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Derek Marsh - One of the best experts on this subject based on the ideXlab platform.
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Electron spin resonance in membrane research: protein–lipid Interactions from challenging beginnings to state of the art
European Biophysics Journal, 2010Co-Authors: Derek MarshAbstract:Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein Interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments.
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protein lipid Interactions with fusobacterium nucleatum major outer membrane protein foma spin label epr and polarized infrared spectroscopy
Biochemistry, 2008Co-Authors: Veerappan Anbazhagan, N Vijay, Jorg H Kleinschmidt, Derek MarshAbstract:FomA, the major outer membrane protein of Fusobacterium nucleatum, was expressed and purified in Escherichia coli and reconstituted from detergent in bilayer membranes of phosphatidylcholines with chain lengths from C(12:0) to C(17:0). The conformation and orientation of membrane-incorporated FomA were determined from polarized, attenuated total reflection, infrared (IR) spectroscopy, and Lipid-Protein Interactions with FomA were characterized by using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled lipids. Approximately 190 residues of membranous FomA are estimated to be in a � -sheet configuration from IR band fitting, which is consistent with a 14-strand transmembrane � -barrel structure. IR dichroism of FomA indicates that the � -strands are tilted by ∼45° relative to the sheet/barrel axis and that the order parameter of the latter displays a discontinuity corresponding to hydrophobic matching with fluid C(13:0) lipid chains. The stoichiometry (Nb ) 23 lipids/monomer) of Lipid-Protein Interaction from EPR demonstrates that FomA is not trimeric in membranes of diC(14:0) phosphatidylcholine and is consistent with a monomeric � -barrel of 14-16 strands. The pronounced selectivity of Interaction found with anionic spin-labeled lipids places basic residues of the protein in the vicinity of the polar-apolar membrane interfaces, consistent with current topology models. Comparison with similar data from the 8- to 22-stranded E. coli outer membrane proteins, OmpA, OmpG, and FhuA, supports the above conclusions.
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Energetics of Hydrophobic Matching in Lipid-Protein Interactions
Biophysical journal, 2008Co-Authors: Derek MarshAbstract:Lipid chain length modulates the activity of transmembrane proteins by mismatch between the hydrophobic span of the protein and that of the lipid membrane. Relative binding affinities of lipids with different chain lengths are used to estimate the excess free energy of Lipid-Protein Interaction that arises from hydrophobic mismatch. For a wide range of integral proteins and peptides, the energy cost is much less than the elastic penalty of fully stretching or compressing the lipid chains to achieve complete hydrophobic matching. The chain length dependences of the free energies of lipid association are described by a model that combines elastic chain extension with a free energy term that depends linearly on the extent of residual mismatch. The excess free energy densities involved lie in the region of 0.5–2.0 kBT.nm−2. Values of this size could arise from exposure of hydrophobic groups to polar portions of the lipid or protein, but not directly to water, or alternatively from changes in tilt of the transmembrane helices that are energetically comparable to those activating mechanosensitive channels. The influence of hydrophobic mismatch on dimerization of transmembrane helices and their transfer between lipid vesicles, and on shifts in chain-melting transitions of lipid bilayers by incorporated proteins, is analyzed by using the same thermodynamic model. Segmental order parameters confirm that elastic lipid chain distortions are insufficient to compensate fully for the mismatch, but the dependence on chain length with tryptophan-anchored peptides requires that the free energy density of hydrophobic mismatch should increase with increasing extent of mismatch.
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Selectivity of Lipid-Protein Interactions with trypsinized Na, K-ATPase studied by spin-label EPR.
Biochimica et biophysica acta, 1998Co-Authors: Ashish Arora, Mikael Esmann, Derek MarshAbstract:The selectivity of the Lipid-Protein Interactions in trypsinised Na, K-ATPase membranes from Squalus acanthias has been determined by using EPR spectroscopy with different lipid probes spin-labelled on the 14-C atom of the fatty acid chain. From measurements at low ionic strength and different pH values, the pattern of selectivity is: (stearic acid)->(phosphatidylserine)->(stearic acid)0>(phosphatidylcholine)+/-, where superscripts indicate the formal electrostatic charge on the lipid headgroup. This is in the same order as that determined with native Na,K-ATPase membranes [M. Esmann, D. Marsh, Biochemistry 24 (1985) 3572-3578]. The selectivity for phosphatidylserine is independent of pH, over the range pH 6.0-9. 0, as found also for native membranes. For membranes trypsinised in the presence of Rb+ ions, and in the presence of Na+ (which allows more extensive proteolysis), the relative association constants, Kr, of all lipids are the same as for control membranes, with the exception of ionised (stearic acid)- that shows the highest specificity. Therefore, both the stoichiometry and the principal determinants of the specificity of Lipid-Protein Interaction are preserved on extensive trypsinisation of Na,K-ATPase membranes. This has implications for the location and arrangement of those amino acid side chains that determine the lipid selectivity of the native Na,K-ATPase.
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Stoichiometry of Lipid-Protein Interaction and integral membrane protein structure
European Biophysics Journal, 1997Co-Authors: Derek MarshAbstract:The stoichiometries of Lipid-Protein Interaction obtained from spin label electron spin resonance experiments with integral membrane proteins are compared with simple geometric models for the intramembranous perimeter that are based on the predicted numbers of transmembrane helices. Deviations from the predicted values provide evidence for oligomerization of the protein in the membrane and/or more complex arrangements of the transmembrane segments.
Peter D Tieleman - One of the best experts on this subject based on the ideXlab platform.
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lipid protein Interactions are a unique property and defining feature of g protein coupled receptors
Biophysical Journal, 2020Co-Authors: Besian I Sejdiu, Peter D TielemanAbstract:Abstract G protein-coupled receptors (GPCRs) are membrane-bound proteins that depend on their lipid environment to carry out their physiological function. Combined efforts from many theoretical and experimental studies on the Lipid-Protein Interaction profile of several GPCRs hint at an intricate relationship of these receptors with their surrounding membrane environment, with several lipids emerging as particularly important. Using coarse-grained molecular dynamics simulations, we explore the Lipid-Protein Interaction profiles of 28 different GPCRs, spanning different levels of classification and conformational states and totaling to 1 ms of simulation time. We find a close relationship with lipids for all GPCRs simulated, in particular, cholesterol and phosphatidylinositol phosphate (PIP) lipids, but the number, location, and estimated strength of these Interactions is dependent on the specific GPCR as well as its conformational state. Although both cholesterol and PIP lipids bind specifically to GPCRs, they utilize distinct mechanisms. Interactions with PIP lipids are mediated by charge-charge Interactions with intracellular loop residues and stabilized by one or both of the transmembrane helices linked by the loop. Interactions with cholesterol, on the other hand, are mediated by a hydrophobic environment, usually made up of residues from more than one helix, capable of accommodating its ring structure and stabilized by Interactions with aromatic and charged/polar residues. Cholesterol binding to GPCRs occurs in a small number of sites, some of which (like the binding site on the extracellular side of transmembrane 6/7) are shared among many class A GPCRs. Combined with a thorough investigation of the local membrane structure, our results provide a detailed picture of GPCR-lipid Interactions. Additionally, we provide an accompanying website to interactively explore the Lipid-Protein Interaction profile of all GPCRs simulated to facilitate analysis and comparison of our data.
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Lipid Protein Interactions Of G Protein Coupled Receptors
Biophysical Journal, 2016Co-Authors: Besian I Sejdiu, Christine Degagne, Valentina Corradi, Peter D TielemanAbstract:G Protein - Coupled Receptors (GPCRs) are a large family of membrane proteins involved in a large array of biochemical processes. Recent studies show that in order to explain the activity of GPCRs, their lipid environment has to be taken into account. Most notably, the Interaction of GPCRs with cholesterol molecules has been the center of focus of several studies conducted in the past few years. To understand this Lipid-Protein dynamics, we have carried out large-scale molecular dynamics simulations, using the MARTINI coarse-grained model, of several biologically important GPCRs (e.g. the human chemokine (CXCR1) receptor involved in breast cancer, and the delta opioid receptor important in pain sensing). Our simulation setup consists of four copies of proteins embedded in a 40nm x 40nm complex lipid bilayer, composed of more than 60 lipid types and simulated for 30 microseconds. Here, we present our results on the lipid organization at different distances from each protein copy, and show their relative lipid enrichment. In addition, we present our results on the lipid distribution and the Interactions of cholesterol with GPCRs. We believe that the simulation setup design we have employed and the long time scales we have achieved offer a unique perspective on the Lipid-Protein Interaction dynamics of GPCRs.
Yoshihisa Kurachi - One of the best experts on this subject based on the ideXlab platform.
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pip3 inhibition of rgs protein and its reversal by ca2 calmodulin mediate voltage dependent control of the g protein cycle in a cardiac k channel
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Masaru Ishii, Atsushi Inanobe, Yoshihisa KurachiAbstract:Regulators of G protein signaling (RGS) accelerate intrinsic GTP hydrolysis on α subunits of trimeric G proteins and play crucial roles in the physiological regulation of G protein-mediated cell signaling. The control mechanisms of the action of RGS proteins per se are poorly clarified, however. We recently showed a physiological mode of action of a RGS protein in cardiac myocytes. The voltage-dependent formation of Ca2+/calmodulin facilitated the GTPase activity of RGS by an unidentified mechanism, which underlay the “relaxation” behavior of G protein-gated K+ (KG) channels. Here we report the mechanism which is the reversal by Ca2+/calmodulin of phosphatidylinositol-3,4,5,-trisphosphate (PIP3)-mediated inhibition of RGS. Purified RGS4 protein alone inhibited GTP-induced KG channel activity in inside-out patches from atrial myocytes. The inhibitory effect of RGS4 was reduced by PIP3 and restored by addition of Ca2+/calmodulin. The intracellular application of anti-PIP3 antibody abolished the RGS-dependent relaxation behavior of KG current in atrial myocytes. This study, therefore, reveals a general physiological control mechanism of RGS proteins by lipid–protein Interaction.
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PIP3 inhibition of RGS protein and its reversal by Ca2+/calmodulin mediate voltage-dependent control of the G protein cycle in a cardiac K+ channel
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Masaru Ishii, Atsushi Inanobe, Yoshihisa KurachiAbstract:Regulators of G protein signaling (RGS) accelerate intrinsic GTP hydrolysis on alpha subunits of trimeric G proteins and play crucial roles in the physiological regulation of G protein-mediated cell signaling. The control mechanisms of the action of RGS proteins per se are poorly clarified, however. We recently showed a physiological mode of action of a RGS protein in cardiac myocytes. The voltage-dependent formation of Ca2+/calmodulin facilitated the GTPase activity of RGS by an unidentified mechanism, which underlay the "relaxation" behavior of G protein-gated K+ (K(G)) channels. Here we report the mechanism which is the reversal by Ca2+/calmodulin of phosphatidylinositol-3,4,5,-trisphosphate (PIP3)-mediated inhibition of RGS. Purified RGS4 protein alone inhibited GTP-induced K(G) channel activity in inside-out patches from atrial myocytes. The inhibitory effect of RGS4 was reduced by PIP3 and restored by addition of Ca2+/calmodulin. The intracellular application of anti-PIP3 antibody abolished the RGS-dependent relaxation behavior of K(G) current in atrial myocytes. This study, therefore, reveals a general physiological control mechanism of RGS proteins by Lipid-Protein Interaction.
Ana Coutinho - One of the best experts on this subject based on the ideXlab platform.
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electrostatically driven lipid protein Interaction answers from fret
Biochimica et Biophysica Acta, 2015Co-Authors: Fabio Fernandes, Ana Coutinho, Manuel Prieto, Luis M S LouraAbstract:Abstract Electrostatics govern the association of a large number of proteins with cellular membranes. In some cases, these proteins present specialized lipid-binding modules or membrane targeting domains while in other cases association is achieved through nonspecific Interaction of unstructured clusters of basic residues with negatively charged lipids. Given its spatial resolution in the nanometer range, Forster resonance energy transfer (FRET) is a powerful tool to give insight into protein–lipid Interactions and provide molecular level information which is difficult to retrieve with other spectroscopic techniques. In this review we present and discuss the basic formalisms of both hetero- and homo-FRET pertinent to the most commonly encountered problems in lipid–protein Interaction studies and highlight some examples of implementations of different FRET methodologies to characterize lipid/protein systems in which electrostatic Interactions play a crucial role. This article is part of a Special Issue entitled: Lipid–protein Interactions.
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Quantifying Lipid-Protein Interaction by fluorescence correlation spectroscopy (FCS).
Methods in Molecular Biology, 2013Co-Authors: Ana M. Melo, Manuel Prieto, Ana CoutinhoAbstract:Fluorescence correlation spectroscopy (FCS) is a powerful method to investigate molecular Interactions based on the variation of diffusion properties at the single-molecule level. This technique allows studying quantitatively the Interaction of fluorescently labeled proteins/peptides with lipid vesicles. Here, we describe how to acquire and analyze FCS partition data in order to accurately determine the protein/peptide partition coefficients between the aqueous and lipid phases. It is shown that the recovery of unbiased partition coefficients from FCS partition curves (fractional amplitude of the bound species versus lipid concentration) requires considering explicitly the Poissonian loading of the lipid vesicles with the fluorescently labeled protein in order to account for the variable liposome brightness in each sample. Additionally, the impact of a trace amount of a fluorescent non-binding component on the partition curves determined by FCS is also discussed.
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Quantifying Protein Binding to Lipid Vesicles Using Fluorescence Correlation Spectroscopy (FCS)
Biophysical Journal, 2011Co-Authors: Ana M. Melo, Ana Coutinho, Manuel PrietoAbstract:Quantitative evaluation of protein binding to liposomes through the determination of their partition coefficient (Kp) is an important step in any Lipid-Protein Interaction study because it allows the calculation of protein interfacial coverage of the lipid vesicles, often the critical parameter controlling the protein membrane binding mode (peripheral vs partial insertion) and/or its oligomerization state. In this study, the application of fluorescence correlation spectroscopy (FCS) technique to investigate the binding of protein molecules to phospholipid vesicles is presented. We have found that the type of fitting function used in the analysis of the experimental AC curves obtained in a partitioning experiment strongly influences the final recovered partition coefficients. We show that if the parameter chosen to establish the partition curves of the fluorescent peptide/ protein is the fractional amplitude associated with the slowest diffusing particles in the system (lipid vesicles) then it is necessary to consider explicitly the statistical (Poissonian) distribution of the fluorescently-labeled protein among the ensemble of lipid vesicles in the fitting procedure to not overestimate the determined Kp values. We have extended this analytical model by considering the presence of a trace amount of free fluorescent dye (nonbinding component) in the system to account for an apparent maximum binding level less than 100% in the experimental partitioning curves obtained for Alexa 488 fluorescently-labeled lysozyme and liposomes prepared with variable anionic phospholipid content. The extreme sensitivity of the FCS technique allowed uncoupling lysozyme partition from the protein-induced liposome aggregation, confirming that lysozyme binding to negatively charged liposomes is dominantly driven by electrostatic Interactions.
Manuel Prieto - One of the best experts on this subject based on the ideXlab platform.
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electrostatically driven lipid protein Interaction answers from fret
Biochimica et Biophysica Acta, 2015Co-Authors: Fabio Fernandes, Ana Coutinho, Manuel Prieto, Luis M S LouraAbstract:Abstract Electrostatics govern the association of a large number of proteins with cellular membranes. In some cases, these proteins present specialized lipid-binding modules or membrane targeting domains while in other cases association is achieved through nonspecific Interaction of unstructured clusters of basic residues with negatively charged lipids. Given its spatial resolution in the nanometer range, Forster resonance energy transfer (FRET) is a powerful tool to give insight into protein–lipid Interactions and provide molecular level information which is difficult to retrieve with other spectroscopic techniques. In this review we present and discuss the basic formalisms of both hetero- and homo-FRET pertinent to the most commonly encountered problems in lipid–protein Interaction studies and highlight some examples of implementations of different FRET methodologies to characterize lipid/protein systems in which electrostatic Interactions play a crucial role. This article is part of a Special Issue entitled: Lipid–protein Interactions.
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Quantifying Lipid-Protein Interaction by fluorescence correlation spectroscopy (FCS).
Methods in Molecular Biology, 2013Co-Authors: Ana M. Melo, Manuel Prieto, Ana CoutinhoAbstract:Fluorescence correlation spectroscopy (FCS) is a powerful method to investigate molecular Interactions based on the variation of diffusion properties at the single-molecule level. This technique allows studying quantitatively the Interaction of fluorescently labeled proteins/peptides with lipid vesicles. Here, we describe how to acquire and analyze FCS partition data in order to accurately determine the protein/peptide partition coefficients between the aqueous and lipid phases. It is shown that the recovery of unbiased partition coefficients from FCS partition curves (fractional amplitude of the bound species versus lipid concentration) requires considering explicitly the Poissonian loading of the lipid vesicles with the fluorescently labeled protein in order to account for the variable liposome brightness in each sample. Additionally, the impact of a trace amount of a fluorescent non-binding component on the partition curves determined by FCS is also discussed.
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Quantifying Protein Binding to Lipid Vesicles Using Fluorescence Correlation Spectroscopy (FCS)
Biophysical Journal, 2011Co-Authors: Ana M. Melo, Ana Coutinho, Manuel PrietoAbstract:Quantitative evaluation of protein binding to liposomes through the determination of their partition coefficient (Kp) is an important step in any Lipid-Protein Interaction study because it allows the calculation of protein interfacial coverage of the lipid vesicles, often the critical parameter controlling the protein membrane binding mode (peripheral vs partial insertion) and/or its oligomerization state. In this study, the application of fluorescence correlation spectroscopy (FCS) technique to investigate the binding of protein molecules to phospholipid vesicles is presented. We have found that the type of fitting function used in the analysis of the experimental AC curves obtained in a partitioning experiment strongly influences the final recovered partition coefficients. We show that if the parameter chosen to establish the partition curves of the fluorescent peptide/ protein is the fractional amplitude associated with the slowest diffusing particles in the system (lipid vesicles) then it is necessary to consider explicitly the statistical (Poissonian) distribution of the fluorescently-labeled protein among the ensemble of lipid vesicles in the fitting procedure to not overestimate the determined Kp values. We have extended this analytical model by considering the presence of a trace amount of free fluorescent dye (nonbinding component) in the system to account for an apparent maximum binding level less than 100% in the experimental partitioning curves obtained for Alexa 488 fluorescently-labeled lysozyme and liposomes prepared with variable anionic phospholipid content. The extreme sensitivity of the FCS technique allowed uncoupling lysozyme partition from the protein-induced liposome aggregation, confirming that lysozyme binding to negatively charged liposomes is dominantly driven by electrostatic Interactions.