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The integrity of the colon and the development of colon cancer depend on the sphingolipid balance in colon epithelial cells. In this review, we summarize the current knowledge on how ceramides and their complex derivatives influence normal colon development and colon cancer development. Ceramides, glucosylceramides and sphingomyelin are essential membrane components and, due to their biophysical properties, can influence the activation of membrane proteins, affecting protein–protein interactions and downstream signalling pathways. Here, we review the cellular mechanisms known to be affected by ceramides and their effects on colon development. We also describe which ceramides are deregulated during colorectal carcinogenesis, the molecular mechanisms involved in ceramide deregulation and how this affects carcinogenesis. Finally, we review new methods that are now state of the art for studying lipid-protein interactions in the physiological environment. [ABSTRACT FROM AUTHOR]

Endocytosis plays a complex role in pathogen-host interactions. It serves as a pathway for pathogens to enter the host cell and acts as a part of the immune defense mechanism. Endocytosis involves the formation of lipid membrane vesicles and the reshaping of the cell membrane, a task predominantly managed by proteins containing BAR (Bin1/Amphiphysin/yeast RVS167) domains. Insights into how BAR domains can remodel and reshape cell membranes provide crucial information on infections and can aid the development of treatment. Aiming at deciphering the roles of the BAR dimers in lipid membrane bending and remodeling, we conducted extensive all-atom molecular dynamics simulations and discovered that the presence of helix kinks divides the BAR monomer into two segments—the "arm segment" and the "core segment"—which exhibit distinct movement patterns. Contrary to the prior hypothesis of BAR domains working as a rigid scaffold, we found that it functions in an "Arms-Hands" mode. These findings enhance the understanding of endocytosis, potentially advancing research on pathogen-host interactions and aiding in the identification of new treatment strategies targeting BAR domains. [ABSTRACT FROM AUTHOR]

The delineation of protein–lipid interfaces is essential for understanding the mechanisms of various membrane-associated processes crucial to plant development and growth, including signalling, trafficking, and membrane transport. Due to their highly dynamic nature, the precise characterization of lipid–protein interactions by experimental techniques is challenging. Molecular dynamics simulations provide a powerful computational alternative with a spatial–temporal resolution allowing the atomistic-level description. In this review, we aim to introduce plant scientists to molecular dynamics simulations. We describe different steps of performing molecular dynamics simulations and provide a broad survey of molecular dynamics studies investigating plant protein–lipid interfaces. Our aim is also to illustrate that combining molecular dynamics simulations with artificial intelligence-based protein structure determination opens up unprecedented possibilities for future investigations of dynamic plant protein–lipid interfaces. [ABSTRACT FROM AUTHOR]

Once regarded as mere membrane building blocks, lipids are now recognized as diverse and intricate players that mold the functions, identities, and responses of cellular membranes. Although the interactions of lipids with integral and peripheral membrane proteins are crucial for their localization, activity, and function, how proteins bind lipids is still far from being thoroughly explored. Describing and characterizing these dynamic protein–lipid interactions is thus essential to understanding the membrane-associated processes. Here we review the current range of experimental techniques employed to study plant protein–lipid interactions, integrating various methods. We summarize the principles, advantages, and limitations of classical in vitro biochemical approaches, including protein–lipid overlays and various liposome binding assays, and complement them with in vivo microscopic techniques centered around the use of genetically encoded lipid sensors and pharmacological or genetic membrane lipid manipulation tools. We also highlight several emerging techniques still awaiting their advancement into plant membrane research and emphasize the need to use complementary experimental strategies as key for elucidating the mechanistic roles of protein–lipid interactions in plant cell biology. [ABSTRACT FROM AUTHOR]

The inward rectifier potassium channel Kir2.1 (KCNJ2) is an important regulator of resting membrane potential in both excitable and non-excitable cells. The functions of Kir2.1 channels are dependent on their lipid environment, including the availability of PI(4,5)P2, secondary anionic lipids, cholesterol and long-chain fatty acids acyl coenzyme A (LC-CoA). Endocannabinoids are a class of lipids that are naturally expressed in a variety of cells, including cardiac, neuronal, and immune cells. While these lipids are identified as ligands for cannabinoid receptors there is a growing body of evidence that they can directly regulate the function of numerous ion channels independently of CBRs. Here we examine the effects of a panel of endocannabinoids on Kir2.1 function and demonstrate that a subset of endocannabinoids can alter Kir2.1 conductance to varying degrees independently of CBRs. Using computational and Surface plasmon resonance analysis, endocannabinoid regulation of Kir2.1 channels appears to be the result of altered membrane properties, rather than through direct protein-lipid interactions. Furthermore, differences in endocannabinoid effects on Kir4.1 and Kir7.1 channels, indicating that endocannabinoid regulation is not conserved among Kir family members. These findings may have broader implications on the function of cardiac, neuronal and/or immune cells. [ABSTRACT FROM AUTHOR]

The paper overviews the results of computational studies of the molecular mechanisms underlying the adaptation of model cell membranes taking place during their interaction with proteins and peptides. We discuss changes in the structural and dynamic parameters of the water–lipid environment, the hydrophobic/hydrophilic organization of the lipid bilayer surface (the so-called "mosaicity"), etc. Taken together, these effects are called the "membrane response" (MR) and constitute the most important ability of the cell membranes to respond specifically and consistently to the incorporation of extraneous agents, primarily proteins and peptides, and their subsequent functioning. The results of the authors' long-term research in the field of molecular modeling of MR processes with various spatial and temporal characteristics are described, from the effects of binding of individual lipid molecules to proteins to changes in the integral macroscopic parameters of membranes. The bulk of the results were obtained using the "dynamic molecular portrait" approach developed by the authors. The biological role of the observed phenomena and potential ways of rationally designing artificial membrane systems with specified MR characteristics are discussed. This, in turn, is important for targeted changes in the activity profile of proteins and peptides exerting action on biomembranes, not least as promising pharmacological agents. [ABSTRACT FROM AUTHOR]

Biological membranes consist of a lipid bilayer in which integral membrane proteins are embedded. Based on the compositional complexity of the lipid species found in membranes, and on their specific and selective interactions with membrane proteins, we recently suggested that membrane bilayers can be best described as "finely-tuned molecular machines." We now discuss one such set of lipid-protein interactions by describing a negative feedback mechanism operating in the de novo sphingolipid biosynthetic pathway, which occurs in the membrane of the endoplasmic reticulum, and describe the atomic interactions between the first enzyme in the pathway, namely serine palmitoyl transferase, and the product of the fourth enzyme in the pathway, ceramide. We explore how hydrogen-bonding and hydrophobic interactions formed between Asn13 and Phe63 in the serine palmitoyl transferase complex and ceramide can influence the ceramide content of the endoplasmic reticulum. This example of finely-tuned biochemical interactions raises intriguing mechanistic questions about how sphingolipids and their biosynthetic enzymes could have evolved, particularly in light of their metabolic co-dependence. [ABSTRACT FROM AUTHOR]

One of the reasons to suggest olive oil consumption for a healthy life is its potential to induce robust lipidomic remodeling through membrane modification by dietary lipids. This remodeling might, in turn, modulate essential lipid-protein interactions while maintaining accurate transmembrane protein/domain orientation. Oleic acid, the primary compound in olive oil, has been suggested as amodulator of ion channel function. In this study, we explored whether this lipid could rescue the trafficking of mutated transmembrane proteins. In our initial approach, we supplemented the cell culturemedium of HEK-293 cells expressing cyclic nucleotide channels tagged using green fluorescent protein (CNG-GFP) with olive oil or oleic acid. In addition to wildtype channels, we also expressed R272Q and R278W mutant channels, two nonfunctional intracellularly retained channels related to retinopathies. We used fluorescence microscopy and patch-clamp in the inside-out configuration to assess changes in the cell localization and function of the tested channels. Our results demonstrated that olive oil and oleic acid facilitated the transport of cyclic nucleotide-gated R272Q mutant channels towards the plasma membrane, rendering them electrophysiologically functional. Thus, our findings reveal a novel property of olive oil as a membrane protein traffic inductor. [ABSTRACT FROM AUTHOR]

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions. Synopsis: Three molecules of cardiolipin, the signature mitochondrial phospholipid, tightly interact with the ADP/ATP carrier (AAC/ANT). This study demonstrates that each cardiolipin-AAC/ANT interaction is structurally and functionally important. Negatively charged mutations into the cardiolipin binding sites of yeast Aac2 disrupted cardiolipin-Aac2 interaction. Aac2 tertiary structure was destabilized in the mutants with disrupted cardiolipin binding. Disruption of cardiolipin binding impaired Aac2 transport activity in a site-specific manner. A disease-associated mutation in human ANT1 compromises ANT1 structure and transport activity due to a disturbed cardiolipin-ANT1 interaction. The binding of three cardiolipin molecules to the mitochondrial ADP/ATP carrier supports the protein's tertiary structure and transport activity. [ABSTRACT FROM AUTHOR]

SLC40A1 is the sole iron export protein reported in mammals. In humans, its dysfunction is responsible for ferroportin disease, an inborn error of iron metabolism transmitted as an autosomal dominant trait and observed in different ethnic groups. As a member of the major facilitator superfamily, SLC40A1 requires a series of conformational changes to enable iron translocation across the plasma membrane. The influence of lipids on protein stability and its conformational changes has been little investigated to date. Here, we combine molecular dynamics simulations of SLC40A1 embedded in membrane bilayers with experimental alanine scanning mutagenesis to analyze the specific role of glycerophospholipids. We identify four basic residues (Lys90, Arg365, Lys366, and Arg371) that are located at the membrane‐cytosol interface and consistently interact with 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE) molecules. These residues surround a network of salt bridges and hydrogens bonds that play a critical role in stabilizing SLC40A1 in its basal outward‐facing conformation. More deeply embedded in the plasma membrane, we identify Arg179 as a charged amino acid residue also tightly interacting with lipid polar heads. This results in a local deformation of the lipid bilayer. Interestingly, Arg179 is adjacent to Arg178, which forms a functionally important salt‐bridge with Asp473 and is a recurrently associated with ferroportin disease when mutated to glutamine. We demonstrate that the two p.Arg178Gln and p.Arg179Thr missense variants have similar functional behaviors. These observations provide insights into the role of phospholipids in the formation/disruption of the SLC40A1 inner gate, and give a better understanding of the diversity of molecular mechanisms of ferroportin disease. [ABSTRACT FROM AUTHOR]

Background and Aims: Cardiopulmonary bypass (CPB) and circulatory arrest (CA) can induce intestinal injury and consequently lead to multiple organ dysfunction. Nitric oxide (NO) has protective effects, but its effect on the intestine has not been studied. The study aimed to investigate intestinal injury variables and prove the intestinal protective effects of exogenous nitric oxide when modelling CPB and CA in an experiment. Methods: The study was performed on sheep (n = 24). There were four groups: CPB, CPB + NO, CPB + CA and CPB + CA + NO. Sheep in NO groups received intraoperative inhalation of NO at a dose of 80 ppm. Groups without NO underwent CPB and CA without NO delivery. Defaecation rate, dynamics of intestinal fatty acid binding protein (i-FABP), coefficient of microviscosity and polarity in the areas of lipid--lipid and protein-lipid interactions of erythrocyte membranes were assessed. One hour after CPB, the intestinal tissue was collected and assessed for tissue concentrations of adenosine triphosphate (ATP) and lactate. Results: The defaecation rate after CPB was higher in the CPB + NO group than in the CPB group. The concentration of i-FABP after CPB was lower in the CPB + NO and CPB + CA + NO groups than in the CPB and CPB + CA groups. Erythrocyte deformability before and after CPB revealed no significant dynamics in groups with NO. The ATP concentration 1 h after CPB was higher in the CPB + NO group than in the CPB group. The morphological picture in groups with NO was better. Conclusion: When modelling CPB and CA, NO had a positive effect on the functional and structural state of the intestine and also maintained erythrocyte deformability. [ABSTRACT FROM AUTHOR]

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo. Understanding principles that govern protein association with extracellular vesicles should expand their potential as a therapeutic modality. Here, the authors show that by localizing proteins to the plasma membrane and lipid rafts, a variety of proteins can be preferentially loaded into extracellular vesicles. [ABSTRACT FROM AUTHOR]

A method is described to deconstruct the network of hydropathic interactions within and between a protein's sidechain and its environment into residue-based three-dimensional maps. These maps encode favorable and unfavorable hydrophobic and polar interactions, in terms of spatial positions for optimal interactions, relative interaction strength, as well as character. In addition, these maps are backbone angle-dependent. After map calculation and clustering, a finite number of unique residue sidechain interaction maps exist for each backbone conformation, with the number related to the residue's size and interaction complexity. Structures for soluble proteins (~749,000 residues) and membrane proteins (~387,000 residues) were analyzed, with the latter group being subdivided into three subsets related to the residue's position in the membrane protein: soluble domain, core-facing transmembrane domain, and lipid-facing transmembrane domain. This work suggests that maps representing residue types and their backbone conformation can be reassembled to optimize the medium-to-high resolution details of a protein structure. In particular, the information encoded in maps constructed from the lipid-facing transmembrane residues appears to paint a clear picture of the protein–lipid interactions that are difficult to obtain experimentally. [ABSTRACT FROM AUTHOR]

Extracting membrane proteins from the hydrophobic environment of the biological membrane, in a physiologically relevant and stable state, suitable for downstream analysis remains a challenge. The traditional route to membrane protein extraction has been to use detergents and the last 15 years or so have seen a veritable explosion in the development of novel detergents with improved properties, making them more suitable for individual proteins and specific applications. There have also been significant advances in the development of encapsulation of membrane proteins in lipid based nanodiscs, either directly from the native membrane using polymers allowing effective capture of the protein and protein‐associated membrane lipids, or via reconstitution of detergent extracted and purified protein into nanodiscs of defined lipid composition. All of these advances have been successfully applied to the study of membrane proteins via a range of techniques and there have been some spectacular membrane protein structures solved. In addition, the first detailed structural and biophysical analyses of membrane proteins retained within a biological membrane have been reported. Here we summarise and review the recent advances with respect to these new agents and systems for membrane protein extraction, reconstitution and analysis. [ABSTRACT FROM AUTHOR]

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Simple Summary: Gap junction channels are composed of an assembly of connexin proteins and allow direct communication between cells. They are highly conserved across vertebrates and form wide pores in cell membranes for the passage of ions and metabolites. Junctional channels are formed from the end-to-end docking of hemichannels, and both junctional channels and hemichannels are vital for many physiological activities. Several medical conditions are associated with problems in gap junction communication, ranging from deafness to fatal cardiac arrhythmias. Many connexin channel diseases can be linked to genetic mutations, and nearly 1000 have been identified in connexin genes. Prior to 2009, atomic-level structural details of gap junction channels were essentially non-existent. This information is critical for understanding channel function and to assess the pathological nature of disease-causing mutations. Fortunately, since 2009, the powerful tools of X-ray crystallography and electron cryomicroscopy have yielded over 50 high-resolution structures of connexin channels. This review aims to provide a comprehensive summary of this astounding 15-year period of structural discovery in the gap junction field. Divided into eight distinct sections, we describe key details found in this compendium of structures, such as conserved features in the design of connexin channels, insights into channel gating and surprises regarding where membrane lipids are bound to the channels. In addition, we highlight areas in which we need more information, such as the structure of highly flexible regions within connexin channels that have so far resisted visualization. Furthermore, targeting connexins for drug discovery is still in its infancy, and much more structural data are needed to pursue this end. Connexins (Cxs) are a family of integral membrane proteins, which function as both hexameric hemichannels (HCs) and dodecameric gap junction channels (GJCs), behaving as conduits for the electrical and molecular communication between cells and between cells and the extracellular environment, respectively. Their proper functioning is crucial for many processes, including development, physiology, and response to disease and trauma. Abnormal GJC and HC communication can lead to numerous pathological states including inflammation, skin diseases, deafness, nervous system disorders, and cardiac arrhythmias. Over the last 15 years, high-resolution X-ray and electron cryomicroscopy (cryoEM) structures for seven Cx isoforms have revealed conservation in the four-helix transmembrane (TM) bundle of each subunit; an αβ fold in the disulfide-bonded extracellular loops and inter-subunit hydrogen bonding across the extracellular gap that mediates end-to-end docking to form a tight seal between hexamers in the GJC. Tissue injury is associated with cellular Ca2+ overload. Surprisingly, the binding of 12 Ca2+ ions in the Cx26 GJC results in a novel electrostatic gating mechanism that blocks cation permeation. In contrast, acidic pH during tissue injury elicits association of the N-terminal (NT) domains that sterically blocks the pore in a "ball-and-chain" fashion. The NT domains under physiologic conditions display multiple conformational states, stabilized by protein–protein and protein–lipid interactions, which may relate to gating mechanisms. The cryoEM maps also revealed putative lipid densities within the pore, intercalated among transmembrane α-helices and between protomers, the functions of which are unknown. For the future, time-resolved cryoEM of isolated Cx channels as well as cryotomography of GJCs and HCs in cells and tissues will yield a deeper insight into the mechanisms for channel regulation. The cytoplasmic loop (CL) and C-terminal (CT) domains are divergent in sequence and length, are likely involved in channel regulation, but are not visualized in the high-resolution X-ray and cryoEM maps presumably due to conformational flexibility. We expect that the integrated use of synergistic physicochemical, spectroscopic, biophysical, and computational methods will reveal conformational dynamics relevant to functional states. We anticipate that such a wealth of results under different pathologic conditions will accelerate drug discovery related to Cx channel modulation. [ABSTRACT FROM AUTHOR]

• Ion and lipid-protein interactions modulate K+ channels structure and function. • Fluorescence-based methods monitor such interactions in mild conditions. • The number and affinity of ion binding events to the selectivity filter is obtained. • The modulating effect of several channel blockers or lipids can also be monitored. • These studies have contributed to define the channel conformational landscape. A variety of equilibrium and non-equilibrium methods have been used in a multidisciplinary approach to study the conformational landscape associated with the binding of different cations to the pore of potassium channels. These binding processes, and the conformational changes resulting therefrom, modulate the functional properties of such integral membrane properties, revealing these permeant and blocking cations as true effectors of such integral membrane proteins. KcsA, a prototypic K+ channel from Streptomyces lividans , has been extensively characterized in this regard. Here, we revise several fluorescence-based approaches to monitor cation binding under different experimental conditions in diluted samples, analyzing the advantages and disadvantages of each approach. These studies have contributed to explain the selectivity, conduction, and inactivation properties of K+ channels at the molecular level, together with the allosteric communication between the two gates that control the ion channel flux, and how they are modulated by lipids. [ABSTRACT FROM AUTHOR]

• Steric trapping allows a reversible control of membrane protein folding. • The method couples unfolding of a biotinylated protein to binding of streptavidin. • The biotin derivatives with a spectroscopic probe allow a major technical advance. • Steric trapping can delineate the folding energy landscape of membrane proteins. Elucidating the folding energy landscape of membrane proteins is essential to the understanding of the proteins' stabilizing forces, folding mechanisms, biogenesis, and quality control. This is not a trivial task because the reversible control of folding is inherently difficult in a lipid bilayer environment. Recently, novel methods have been developed, each of which has a unique strength in investigating specific aspects of membrane protein folding. Among such methods, steric trapping is a versatile strategy allowing a reversible control of membrane protein folding with minimal perturbation of native protein–water and protein–lipid interactions. In a nutshell, steric trapping exploits the coupling of spontaneous denaturation of a doubly biotinylated protein to the simultaneous binding of bulky monovalent streptavidin molecules. This strategy has been evolved to investigate key elements of membrane protein folding such as thermodynamic stability, spontaneous denaturation rates, conformational features of the denatured states, and cooperativity of stabilizing interactions. In this review, we describe the critical methodological advancement, limitation, and outlook of the steric trapping strategy. [ABSTRACT FROM AUTHOR]

Serum amyloid A (SAA) is a highly conserved acute‐phase protein that plays roles in activating multiple pro‐inflammatory pathways during the acute inflammatory response and is commonly used as a biomarker of inflammation. It has been linked to beneficial roles in tissue repair through improved clearance of lipids and cholesterol from sites of damage. In patients with chronic inflammatory diseases, elevated levels of SAA may contribute to increased severity of the underlying condition. The majority of circulating SAA is bound to lipoproteins, primarily high‐density lipoprotein (HDL). Interaction with HDL not only stabilizes SAA but also alters its functional properties, likely through altered accessibility of protein–protein interaction sites on SAA. While high‐resolution structures for lipid‐free, or apo‐, forms of SAA have been reported, their relationship with the HDL‐bound form of the protein, and with other possible mechanisms of SAA binding to lipids, has not been established. Here, we have used multiple biophysical techniques, including SAXS, TEM, SEC‐MALS, native gel electrophoresis, glutaraldehyde crosslinking, and trypsin digestion to characterize the lipid‐free and lipid‐bound forms of SAA. The SAXS and TEM data show the presence of soluble octamers of SAA with structural similarity to the ring‐like structures reported for lipid‐free ApoA‐I. These SAA octamers represent a previously uncharacterized structure for lipid‐free SAA and are capable of scaffolding lipid nanodiscs with similar morphology to those formed by ApoA‐I. The SAA–lipid nanodiscs contain four SAA molecules and have similar exterior dimensions as the lipid‐free SAA octamer, suggesting that relatively few conformational rearrangements may be required to allow SAA interactions with lipid‐containing particles such as HDL. This study suggests a new model for SAA–lipid interactions and provides new insight into how SAA might stabilize protein‐lipid nanodiscs or even replace ApoA‐I as a scaffold for HDL particles during inflammation. [ABSTRACT FROM AUTHOR]

Introduction: This study aims to explore the important factors affecting the characteristics of different parts of pork. Methods: Lipidomics and proteomics methods were used to analyze DAL (differential lipids) and DAPs (differential proteins) in five different parts (longissimus dorsi, belly meat, loin, forelegs and buttocks) of Duhua pig (Duroc × Guangdong small spotted pig), to identify potential pathways affecting meat quality, investigating fat deposition in pork and its lipid-protein interactions. Results: The results show that TG (triglyceride) is the lipid subclass with the highest proportion in muscle, and the pathway with the most significantly enriched lipids is GP. DAP clustered on several GO terms closely related to lipid metabolism and lipogenesis (lipid binding, lipid metabolism, lipid transport, and lipid regulation). In KEGG analysis, there are two main DAP aggregation pathways related to lipid metabolism, namely Fatty acid degradation and oxidative phosphorylation. In PPI analysis, we screened out 31 core proteins, among which NDUFA6, NDUFA9 and ACO2 are the most critical. Discussion: PC (phosphatidylcholine) is regulated by SNX5, THBS1, ANXA7, TPP1, CAVIN2, and VDAC2 in the phospholipid binding pathway. TG is regulated by AUH/HADH/ACADM/ACADL/HADHA in the lipid oxidation and lipid modification pathways. Potential biomarkers are rich in SFA, MUFA and PUFA respectively, the amounts of SFA, MUFA and PUFA in the lipid measurement results are consistent with the up- and down-regulation of potential biomarker lipids. This study clarified the differences in protein and lipid compositions in different parts of Duhua pigs and provided data support for revealing the interactions between pork lipids and proteins. These findings provide contributions to the study of intramuscular fat deposition in pork from a genetic and nutritional perspective. [ABSTRACT FROM AUTHOR]

Biological phenomena, from enzymatic catalysis to synaptic transmission, originate in the structural transformations of biomolecules and biomolecular assemblies in liquid water. However, directly imaging these nanoscopic dynamics without probes or labels has been a fundamental methodological challenge. Here, we developed an approach for "electron videography"--combining liquid phase electron microscopy with molecular modeling--with which we filmed the nanoscale structural fluctuations of individual, suspended, and unlabeled membrane protein nanodiscs in liquid. Systematic comparisons with biochemical data and simulation indicate the graphene encapsulation involved can afford sufficiently reduced effects of the illuminating electron beam for these observations to yield quantitative fingerprints of nanoscale lipid-protein interactions. Our results suggest that lipid-protein interactions delineate dynamically modified membrane domains across unexpectedly long ranges. Moreover, they contribute to the molecular mechanics of the nanodisc as a whole in a manner specific to the protein within. Overall, this work illustrates an experimental approach to film, quantify, and understand biomolecular dynamics at the nanometer scale. [ABSTRACT FROM AUTHOR]

All plasma membranes exhibit asymmetry, both in lipid distribution and protein topology. Cells invest significant free energy to uphold this asymmetry. Despite its critical physiological role, our quantitative understanding of membrane asymmetry is limited, due primarily to insufficient in vitro methods for creating asymmetric model membranes. New experimental protocols now allow for the preparation of vesicles with tailored, asymmetric lipid distributions. Leaflets with distinct lipid compositions can affect each other's properties, such as lateral diffusion, hydrocarbon chain ordering, and bending elasticity. Integral membrane proteins not only adapt to lipid asymmetry but also exhibit asymmetric topological orientations, which in turn can depend on lipid composition. Plasma membranes utilize free energy to maintain highly asymmetric, non-equilibrium distributions of lipids and proteins between their two leaflets. In this review we discuss recent progress in quantitative research enabled by using compositionally controlled asymmetric model membranes. Both experimental and computational studies have shed light on the nuanced mechanisms that govern the structural and dynamic coupling between compositionally distinct bilayer leaflets. This coupling can increase the membrane bending rigidity and induce order – or lipid domains – across the membrane. Furthermore, emerging evidence indicates that integral membrane proteins not only respond to asymmetric lipid distributions but also exhibit intriguing asymmetric properties themselves. We propose strategies to advance experimental research, aiming for a deeper, quantitative understanding of membrane asymmetry, which carries profound implications for cellular physiology. [ABSTRACT FROM AUTHOR]

RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria. They are secreted as water-soluble proteins that are able to bind to the host cell membrane and insert hydrophobic segments into the lipid bilayer that ultimately contribute to the formation of transmembrane pores. Ion diffusion through these pores leads then to cytotoxic and cytolytic effects on the hosts. Several reports have evidenced that the binding of several RTX toxins to the target cell membrane may take place through a high-affinity interaction with integrins of the β2 family that is highly expressed in immune cells of the myeloid lineage. However, at higher toxin doses, cytotoxicity by most RTX toxins has been observed also on β2-deficient cells in which toxin binding to the cell membrane has been proposed to occur through interaction with glycans of glycosylated lipids or proteins present in the membrane. More recently, cumulative pieces of evidence show that membrane cholesterol is essential for the mechanism of action of several RTX toxins. Here, we summarize the most important aspects of the RTX toxin interaction with the target cell membrane, including the cholesterol dependence, the recent identification in the sequences of several RTX toxins of linear motifs coined as the Cholesterol Recognition/interaction Amino acid Consensus (CRAC), and the reverse or mirror CARC motif, which is involved in the toxin–cholesterol interaction. [ABSTRACT FROM AUTHOR]

Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily conserved family of membrane proteins whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila. TMC1 and TMC2 are components of ion channel complexes, but the molecular features that tune these complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here, we present the single-particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans. The complex is composed of two copies of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex highlights conserved protein-lipid interactions, as well as a p-helical structural motif in the pore-forming helices, that together suggest a mechanism for TMC-mediated mechanosensory transduction. [ABSTRACT FROM AUTHOR]

Using the gramicidin A channel as a molecular probe, we show that tubulin binding to planar lipid membranes changes the channel kinetics—seen as an increase in the lifetime of the channel dimer—and thus points towards modification of the membrane's mechanical properties. The effect is more pronounced in the presence of non-lamellar lipids in the lipid mixture used for membrane formation. To interpret these findings, we propose that tubulin binding redistributes the lateral pressure of lipid packing along the membrane depth, making it closer to the profile expected for lamellar lipids. This redistribution happens because tubulin perturbs the lipid headgroup spacing to reach the membrane's hydrophobic core via its amphiphilic α-helical domain. Specifically, it increases the forces of repulsion between the lipid headgroups and reduces such forces in the hydrophobic region. We suggest that the effect is reciprocal, meaning that alterations in lipid bilayer mechanics caused by membrane remodeling during cell proliferation in disease and development may also modulate tubulin membrane binding, thus exerting regulatory functions. One of those functions includes the regulation of protein–protein interactions at the membrane surface, as exemplified by VDAC complexation with tubulin. [ABSTRACT FROM AUTHOR]

Vacuolar ATPases (V-ATPases), proton pumps composed of 16 subunits, are necessary for a variety of cellular functions. Subunit "a" has four isoforms, a1–a4, each with a distinct cellular location. We identified a phosphoinositide (PIP) interaction motif, KXnK(R)IK(R), conserved in all four isoforms, and hypothesize that a/PIP interactions regulate V-ATPase recruitment/retention to different organelles. Among the four isoforms, a2 is enriched on Golgi with a2 mutations in the PIP motif resulting in cutis laxa. We hypothesize that the hydrophilic N-terminal (NT) domain of a2 contains a lipid-binding domain, and mutations in this domain prevent interaction with Golgi-enriched PIPs, resulting in cutis laxa. We recreated the cutis laxa-causing mutation K237_V238del, and a double mutation in the PIP-binding motif, K237A/V238A. Circular dichroism confirmed that there were no protein structure alterations. Pull-down assays with PIP-enriched liposomes revealed that wildtype a2NT preferentially binds phosphatidylinositol 4-phosphate (PI(4)P), while mutants decreased binding to PI(4)P. In HEK293 cells, wildtype a2NT was localized to Golgi and co-purified with microsomal membranes. Mutants reduced Golgi localization and membrane association. Rapamycin depletion of PI(4)P diminished a2NT-Golgi localization. We conclude that a2NT is sufficient for Golgi retention, suggesting the lipid-binding motif is involved in V-ATPase targeting and/or retention. Mutational analyses suggest a molecular mechanism underlying how a2 mutations result in cutis laxa. [ABSTRACT FROM AUTHOR]

The cell is separated from the external environment by a lipid bilayer. In this biological interface, phospholipids and proteins play relevant roles on both the outer and inner sides. Understanding lipid–protein interactions is important to advance our knowledge of the relation between membrane properties and cell's functions in order to reveal the driving forces behind membrane-related processes and to develop new biomedical applications. To this end, one strategy is to study lipid–protein interactions by determining thermodynamic properties of lipid membrane models in interactions with proteins. In this work, we present a systematic biophysical investigation exploring how changes in membrane properties promote the interaction of proteins with the lipid bilayer. We studied the interaction of ovalbumin (OVA) and lysozyme (LYZ) with DMPC/sphingomyelin/cholesterol (MSC) membranes (using large unilamellar vesicles, LUVs, as membrane models) by differential scanning calorimetry (DSC) and dynamic light scattering (DLS). Our results show that lipid bilayer composition, large incubation time periods, and the membrane phase are determinants to induce nonspecific lipid–protein interactions at the level of the lipid bilayer-water interface, significantly modifying the membrane thermal response. We demonstrated that cholesterol and sphingomyelin not only rule the thermodynamic properties of pure lipid membranes but also regulate the cooperativity and interactions in lipid/protein systems. Our results contribute to a better understanding of the driving forces of lipid–protein interactions in the bilayer and could be of practical reference for the designing and development of new applications related to biomembrane interactions, for example, smart drug delivery systems. [ABSTRACT FROM AUTHOR]

Fluorescence spectroscopy provides numerous methodological tools for structural and functional studies of biological macromolecules and their complexes. All fluorescence‐based approaches require either existence of an intrinsic probe or an introduction of an extrinsic one. Moreover, studies of complex systems often require an additional introduction of a specific quencher molecule acting in combination with a fluorophore to provide structural or thermodynamic information. Here, we review the fundamentals and summarize the latest progress in applications of different classes of fluorescent probes and their specific quenchers, aimed at studies of protein folding and protein‐membrane interactions. Specifically, we discuss various environment‐sensitive dyes, FRET probes, probes for short‐distance measurements, and several probe‐quencher pairs for studies of membrane penetration of proteins and peptides. The goals of this review are: (a) to familiarize the readership with the general concept that complex biological systems often require both a probe and a quencher to decipher mechanistic details of functioning and (b) to provide example of the immediate applications of the described methods. [ABSTRACT FROM AUTHOR]

Protein-lipid interactions are crucial for various cellular biological processes like intracellular signaling, membrane transport, and cytoskeletal dynamics. Therefore, studying these interactions is essential to understand and unravel their specific functions. Nevertheless, the interacting proteins of many lipids are poorly understood and still require systematic study. Liposomes are the most well-known and familiar biomimetic systems used to study protein-lipid interactions. Although liposomes have been widely used for studying protein-lipid interactions in classical methods such as the co-flotation assay (CFA), co-sedimentation assay (CSA), and flow cytometric assay (FCA), an overview of their current applications and developments in high-throughput methods is not yet available. Here, we summarize the liposome development in low and high-throughput methods to study protein-lipid interactions. Besides, a constructive comment for each platform is presented to stimulate the advancement of these technologies in the future. [ABSTRACT FROM AUTHOR]

Three-dimensional structure determination and prediction of proteins with intrinsically disordered regions, unstructured regions, conformational flexibility, and lacking homologous structures are challenging. We previously predicted and refined an in silico structure of a plant cellulose synthase from cotton (GhCESA1), and more recently, cryo-electron microscopy (cryo-EM) has resolved a majority of the lengths of two CESA structures from poplar (PttCESA8) and cotton (GhCESA7). However, 26–30% of these cryo-EM structures remain unresolved, including the N-terminal domain, half of the class-specific region, the gating loop region, and the C-terminal domain. Here, we describe the generation and evaluation of a full-length hybrid GhCESA1 model based on this cryo-EM PttCESA8 structure, with unresolved regions completed using this in silico refined GhCESA1 model. All-atom molecular dynamics simulations and subsequent energy minimizations were performed for the in silico and hybrid GhCESA1 models in a lipid bilayer-water-ion environment, and structural stability, dynamics, energetics, contacts, and quality were evaluated. The unresolved regions were found to be the most dynamic, in agreement with their poor electron density with cryo-EM. The hybrid model exhibited a higher total secondary structure content, more favorable intra-protein and protein-lipid interaction energies, and improved quality metrics. Moreover, hydrogen bonding was revealed to be a primary mechanism for intra-protein and protein-lipid contacts. These results demonstrate that in silico structure prediction and refinement may be useful to augment experimental structure determination, especially for disordered and unstructured regions. This hybrid model can serve as a steppingstone to derive full-length homology models of other CESAs found in more experimentally tractable organisms. [ABSTRACT FROM AUTHOR]

Ligand-gated ion channels transduce electrochemical signals in neurons and other excitable cells. Aside from canonical ligands, phospholipids are thought to bind specifically to the transmembrane domain of several ion channels. However, structural details of such lipid contacts remain elusive, partly due to limited resolution of these regions in experimental structures. Here, we discovered multiple lipid interactions in the channel GLIC by integrating cryo-electron microscopy and large-scale molecular simulations. We identified 25 bound lipids in the GLIC closed state, a conformation where none, to our knowledge, were previously known. Three lipids were associated with each subunit in the inner leaflet, including a buried interaction disrupted in mutant simulations. In the outer leaflet, two intrasubunit sites were evident in both closed and open states, while a putative intersubunit site was preferred in open-state simulations. This work offers molecular details of GLIC-lipid contacts particularly in the ill-characterized closed state, testable hypotheses for state-dependent binding, and a multidisciplinary strategy for modeling protein-lipid interactions. [ABSTRACT FROM AUTHOR]

Structural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5’-diphosphate and vanadate. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as –100 kJ/mol at 298 K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery. [ABSTRACT FROM AUTHOR]

Protein kinases (PKs) are proteins at the core of cellular signalling and are thereby responsible for most cellular physiological processes and their regulations. As for all intracellular proteins, PKs are subjected to Brownian thermal energy that tends to homogenise their distribution throughout the volume of the cell. To access their substrates and perform their critical functions, PK localisation is therefore tightly regulated in space and time, relying upon a range of clustering mechanisms. These include post-translational modifications, protein-protein and protein-lipid interactions, as well as liquid-liquid phase separation, allowing spatial restriction and ultimately regulating access to their substrates. In this review, we will focus on key mechanisms mediating PK nanoclustering in physiological and pathophysiological processes. We propose that PK nanoclusters act as a cellular quantal unit of signalling output capable of integration and regulation in space and time. We will specifically outline the various super-resolution microscopy approaches currently used to elucidate the composition and mechanisms driving PK nanoscale clustering and explore the pathological consequences of altered kinase clustering in the context of neurodegenerative disorders, inflammation, and cancer. [ABSTRACT FROM AUTHOR]

Phenol-soluble modulins (PSMs) are key virulence factors of S. aureus, and they comprise the structural scaffold of biofilm as they self-assemble into functional amyloids. They have been shown to interact with cell membranes as they display toxicity towards human cells through cell lysis, with αPSM3 being the most cytotoxic. In addition to causing cell lysis in mammalian cells, PSMs have also been shown to interact with bacterial cell membranes through antimicrobial effects. Here, we present a study on the effects of lipid bilayers on the aggregation mechanism of αPSM using chemical kinetics to study the effects of lipid vesicles on the aggregation kinetics and using circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM) to investigate the corresponding secondary structure of the aggregates. We found that the effects of lipid bilayers on αPSM aggregation were not homogeneous between lipid type and αPSM peptides, although none of the lipids caused changes in the dominating aggregation mechanism. In the case of αPSM3, all types of lipids slowed down aggregation to a varying degree, with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) having the most pronounced effect. For αPSM1, lipids had opposite effects, where DOPC decelerated aggregation and lipopolysaccharide (LPS) accelerated the aggregation, while 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG) had no effect. For αPSM4, both DOPG and LPS accelerated the aggregation, but only at high concentration, while DOPC showed no effect. None of the lipids was capable of inducing aggregation of αPSM2. Our data reveal a complex interaction pattern between PSMs peptides and lipid bilayers that causes changes in the aggregation kinetics by affecting different kinetic parameters along with only subtle changes in morphology. [ABSTRACT FROM AUTHOR]

NMR is a noninvasive, molecular-level spectroscopic technique widely used for chemical characterization. However, it lacks the sensitivity to probe the small number of spins at surfaces and interfaces. Here, we use nitrogen vacancy (NV) centers in diamond as quantum sensors to optically detect NMR signals from chemically modified thin films. To demonstrate the method's capabilities, aluminum oxide layers, common supports in catalysis and materials science, are prepared by atomic layer deposition and are subsequently functionalized by phosphonate chemistry to form self-assembled monolayers. The surface NV-NMR technique detects spatially resolved NMR signals from the monolayer, indicates chemical binding, and quantifies molecular coverage. In addition, it can monitor in real time the formation kinetics at the solid-liquid interface. With our approach, we show that NV quantum sensors are a surface-sensitive NMR tool with femtomole sensitivity for in situ analysis in catalysis, materials, and biological research.

Biological membranes that play major roles in diverse functions are composed of numerous lipids and proteins, making them an important target for coarse-grained (CG) molecular dynamics (MD) simulations. Recently, we have developed the CG implicit solvent lipid force field (iSoLF) that has a resolution compatible with the widely used Cα protein representation [D. Ugarte La Torre and S. Takada, J. Chem. Phys. 153, 205101 (2020)]. In this study, we extended it and developed a lipid–protein interaction model that allows the combination of the iSoLF and the Cα protein force field, AICG2+. The hydrophobic–hydrophilic interaction is modeled as a modified Lennard-Jones potential in which parameters were tuned partly to reproduce the experimental transfer free energy and partly based on the free energy profile normal to the membrane surface from previous all-atom MD simulations. Then, the obtained lipid–protein interaction is tested for the configuration and placement of transmembrane proteins, water-soluble proteins, and peripheral proteins, showing good agreement with prior knowledge. The interaction is generally applicable and is implemented in the publicly available software, CafeMol. [ABSTRACT FROM AUTHOR]

The Prion protein is the molecular hallmark of the incurable prion diseases affecting mammals, including humans. The protein-only hypothesis states that the misfolding, accumulation, and deposition of the Prion protein play a critical role in toxicity. The cellular Prion protein (PrPC) anchors to the extracellular leaflet of the plasma membrane and prefers cholesterol- and sphingomyelin-rich membrane domains. Conformational Prion protein conversion into the pathological isoform happens on the cell surface. In vitro and in vivo experiments indicate that Prion protein misfolding, aggregation, and toxicity are sensitive to the lipid composition of plasma membranes and vesicles. A picture of the underlying biophysical driving forces that explain the effect of Prion protein - lipid interactions in physiological conditions is needed to develop a structural model of Prion protein conformational conversion. To this end, we use molecular dynamics simulations that mimic the interactions between the globular domain of PrPC anchored to model membrane patches. In addition, we also simulate the Doppel protein anchored to such membrane patches. The Doppel protein is the closest in the phylogenetic tree to PrPC, localizes in an extracellular milieu similar to that of PrPC, and exhibits a similar topology to PrPC even if the amino acid sequence is only 25% identical. Our simulations show that specific protein-lipid interactions and conformational constraints imposed by GPI anchoring together favor specific binding sites in globular PrPC but not in Doppel. Interestingly, the binding sites we found in PrPC correspond to prion protein loops, which are critical in aggregation and prion disease transmission barrier (β2-α2 loop) and in initial spontaneous misfolding (α2-α3 loop). We also found that the membrane re-arranges locally to accommodate protein residues inserted in the membrane surface as a response to protein binding.

Background: TMC1, which encodes transmembrane channel-like protein 1, forms the mechanoelectrical transduction (MET) channel in auditory hair cells, necessary for auditory function. TMC1 variants are known to cause autosomal dominant (DFNA36) and autosomal recessive (DFNB7/11) non-syndromic hearing loss, but only a handful of TMC1 variants underlying DFNA36 have been reported, hampering analysis of genotype-phenotype correlations. Methods: In this study, we retrospectively reviewed 338 probands in an in-house database of genetic hearing loss, evaluating the clinical phenotypes and genotypes of novel TMC1 variants associated with DFNA36. To analyze the structural impact of these variants, we generated two structural models of human TMC1, utilizing the Cryo-EM structure of C. elegans TMC1 as a template and AlphaFold protein structure database. Specifically, the lipid bilayer-embedded protein database was used to construct membrane-embedded models of TMC1. We then examined the effect of TMC1 variants on intramolecular interactions and predicted their potential pathogenicity. Results: We identified two novel TMC1 variants related to DFNA36 (c.1256T > C:p.Phe419Ser and c.1444T > C:p.Trp482Arg). The affected subjects had bilateral, moderate, late-onset, progressive sensorineural hearing loss with a down-sloping configuration. The Phe419 residue located in the transmembrane domain 4 of TMC1 faces outward towards the channel pore and is in close proximity to the hydrophobic tail of the lipid bilayer. The non-polar-to-polar variant (p.Phe419Ser) alters the hydrophobicity in the membrane, compromising protein-lipid interactions. On the other hand, the Trp482 residue located in the extracellular linker region between transmembrane domains 5 and 6 is anchored to the membrane interfaces via its aromatic rings, mediating several molecular interactions that stabilize the structure of TMC1. This type of aromatic ring-based anchoring is also observed in homologous transmembrane proteins such as OSCA1.2. Conversely, the substitution of Trp with Arg (Trp482Arg) disrupts the cation-π interaction with phospholipids located in the outer leaflet of the phospholipid bilayer, destabilizing protein-lipid interactions. Additionally, Trp482Arg collapses the CH-π interaction between Trp482 and Pro511, possibly reducing the overall stability of the protein. In parallel with the molecular modeling, the two mutants degraded significantly faster compared to the wild-type protein, compromising protein stability. Conclusions: This results expand the genetic spectrum of disease-causing TMC1 variants related to DFNA36 and provide insight into TMC1 transmembrane protein-lipid interactions. [ABSTRACT FROM AUTHOR]

The disruption of cell membranes by tau and amylin oligomers is linked to amyloid diseases such as Alzheimer's and diabetes, respectively. The recent studies suggest that misfolded tau and amylin can form neurotoxic hetero-oligomers that are structurally different from homo-oligomers. However, the molecular interactions of these hetero-oligomers with the neuronal membranes remain unclear. Using MD simulations, we have investigated the binding behaviors, membrane disruption, and protein folding of hetero-oligomers on a raft membrane containing phase-separated lipid nanodomains like those found in neurons. We discovered that the hetero-oligomers bind to the liquid-order and liquid-disorder phase boundaries of the raft membrane. The major lipid-binding sites of these interactions include the L16 and I26 residues of amylin and the N-terminal of tau. Strong disruptions of the raft domain size by the hetero-tetramer were detected. Furthermore, the hetero-dimer disrupted the saturated phospholipid orientational order to a greater extent than the individual tau or amylin monomer. In addition, the constituent tau more strongly promoted the alpha-helix to the beta-sheet transition of the constituent amylin within the hetero-dimer when compared with the amylin monomer alone. Our results provide new molecular insights into understanding the neurotoxicity of the hetero-oligomers associated with the cross-talk between amyloid diseases. [ABSTRACT FROM AUTHOR]

Azurin protein potentially plays an important role as an anti-cancer therapeutic agent, particularly in treating breast cancer in experiments and showing without having a negative effect on normal cells. Although the interaction mechanism between protein and lipid membrane is complicated, it can be modeled as protein-lipid interaction. Since the all-atom (AA) model simulation is cost computing, we apply a coarse-grained (CG-MARTINI) model to calculate the protein-lipid interaction. We investigate the binding free energy value dependency by varying the windows separation and electrostatic scale parameters. After scaling the electrostatic interactions by a factor of 0.04, the best result in terms of free energy is -140.831 kcal/mol, while after window-separation optimization, it reaches -71.859 kcal/mol. This scaling was necessary because the structures from the CG MARTINI model have a higher density than the corresponding all-atom structures. We thus postulate that electrostatic interactions should be scaled down in this case of CG-MARTINI simulations. [ABSTRACT FROM AUTHOR]

One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point need to either cross or just interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions with novel molecules -both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopolysaccharides, membrane proteins and other components, constructing relevant synthetic liposome-based models of the membrane is both difficult and expensive. We here propose to let the bacteria do the hard work for us. Bacterial extracellular vesicles (bEVs) are naturally secreted by Gram-negative and Gram-positive bacteria, playing a role in communication between bacteria, as virulence factors, molecular transport or being a part of the antimicrobial resistance mechanism. bEVs consist of the bacterial outer membrane and thus inherit many components and properties of the native outer cell envelope. In this work, we have isolated and characterized bEVs from one Escherichia coli mutant and three clinical strains of the ESKAPE pathogens Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The bEVs were shown to be representative models for the bacterial membrane in terms of lipid composition with speciesstrain specific variations. The bEVs were further used to probe the interactions between bEV and antimicrobial peptides (AMPs) as model compounds by Surface Plasmon Resonance (SPR) and provide proof-of-principle that bEVs can be used as an easily accessible and highly realistic model for the bacterial surface in interaction studies. This further enables direct monitoring of the effect induced by antibiotics, or the response to host-pathogen interactions.

The problems arising from the accumulation of plastic have led to comprehensive research for bio-based packaging materials. Proteins are a promising alternative to traditional plastic because of their great barrier properties and multifunctional properties. However, the hydrophilic nature of proteins limits their wide application for the packaging of low and medium-moisture content foods. Distinct functional groups of proteins allow various modifications to obtain hydrophobic films that meet the requirements of suitable packaging. Besides, the valorization of agro-industrial by-products is considered a promising solution for obtaining packaging materials. Herein, protein-rich agro-industrial by-products and various hydrophobisation techniques including blending, emulsification, grafting, nanocomposite, cross-linking, plasticizer alternatives, and physical treatments for the fabrication of protein films are comprehensively investigated. The challenges and prospects are also evaluated. Hydrophobisation is generally based on reducing the hydrophilic groups, compacting the protein network, or incorporating hydrophobic groups. It mostly resulted in a protein matrix with low water sensitivity, high tensile strength, and low elongation. However, lipids incorporation into the protein matrix may overcome the brittleness of hydrophobised protein films. Physical treatments by unfolding protein chains make functional groups accessible for further treatment. Nevertheless, commercialization of protein-based packaging is still in its early stages, although there have been some recent advancements including water-soluble pouches and coating materials. Further research is still needed on optimizing the current techniques or utilizing emerging technologies such as 3D printing, plasma deposition, corona discharge, and pulsed light to accelerate the commercialization of hydrophobic protein films. [Display omitted] • Sensitivity to moisture is one of the main drawbacks of protein packaging. • Various protein-rich industrial by-products can be used for sustainable packaging. • Physical and thermal pre-treatments are beneficial for protein hydrophobisation. • Protein-lipid interaction is the most efficient hydrophobisation approach. • Food competition, safety concerns, and commercialization are the most challenges. [ABSTRACT FROM AUTHOR]

High salinity in agricultural lands is one of the predominant issues limiting agricultural yields. Plants have developed several mechanisms to withstand salinity stress, but the mechanisms are not effective enough for most crops to prevent and persist the salinity stress. Plant salt tolerance pathways involve membrane proteins that have a crucial role in sensing and mitigating salinity stress. Due to a strategic location interfacing two distinct cellular environments, membrane proteins can be considered checkpoints to the salt tolerance pathways in plants. Related membrane proteins functions include ion homeostasis, osmosensing or ion sensing, signal transduction, redox homeostasis, and small molecule transport. Therefore, modulating plant membrane proteins' function, expression, and distribution can improve plant salt tolerance. This review discusses the membrane protein–protein and protein–lipid interactions related to plant salinity stress. It will also highlight the finding of membrane protein–lipid interactions from the context of recent structural evidence. Finally, the importance of membrane protein–protein and protein–lipid interaction is discussed, and a future perspective on studying the membrane protein–protein and protein–lipid interactions to develop strategies for improving salinity tolerance is proposed. Significance statement: Membrane proteins play a crucial role in plant salt tolerance, from sensing to mitigating stress. Many membrane proteins are currently known to participate directly in different cellular processes during stress. Salt stress also affects lipids and their content in the membrane. Because lipids are indispensable for membrane protein activity, it is essential to identify the critical lipid molecules involved in regulating membrane protein function. The current understanding of membrane protein–protein interaction and membrane protein–lipid interaction is limited. This review attempts to summarize the currently known membrane protein–protein interaction and membrane protein–lipid interaction in plant salt stress and advocates methodology that may be proven effective in studying membrane–protein interaction. [ABSTRACT FROM AUTHOR]

Polymer-encapsulated nanodiscs enable membrane proteins to be investigated within a native-like lipid-bilayer environment. Unlike other bilayer-based membrane mimetics, these nanodiscs are equilibrium structures that permit lipid exchange on experimentally relevant timescales. Therefore, examining the kinetics and mechanisms of lipid exchange is of great interest. Since the high charge densities of existing anionic polymers can interfere with protein–protein and protein–lipid interactions as well as charge-sensitive analysis techniques, electroneutral nanodisc-forming polymers have been recently introduced. However, it has remained unclear how the electroneutrality of these polymers affects the lipid-exchange behavior of the nanodiscs. Here, we use time-resolved Förster resonance energy transfer to study the kinetics and the mechanisms of lipid exchange among nanodiscs formed by the electroneutral polymer Sulfo-DIBMA. We also examine the role of coulombic repulsion and specific counterion association in lipid exchange. Our results show that Sulfo-DIBMA nanodiscs exchange lipids on a similar timescale as DIBMA nanodiscs. In contrast with nanodiscs made from polyanionic DIBMA, however, the presence of mono- and divalent cations does not influence lipid exchange among Sulfo-DIBMA nanodiscs, as expected from their electroneutrality. The robustness of Sulfo-DIBMA nanodiscs against varying ion concentrations opens new possibilities for investigating charge-sensitive processes involving membrane proteins. [ABSTRACT FROM AUTHOR]

Membrane contact sites are defined as regions of close proximity between two membranes; this association is mediated by protein-protein and/or protein-lipid interactions. Contact sites are often involved in lipid transport, but also can perform other functions. Peroxisomal membrane contact sites have obtained little attention compared to those of other cell organelles. However, recent studies resulted in a big leap in our knowledge of the occurrence, composition and function of peroxisomal contact sites. Studies in yeast strongly contributed to this progress. In this Review, we present an overview of our current knowledge on peroxisomal membrane contact sites in various yeast species, including Hansenula polymorpha, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica. Yeast peroxisomes form contacts with almost all other cellular organelles and with the plasma membrane. The absence of a component of a yeast peroxisomal contact site complex results in a range of peroxisomal phenotypes, including metabolic and biogenesis defects and alterations in organelle number, size or position. [ABSTRACT FROM AUTHOR]

In the present study, the dependency of purple membrane (PM) dielectric responses on the wavelength of light in the range 380–750 nm has showed meaningful changes about the rotation of PM in suspension and about the rotation of bacteriorhodopsin (bR) trimer inside PM, as well. The action spectrum of PM random walk substantiates the existence of two states of bR. One of them (blue edge-state) lies at the blue edge and the other (red edge-state) at the red edge of the visible absorption of bR. The results might bear on correlation of these bands to some bR photocycle intermediates or bR photoproducts. The results implicate the protein–chromophore interactions that eventually underlie protein–lipid interactions. Disrupting the protein–lipid contact during the illumination with light of wavelength in ranges of (410–470 nm) and (610–720 nm) has resulted in emergence of distinct dielectric dispersion at 0.06–0.08 MHz which is comparable to the size of bR trimer or monomer.The work reports on the chromatic adaptation of bR in view of the dielectric spectral parameters of PM. It aimed to explore a correlation seemingly found between the light wavelength and the relaxations of bR trimer inside PM. Changes in rotational diffusion of bR trimer upon blue and red light illumination can influence the three dimensional data storage based on bR, which may implicate bR in bioelectronics. [ABSTRACT FROM AUTHOR]

Cell membrane rafts form signaling platforms on the cell surface, controlling numerous protein–protein and lipid–protein interactions. Bacteria invading eukaryotic cells trigger cell signaling to induce their own uptake by non-phagocytic cells. The aim of this work was to reveal the involvement of membrane rafts in the penetration of the bacteria Serratia grimesii and Serratia proteamaculans into eukaryotic cells. Our results show that the disruption of membrane rafts by MβCD in the three cell lines tested, M-HeLa, MCF-7 and Caco-2, resulted in a time-dependent decrease in the intensity of Serratia invasion. MβCD treatment produced a more rapid effect on the bacterial susceptibility of M-HeLa cells compared to other cell lines. This effect correlated with a faster assembly of the actin cytoskeleton upon treatment with MβCD in M-HeLa cells in contrast to that in Caco-2 cells. Moreover, the 30 min treatment of Caco-2 cells with MβCD produced an increase in the intensity of S. proteamaculans invasion. This effect correlated with an increase in EGFR expression. Together with the evidence that EGFR is involved in S. proteamaculans invasion but not in S. grimesii invasion, these results led to the conclusion that an increase in EGFR amount on the plasma membrane with the undisassembled rafts of Caco-2 cells after 30 min of treatment with MβCD may increase the intensity of S. proteamaculans but not of S. grimesii invasion. Thus, the MβCD-dependent degradation of lipid rafts, which enhances actin polymerization and disrupts signaling pathways from receptors on the host cell's surface, reduces Serratia invasion. [ABSTRACT FROM AUTHOR]