the concomitant disruption of the overall membrane organization. These results support the improvement of Labyrinthopeptins as therapeutic molecules, opening up new opportunities for future medical advances.