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Organization of biological membranes. (A) Classification of membrane proteins. Lipid-anchored proteins (green) are covalently linked with the membrane through lipids. Integral membrane proteins (blue) penetrate the phospholipid bilayer. Peripheral membrane proteins (blue and red) are associated with the membrane (red) or with integral membrane proteins (blue). (B) Domain formation in biological membranes. Lipid rafts are enriched in cholesterol, glycosphingolipids and proteins. (C) The lipid composition determines membrane thickness. Integral membrane proteins are perfectly embedded in the membrane when membrane thickness (d HT ) and length of the transmembrane domain of integral membrane proteins (d TM ) match. (D) Hydrophobic mismatch occurs when the length of the transmembrane domain (d TM ) and the hydrophobic thickness of the bilayer (d HT ) do not match. Negative (top panel) and positive (bottom panel) mismatch are shown.
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Biological membranes are selectively permeable barriers important for cell organization and compartmentalization. Their organisation strongly depends on the lipids that constitute the lipid bilayer as well as the proteins that reside in the membrane. Unravelling the organisation of biological membranes is therefore of great importance to understand...
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... groups (Fahy et al., 2009(Fahy et al., , 2005). One of the characteristics of phospholipids is their ability to spontaneously form lipid bilayers in aqueous solutions (Watson, 2015). In these lipid bilayers, the hydrophilic head groups face the aqueous surrounding and the hydrophobic fatty acyl chains form the hydrophobic core of the membrane (Fig. ...
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... proteins carry out important cellular processes such as transport of ions and other compounds across the membrane, catalysis of chemical reactions and mediation between the cell and its environment. There are three types of proteins that associate with lipid membranes: peripheral (extrinsic), integral (intrinsic) and lipid-anchored proteins ( Fig. 1A). Peripheral proteins are directly attached to the lipid membrane or to integral membrane proteins and consequently do not interact with the hydrophobic core of the phospholipid bilayer. Their association with the membrane is mediated through electrostatic or other non-covalent interactions with the phospholipid head groups. Integral ...
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... existence of specific domains is an important aspect of membrane organization: The proteins that reside in the membranes are important for cellular function and, in order to coordinate their function, lipid bilayers often segregate into platforms of action -so-called lipid rafts (Fig. 1B). Lipid rafts were initially defined as detergent-resistant glycolipid-enriched (DRG) membrane fractions with a high content of cholesterol, glycosphingolipids, sphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins ( Brown and Rose, 1992). The lipid raft hypothesis proposes that sphingolipids, sterols and specific ...
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... proteins (Lee, 2011). Membrane thickness depends on length and saturation of the fatty acyl chains as well as membrane packing and composition ( Klose et al., 2013). When thickness of the hydrophobic membrane core and length of the membrane-spanning domain of an integral membrane protein match, a protein is perfectly embedded in the lipid bilayer (Fig. 1C). However, if the thickness of the membrane is not consistent with the transmembrane-spanning domain of the protein, hydrophobic mismatch occurs (Fig. 1D). More precisely, when the transmembrane-spanning domain of the protein is too long or too short to match the hydrophobic core of the membrane, positive or negative hydrophobic ...
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... al., 2013). When thickness of the hydrophobic membrane core and length of the membrane-spanning domain of an integral membrane protein match, a protein is perfectly embedded in the lipid bilayer (Fig. 1C). However, if the thickness of the membrane is not consistent with the transmembrane-spanning domain of the protein, hydrophobic mismatch occurs (Fig. 1D). More precisely, when the transmembrane-spanning domain of the protein is too long or too short to match the hydrophobic core of the membrane, positive or negative hydrophobic mismatch is ...
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Biological membranes display a staggering complexity of lipids and proteins orchestrating cellular functions. Superior analytical tools coupled with numerous functional cellular screens have enabled us to query their role in cellular signalling, trafficking, guiding protein structure and function–all of which rely on the dynamic membrane lipid prop...
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... The unique local lipid composition surrounding a membrane protein, including lipids present in annular and non-annular roles, has been termed the "lipid fingerprint" of a protein [7]. It has been shown through coarse-grained molecular dynamics (CG MD) simulations that different membrane proteins have distinct lipid fingerprints, an observation supported by analysis of protein-lipid interactions using mass spectrometry [8,9]. Coarsegrained molecular dynamics is a powerful tool for investigating this phenomenon, capable of achieving timescales that would be beyond the scope of all atom simulations, whilst still faithfully reproducing lipid-binding patterns observed in experimentally determined structures [10]. ...
Transient receptor potential canonical 3 (TRPC3) channel belongs to the superfamily of transient receptor potential (TRP) channels which mediate Ca2+ influx into the cell. These channels constitute essential elements of cellular signalling and have been implicated in a wide range of diseases. TRPC3 is primarily gated by lipids and its surface expression has been shown to be dependent on cholesterol, yet a comprehensive exploration of its interaction with this lipid has thus far not emerged. Here, through 80 µs of coarse-grained molecular dynamics simulations, we show that cholesterol interacts with multiple elements of the transmembrane machinery of TRPC3. Through our approach, we identify an annular binding site for cholesterol on the pre-S1 helix and a non-annular site at the interface between the voltage-sensor-like domain and pore domains. Here, cholesterol interacts with exposed polar residues and possibly acts to stabilise the domain interface.
... The most commonly used ionization technique for the analysis of membrane protein complexes today is nano-Electrospray Ionization (nESI). A good overview of the applicability of nESI can be found in recent reviews on the subject [1][2][3][4][5][6][7][8][9][10][11][12][13][14] and is beyond the scope of this review. Laser Induced Liquid Bead Ion Desorption (LILBID) MS is another soft ionization technique, which was introduced in 2006 [15] and was among the first MS methods to be applied to membrane proteins [16]. ...
Structural Biology has moved beyond the aim of simply identifying the components of a cellular subsystem towards analysing the dynamics and interactions of multiple players within a cell. This focal shift comes with additional requirements for the analytical tools used to investigate these systems of increased size and complexity, such as Native Mass Spectrometry, which has always been an important tool for structural biology. Scientific advance and recent developments, such as new ways to mimic a cell membrane for a membrane protein, have caused established methods to struggle to keep up with the increased demands. In this review, we summarize the possibilities, which Laser Induced Liquid Bead Ion Desorption (LILBID) mass spectrometry offers with regard to the challenges of modern structural biology, like increasingly complex sample composition, novel membrane mimics and advanced structural analysis, including next neighbor relations and the dynamics of complex formation.
... This in turn can help us to identify targets to inhibit this process, which could be of use in the development of strategies to prevent food spoilage. Mass spectrometry is a powerful tool for the identification and quantification of membrane protein composition [7]. However, membrane proteins are mostly hydrophobic and less abundant compared to the largely abundant spore coat and cytosolic proteins [8]. ...
Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.
... Molecular dynamics (MD) simulations have reached time and length scales, including with standard atomistic models and with increasingly sophisticated larger-scale models, where they can be used for meaningful investigations of membrane systems of direct biological and biomedical interest . At the same time, a rapidly growing number of experimentally determined membrane protein structures, primarily by cryo-EM (Cheng 2018; Thonghin et al. 2018), is identifying lipid or surfactant density associated with membrane proteins (Duncan et al. 2020;Sun and Gennis 2019;Thompson and Baenziger 2020), and lipidomics approaches have made a major leap in their ability to identify lipid compositions in complex membranes in general (Lorent et al. 2020;Symons et al. 2021) and, in some cases, lipids associated with membrane proteins (Bolla et al. 2019;Frick and Schmidt 2019;Sun and Gennis 2019;Teo et al. 2019;van 't Klooster et al. 2020). One of the longterm aims of our group is to understand the diverse roles lipid-protein interactions play in biological processes in both qualitative and quantitative terms, from a fundamental biological perspective and to use an improved understanding of lipid-protein interactions in biomedical applications. ...
Lipid-protein interactions play an important direct role in the function of many membrane proteins. We argue they are key players in membrane structure, modulate membrane proteins in more subtle ways than direct binding, and are important for understanding the mechanism of classes of hydrophobic drugs. By directly comparing membrane proteins from different families in the same, complex lipid mixture, we found a unique lipid environment for every protein. Extending this work, we identified both differences and similarities in the lipid environment of GPCRs, dependent on which family they belong to and in some cases their conformational state, with particular emphasis on the distribution of cholesterol. More recently, we have been studying modes of coupling between protein conformation and local membrane properties using model proteins. In more applied approaches, we have used similar methods to investigate specific hypotheses on interactions of lipid and lipid-like molecules with ion channels. We conclude this perspective with some considerations for future work, including a new more sophisticated coarse-grained force field (Martini 3), an interactive visual exploration framework, and opportunities to improve sampling.
... A promising arena for such experiments is enhancing the sensitivity of scrutinizing mixed assemblies. 39 This includes nanoscopic Bottom, lipid micelles, because of their nanoscopic dimensions, when accreting in highly heterogeneous environments will show high compositional variability leading to a large combinatorial number of aptamers. This variation-generating process, when occurring in many instances of mutuallycatalytic assemblies, could jump-start selection. ...
A widespread dogma asserts that life could not have emerged without biopolymers - RNA and proteins. However, the widely acknowledged implausibility of a spontaneous appearance and proliferation of these complex molecules in primordial messy chemistry casts doubt on this scenario. A proposed alternative is "Lipid-First", based on the evidence that lipid assemblies may spontaneously emerge in heterogeneous environments, and are shown to undergo growth and fission, and to portray autocatalytic self-copying. What seems undecided is whether lipid assemblies have protein-like capacities for stereospecific interactions, a sine qua non of life processes. This Viewpoint aims to alleviate such doubts, pointing to growing experimental evidence that lipid aggregates possess dynamic surface configurations capable of stereospecific molecular recognition. Such findings help support a possible key role of lipids in seeding life's origin.
... Gangliosides are generally responsible for proliferation and transmembrane signaling. [30] They reside in the lipid raft of the membrane [31] ( Figure 7f) and are found to be lower with adipogenesis, [32] which is consistent with our observation. In stem cells cultured in stromal media, we found many glycerophospholipids such Cardiolipins prevent lipid droplet formation by increasing energy consumption. ...
Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective treatment requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (<0.001% of the number of cells). Here, a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping-based machine learning analysis is reported to distinguish differentiating human adipose-derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enables identifying differentiated cells at single-cell resolution. The label-free HSI method is compared with the standard techniques such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. HSI is successfully used to assess the abundance of adipocytes derived from transplanted cells in a transgenic mice model. Further, Raman microscopy and multiphoton-based metabolic imaging is performed to provide complementary information for the functional imaging of the hASCs. Finally, the HSI method is validated using matrix-assisted laser desorption/ionization-mass spectrometry imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution.
... Membrane proteins naturally co-purify with their surrounding or stably-bound lipids; associated lipids as well as the interactions they form with the membrane proteins are then often identified by MS [16]. However, the protein-lipid interactions of peripheral membrane proteins, which dynamically interact with the lipid membrane, are difficult to capture. ...
Detergents are commonly employed for solubilization and stabilization of integral membrane proteins. Their effects on the charge states of detergent-solubilized membrane proteins in native mass spectrometry measurements have previously been described. These effects can be mediated through the transmembrane region directly interacting with the detergent micelle or through the soluble domains of the proteins getting in close contact with detergent micelles during electrospray ionization. Here, we explore the effects of detergent micelles on soluble proteins during native mass spectrometry aiming at distinguishing these two events. Specifically, we employ two non-ionic and one zwitterionic detergents as well as a variety of standard proteins differing in size, oligomeric state and surface hydrophobicity, and evaluate the observed charge states in the absence and presence of detergents. Using ion mobility-mass spectrometry, we assess the proteins’ stability as well as the ability to maintain non-covalent interactions with ligands. Finally, we examine lipid transfer from mixed detergent-lipid micelles containing the various detergents to soluble proteins. In summary, we found that C8E4 detergent reduces the proteins’ charge states, stabilizes the proteins and facilitates lipid transfer.
... Mass spectrometry (MS) is another common method to study membrane dynamic interactions (Konijnenberg et al., 2014;Gupta et al., 2017Gupta et al., , 2018Bolla et al., 2019;Frick and Schmidt, 2019). MS does not provide high-resolution target structure, but it can measure the mass and stoichiometry of the complex (Pyle et al., 2018;Frick and Schmidt, 2019). ...
... Mass spectrometry (MS) is another common method to study membrane dynamic interactions (Konijnenberg et al., 2014;Gupta et al., 2017Gupta et al., , 2018Bolla et al., 2019;Frick and Schmidt, 2019). MS does not provide high-resolution target structure, but it can measure the mass and stoichiometry of the complex (Pyle et al., 2018;Frick and Schmidt, 2019). Among different types of MS, hydrogen-deuterium exchange MS (HDX-MS) has gained much attention for studying transient (millisecond range) protein conformational changes (Rist et al., 2005;Giladi and Khananshvili, 2020). ...
... However, affected by the choice of detergent, the obtained results may not represent the native structure of membrane complexes. Membrane-mimic lipid nanostructures, such as picodiscs, nanodiscs, styrene maleic acid lipid particles, bicelles, and liposomes (Grinkova et al., 2010;Dürr et al., 2012;Marty et al., 2016;Frick and Schmidt, 2019), have been developed to alleviate this problem, while the effect of these artificial lipid structures should be still carefully considered. ...
Short-lived cell membrane complexes play a key role in regulating cell signaling and communication. Many of these complexes are formed based on low-affinity and transient interactions among various lipids and proteins. New techniques have emerged to study these previously overlooked membrane transient interactions. Exciting functions of these transient interactions have been discovered in cellular events such as immune signaling, host–pathogen interactions, and diseases such as cancer. In this review, we have summarized current experimental methods that allow us to detect and analyze short-lived cell membrane protein–protein, lipid–protein, and lipid–lipid interactions. These methods can provide useful information about the strengths, kinetics, and/or spatial patterns of membrane transient interactions. However, each method also has its own limitations. We hope this review can be used as a guideline to help the audience to choose proper approaches for studying membrane transient interactions in different membrane trafficking and cell signaling events.
... Gangliosides are generally responsible for proliferation and transmembrane signaling [61]. They reside in the lipid raft of the membrane [62] (Figure 7f) and are found to be lower with adipogenesis [63][64][65], which is consistent [68,69]. Tri(acyl|alkyl)glycerols (TG) are also abundantly found inside the lipid droplets (Figure 7c). ...
Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective therapy requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (< 0.001% of the number of cells). Here, we report a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping (SAM)-based machine learning analysis to distinguish differentiating human adipose derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enabled identification of differentiated cells at single cell resolution. The label-free HSI method was compared with the standard methods such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. Further, we performed Raman microscopy and multiphoton-based metabolic imaging to provide complimentary information for the functional imaging of the hASCs. Finally, the HSI method was validated using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution. This could provide a powerful tool to assess the safety and efficacy of stem cell-based regenerative therapies.
... The lipid is either treated online or offline, and the double bonds undergo chemical reactions that create distinct fragments, which are then detected in the spectrometer. [46][47][48][49][50] dynamics, which reveal not only the class of lipids present in the sample but also their proportions. Frick et al. showed that lipids could be introduced into the MS analysis in the form of lipid vesicles, which have a better representation of lipid bilayer composition than extracts that have been treated with harsh organic solvents. ...
... 49 An important goal for MS lipidome analysis is the study of lipid-membrane protein interactions. 50 Liposome formation offers a potential ''vehicle'' for insoluble membrane-bound molecules, facilitating their detection by MS. Using direct infusion to ESI, experiments with liposomes were performed, and gave MS spectra that corresponded to present lipid ratios. ...
Lipids remain one of the most enigmatic classes of biological molecules. Whereas lipids are well known to form basic units of membrane structure and energy storage, deciphering the exact roles and biological interactions of distinct lipid species has proven elusive. How these building blocks are synthesized, trafficked, and stored are also questions that require closer inspection. This tutorial review covers recent advances on the preparation, derivatization, and analysis of lipids. In particular, we describe several chemical approaches that form part of a powerful toolbox for controlling and characterizing lipid structure. We believe these tools will be helpful in numerous applications, including the study of lipid–protein interactions and the development of novel drug delivery systems.
