The Experts below are selected from a list of 249 Experts worldwide ranked by ideXlab platform
Derek Marsh - One of the best experts on this subject based on the ideXlab platform.
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distinct populations in Spin Label epr spectra from nitroxides
Journal of Physical Chemistry B, 2018Co-Authors: Derek MarshAbstract:Two-component nitroxide Spin-Label electron paramagnetic presonance (EPR) spectra are important to the analysis of lipid-protein interactions, phase separation in lipid membranes, and conformational changes in proteins. A paper published in this journal offers an interpretation of such spectra based on simulations with single-site models. It is not possible to reproduce those simulations published in Meirovitch, E.; [Analysis of Protein Lipid Interactions Based on Model Simulations of Electron Spin Resonance Spectra. J. Phys. Chem. 1984, 88, 3454–3465] that might conceivably be taken to resemble two-component line shapes, when using the motional model and parameters given in that paper. Instead of the apparent two components, the spectra resemble single-component powder patterns expected from axially anisotropic, partial motional-averaging (a situation familiar for chain-Labeled lipids in nonaligned fluid membranes). This is because: (i) the nitroxide z-axis is inclined at a fixed angle to the principal d...
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ether linked lipids Spin Label epr and Spin echoes
Chemistry and Physics of Lipids, 2018Co-Authors: Maria Oranges, Derek Marsh, Rita Guzzi, Rosa BartucciAbstract:Abstract Electron Spin echo envelope modulation (ESEEM) and conventional electron paramagnetic resonance (EPR) of site-specifically Spin-Labelled phospholipids are used to investigate the effect of ether-linked chains on the water-penetration and polarity profiles, as well as the phase behaviour and chain flexibility profiles, of phospholipid membranes. D2O-ESEEM reveals that water exposure of the terminal methyl groups in the interdigitated phase of dihexadecyl phosphatidylcholine (DHPC) is comparable to that of the methylene groups at the polar head-group end of the chains. Similarly, an uniform transmembrane polarity profile is obtained from the dependence of the outer 14N-hyperfine splitting on the Spin-Label position along the chain in frozen interdigitated DHPC dispersions. Two-component conventional EPR spectra of Spin Labels at the terminal methyl end of the chain reveal that the intermediate gel phase above the pretransition of DHPC contains components in which the lipid chains are interdigitated. The polarity and chain-flexibility profiles in the fluid Lα-phase of DHPC with ether-linked chains are shifted outwards, towards the polar-apolar interface, as compared with that of dihexadecanoyl phosphatidylcholine (DPPC) with ester-linked chains. Also, the polarity profile of DHPC is shifted upwards, to higher polarities. These differences reflect those in hydrocarbon thickness and area/lipid molecule reported by x-ray diffraction for the Lα-phases of the two lipids.
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nuclear Spin lattice relaxation in nitroxide Spin Label epr
Journal of Magnetic Resonance, 2016Co-Authors: Derek MarshAbstract:Nuclear relaxation is a sensitive monitor of rotational dynamics in Spin-Label EPR. It also contributes competing saturation transfer pathways in T1-exchange spectroscopy, and the determination of paramagnetic relaxation enhancement in site-directed Spin Labelling. A survey shows that the definition of nitrogen nuclear relaxation rate Wn commonly used in the CW-EPR literature for 14N-nitroxyl Spin Labels is inconsistent with that currently adopted in time-resolved EPR measurements of saturation recovery. Redefinition of the normalised 14N Spin-lattice relaxation rate, b=Wn/(2We), preserves the expressions used for CW-EPR, whilst rendering them consistent with expressions for saturation recovery rates in pulsed EPR. Furthermore, values routinely quoted for nuclear relaxation times that are deduced from EPR spectral diffusion rates in 14N-nitroxyl Spin Labels do not accord with conventional analysis of Spin-lattice relaxation in this three-level system. Expressions for CW-saturation EPR with the revised definitions are summarised. Data on nitrogen nuclear Spin-lattice relaxation times are compiled according to the three-level scheme for 14N-relaxation: T1n=1/Wn. Results are compared and contrasted with those for the two-level 15N-nitroxide system.
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heterogeneity of protein substates visualized by Spin Label epr
Biophysical Journal, 2014Co-Authors: Rita Guzzi, Rosa Bartucci, Derek MarshAbstract:The energy landscape of proteins is characterized by a hierarchy of substates, which give rise to conformational heterogeneity at low temperatures. In multiply Spin-Labeled membranous Na,K-ATPase, this heterogeneous population of conformations is manifest by strong inhomogeneous broadening of the electron paramagnetic resonance (EPR) line shapes and nonexponential Spin-echo decays, which undergo a transition to homogeneous broadening and exponential relaxation at higher temperatures (previous study). In this study, we apply these EPR methods to small water-soluble proteins, of the type for which the existence of conformational substates is well established. Both a-helical and b-sheet aqueous proteins that are Spin-Labeled on a single cysteine residue display Spin-echo decays with a single phase-memory time T2M and conventional EPR line shapes with predominantly homogeneous broadening, over a broad range of temperatures from 77 K to ~ 250 K or higher. Above ~ 200 K, the residual inhomogeneous broadening is reduced almost to zero. In contrast, both the proteins and the Spin Label alone, when in a glycerol-water mixture below the glass transition, display heterogeneity in Spin-echo phase- memory time and a stronger inhomogeneous broadening of the conventional line shapes, similar to multiply Spin-Labeled membranous Na,K-ATPase below 200 K. Above 200 K (or the glass transition), a single phase-memory time and predominantly homogeneous broadening are found in both Spin-Label systems. The results are discussed in terms of solvent-mediated protein transitions, the ability of single Spin-Label sites to detect conformational heterogeneity, and the desirability of exploring multiple sites for proteins with the size and complexity of the Na,K-ATPase.
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Spin-Label EPR for Determining Polarity and Proticity in Biomolecular Assemblies: Transmembrane Profiles.
Applied magnetic resonance, 2009Co-Authors: Derek MarshAbstract:Hyperfine couplings and g-values of nitroxyl Spin Labels are sensitive to polarity and hydrogen bonding in the environment probed. The dependences of these electronic paramagnetic resonance (EPR) properties on environmental dielectric permittivity and proticity are reviewed. Calibrations are given, in terms of the Block-Walker reaction field and local proton donor concentration, for the nitroxides that are commonly used in Spin Labeling of lipids and proteins. Applications to studies of the transverse polarity profiles in lipid bilayers, which constitute the permeability barrier of biological membranes, are reviewed. Emphasis is given to parallels with the permeation profiles of oxygen and nitric oxide that are determined from Spin-Label relaxation enhancements by using nonlinear continuous-wave EPR and saturation recovery EPR, and with permeation profiles of D(2)O that are determined by using (2)H electron Spin echo envelope modulation spectroscopy.
Valérie Belle - One of the best experts on this subject based on the ideXlab platform.
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A Bioresistant Nitroxide Spin Label for In-Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies
Angewandte Chemie International Edition, 2018Co-Authors: Ganesan Karthikeyan, Bruno Guigliarelli, Alessio Bonucci, Gilles Casano, Guillaume Gerbaud, Sébastien Abel, Virginie Thomé, Laurent Kodjabachian, Axel Magalon, Valérie BelleAbstract:Approaching protein structural dynamics and protein-protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site-directed Spin Labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide-based Spin Labels for in-cell studies has represented a major hurdle because of their short persistence in the cellular context. The design and synthesis of the first maleimido-proxyl-based Spin Label (M-TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR is presented. In particular, the extended lifetime of M-TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells.
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Amplitude of pancreatic lipase lid opening in solution and identification of Spin Label conformational subensembles by combining continuous wave and pulsed EPR spectroscopy and molecular dynamics.
Biochemistry, 2010Co-Authors: Sébastien Ranaldi, Valérie Belle, Mireille Woudstra, Raphaël Bourgeas, Bruno Guigliarelli, Philippe Roche, Hervé Vezin, Frédéric Carrière, André FournelAbstract:The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two Spin Labels grafted on this enzyme. One Spin Label was introduced at a rigid position in HPL where an accessible cysteine residue (C181) naturally occurs. A second Spin Label was covalently bound to the mobile lid after introducing a cysteine residue at position 249 by site-directed mutagenesis. Double electron−electron resonance (DEER) experiments allowed the estimation of a distance of 19 ± 2 A between the Spin Labels when biLabeled HPL was alone in a frozen solution, i.e., with the lid in the closed conformation. A magnetic interaction was however detected by continuous wave EPR experiments, suggesting that a fraction of biLabeled HPL contained Spin Labels separated by a shorter distance. These results could be interpreted by the presence of two conformational subensembles for the Spin Label lateral chain at position 249 when the lid was closed. The ...
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Amplitude of pancreatic lipase lid opening in solution and identification of Spin Label conformational subensembles by combining continuous wave and pulsed EPR spectroscopy and molecular dynamics
Biochemistry, 2010Co-Authors: Sébastien Ranaldi, Valérie Belle, Mireille Woudstra, Raphaël Bourgeas, Bruno Guigliarelli, Philippe Roche, Hervé Vezin, Frédéric Carrière, André FournelAbstract:The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two Spin Labels grafted on this enzyme. One Spin Label was introduced at a rigid position in HPL where an accessible cysteine residue (C181) naturally occurs. A second Spin Label was covalently bound to the mobile lid after introducing a cysteine residue at position 249 by site-directed mutagenesis. Double electron−electron resonance (DEER) experiments allowed the estimation of a distance of 19 ± 2 Å between the Spin Labels when biLabeled HPL was alone in a frozen solution, i.e., with the lid in the closed conformation. A magnetic interaction was however detected by continuous wave EPR experiments, suggesting that a fraction of biLabeled HPL contained Spin Labels separated by a shorter distance. These results could be interpreted by the presence of two conformational subensembles for the Spin Label lateral chain at position 249 when the lid was closed. The existence of these conformational subensembles was revealed by molecular dynamics experiments and confirmed by the simulation of the EPR spectrum. When the lid opening was induced by the addition of bile salts and colipase, a larger distance of 43 ± 2 Å between the two Spin Labels was estimated from DEER experiments. The distances measured between the Spin Labels grafted at positions 181 and 249 were in good agreement with those estimated from the known X-ray structures of HPL in the closed and open conformations, but for the first time, the amplitude of the lid opening was measured in solution or in a frozen solution in the presence of amphiphiles.
Rosa Bartucci - One of the best experts on this subject based on the ideXlab platform.
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ether linked lipids Spin Label epr and Spin echoes
Chemistry and Physics of Lipids, 2018Co-Authors: Maria Oranges, Derek Marsh, Rita Guzzi, Rosa BartucciAbstract:Abstract Electron Spin echo envelope modulation (ESEEM) and conventional electron paramagnetic resonance (EPR) of site-specifically Spin-Labelled phospholipids are used to investigate the effect of ether-linked chains on the water-penetration and polarity profiles, as well as the phase behaviour and chain flexibility profiles, of phospholipid membranes. D2O-ESEEM reveals that water exposure of the terminal methyl groups in the interdigitated phase of dihexadecyl phosphatidylcholine (DHPC) is comparable to that of the methylene groups at the polar head-group end of the chains. Similarly, an uniform transmembrane polarity profile is obtained from the dependence of the outer 14N-hyperfine splitting on the Spin-Label position along the chain in frozen interdigitated DHPC dispersions. Two-component conventional EPR spectra of Spin Labels at the terminal methyl end of the chain reveal that the intermediate gel phase above the pretransition of DHPC contains components in which the lipid chains are interdigitated. The polarity and chain-flexibility profiles in the fluid Lα-phase of DHPC with ether-linked chains are shifted outwards, towards the polar-apolar interface, as compared with that of dihexadecanoyl phosphatidylcholine (DPPC) with ester-linked chains. Also, the polarity profile of DHPC is shifted upwards, to higher polarities. These differences reflect those in hydrocarbon thickness and area/lipid molecule reported by x-ray diffraction for the Lα-phases of the two lipids.
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heterogeneity of protein substates visualized by Spin Label epr
Biophysical Journal, 2014Co-Authors: Rita Guzzi, Rosa Bartucci, Derek MarshAbstract:The energy landscape of proteins is characterized by a hierarchy of substates, which give rise to conformational heterogeneity at low temperatures. In multiply Spin-Labeled membranous Na,K-ATPase, this heterogeneous population of conformations is manifest by strong inhomogeneous broadening of the electron paramagnetic resonance (EPR) line shapes and nonexponential Spin-echo decays, which undergo a transition to homogeneous broadening and exponential relaxation at higher temperatures (previous study). In this study, we apply these EPR methods to small water-soluble proteins, of the type for which the existence of conformational substates is well established. Both a-helical and b-sheet aqueous proteins that are Spin-Labeled on a single cysteine residue display Spin-echo decays with a single phase-memory time T2M and conventional EPR line shapes with predominantly homogeneous broadening, over a broad range of temperatures from 77 K to ~ 250 K or higher. Above ~ 200 K, the residual inhomogeneous broadening is reduced almost to zero. In contrast, both the proteins and the Spin Label alone, when in a glycerol-water mixture below the glass transition, display heterogeneity in Spin-echo phase- memory time and a stronger inhomogeneous broadening of the conventional line shapes, similar to multiply Spin-Labeled membranous Na,K-ATPase below 200 K. Above 200 K (or the glass transition), a single phase-memory time and predominantly homogeneous broadening are found in both Spin-Label systems. The results are discussed in terms of solvent-mediated protein transitions, the ability of single Spin-Label sites to detect conformational heterogeneity, and the desirability of exploring multiple sites for proteins with the size and complexity of the Na,K-ATPase.
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interaction of human serum albumin with membranes containing polymer grafted lipids Spin Label esr studies in the mushroom and brush regimes
Biochimica et Biophysica Acta, 2002Co-Authors: Rosa Bartucci, Derek Marsh, Manuela Pantusa, Luigi SportelliAbstract:The adsorption of human serum albumin (HSA) to dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was studied as a function of content and headgroup size of the polymer lipid. In the absence of protein, conversion from the low-density mushroom regime to the high-density brush regime of polymer-lipid content is detected by the change in ESR outer hyperfine splitting, 2Amax, of chain Spin-Labelled phosphatidylcholine in gel-phase membranes. The values of 2Amax remain constant in the mushroom regime, but decrease on entering the brush regime. Conversion between the two regimes occurs at mole fractions XPEG(m !b)c0.04, 0.01–0.02 and 0.005–0.01 for PEG-DPPE with mean PEG molecular masses of 350, 2000 and 5000 Da, respectively, as expected theoretically. Adsorption of HSA to DPPC membranes is detected as a decrease of the Spin Label 2Amax hyperfine splitting in the gel phase. Saturation is obtained at a protein/lipid ratio of ca. 1:1 w/w. In the presence of polymer-grafted lipids, HSA adsorbs to DPPC membranes only in the mushroom regime, irrespective of polymer length. In the brush regime, the Spin-Label values of 2Amax are unchanged in the presence of protein. Even in the mushroom regime, protein adsorption progressively becomes strongly attenuated as a result of the steric stabilization exerted by the polymer lipid. These results are in agreement with theoretical estimates of the lateral pressure exerted by the grafted polymer in the brush and mushroom regimes, respectively. D 2002 Elsevier Science B.V. All rights reserved.
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lipid membrane expansion and micelle formation by polymer grafted lipids scaling with polymer length studied by Spin Label electron Spin resonance
Biophysical Journal, 2001Co-Authors: Giuseppina Montesano, Derek Marsh, Rosa Bartucci, Salvatore Belsito, Luigi SportelliAbstract:Spin-Label electron Spin resonance (ESR) spectroscopy and auxiliary optical density measurements are used to study lipid dispersions of N-poly(ethylene glycol)-dipalmitoyl phosphatidylethanolamine (PEG:5000-DPPE) mixed with dipalmitoyl phosphatidylcholine (DPPC). PEG:5000-DPPE bears a large hydrophilic polymer headgroup (with approximately 114 oxyethylene monomers) and is commonly used for steric stabilization of liposomes used in drug delivery. Comparison is made with results from mixtures of DPPC with polymer lipids bearing shorter headgroups (approximately 45 and 8 oxyethylene monomers). ESR spectra of phosphatidylcholine Spin-Labeled on the 5-C atom position of the sn-2 chain are shown to reflect the area expansion of the lipid membranes by the lateral pressure exerted in the polymer brush, in a way that is consistent with theory. The lipid chain packing density at the onset of micelle formation is the same for all three PEG-lipids, although the mole fraction at which this occurs differs greatly. The mole fraction at onset scales inversely with the size of the polymer headgroup, where the experimental exponent of 0.7 is close to theoretical predictions (viz. 0.55-0.6). The mole fraction of PEG-lipid at completion of micelle formation is more weakly dependent on polymer size, which conforms with theoretical predictions. At high mole fractions of PEG:5000-DPPE the dependence of lipid packing density on mole fraction is multiphasic, which differs qualitatively from the monotonic decrease in packing density found with the shorter polymer lipids. Lipid Spin-Label ESR is an experimental tool that complements theoretical analysis using polymer models combined with the lipid equation of state.
André Fournel - One of the best experts on this subject based on the ideXlab platform.
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Amplitude of pancreatic lipase lid opening in solution and identification of Spin Label conformational subensembles by combining continuous wave and pulsed EPR spectroscopy and molecular dynamics.
Biochemistry, 2010Co-Authors: Sébastien Ranaldi, Valérie Belle, Mireille Woudstra, Raphaël Bourgeas, Bruno Guigliarelli, Philippe Roche, Hervé Vezin, Frédéric Carrière, André FournelAbstract:The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two Spin Labels grafted on this enzyme. One Spin Label was introduced at a rigid position in HPL where an accessible cysteine residue (C181) naturally occurs. A second Spin Label was covalently bound to the mobile lid after introducing a cysteine residue at position 249 by site-directed mutagenesis. Double electron−electron resonance (DEER) experiments allowed the estimation of a distance of 19 ± 2 A between the Spin Labels when biLabeled HPL was alone in a frozen solution, i.e., with the lid in the closed conformation. A magnetic interaction was however detected by continuous wave EPR experiments, suggesting that a fraction of biLabeled HPL contained Spin Labels separated by a shorter distance. These results could be interpreted by the presence of two conformational subensembles for the Spin Label lateral chain at position 249 when the lid was closed. The ...
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Amplitude of pancreatic lipase lid opening in solution and identification of Spin Label conformational subensembles by combining continuous wave and pulsed EPR spectroscopy and molecular dynamics
Biochemistry, 2010Co-Authors: Sébastien Ranaldi, Valérie Belle, Mireille Woudstra, Raphaël Bourgeas, Bruno Guigliarelli, Philippe Roche, Hervé Vezin, Frédéric Carrière, André FournelAbstract:The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two Spin Labels grafted on this enzyme. One Spin Label was introduced at a rigid position in HPL where an accessible cysteine residue (C181) naturally occurs. A second Spin Label was covalently bound to the mobile lid after introducing a cysteine residue at position 249 by site-directed mutagenesis. Double electron−electron resonance (DEER) experiments allowed the estimation of a distance of 19 ± 2 Å between the Spin Labels when biLabeled HPL was alone in a frozen solution, i.e., with the lid in the closed conformation. A magnetic interaction was however detected by continuous wave EPR experiments, suggesting that a fraction of biLabeled HPL contained Spin Labels separated by a shorter distance. These results could be interpreted by the presence of two conformational subensembles for the Spin Label lateral chain at position 249 when the lid was closed. The existence of these conformational subensembles was revealed by molecular dynamics experiments and confirmed by the simulation of the EPR spectrum. When the lid opening was induced by the addition of bile salts and colipase, a larger distance of 43 ± 2 Å between the two Spin Labels was estimated from DEER experiments. The distances measured between the Spin Labels grafted at positions 181 and 249 were in good agreement with those estimated from the known X-ray structures of HPL in the closed and open conformations, but for the first time, the amplitude of the lid opening was measured in solution or in a frozen solution in the presence of amphiphiles.
Olav Schiemann - One of the best experts on this subject based on the ideXlab platform.
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ox slim synthesis of and site specific Labelling with a highly hydrophilic trityl Spin Label
Chemistry: A European Journal, 2021Co-Authors: Nico Fleck, Caspar A Heubach, Tobias Hett, Sebastian Spicher, Stefan Grimme, Olav SchiemannAbstract:The combination of pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) with site-directed Spin Labelling is a powerful tool in structural biology. Rational design of trityl-based Spin Labels has enabled studying biomolecular structures at room temperature and within cells. However, most current trityl Spin Labels suffer either from aggregation with proteins due to their hydrophobicity, or from bioconjugation groups not suitable for in-cell measurements. Therefore, we introduce here the highly hydrophilic trityl Spin Label Ox-SLIM. Engineered as a short-linked maleimide, it combines the most recent developments in one single molecule, as it does not aggregate with proteins, exhibits high resistance under in-cell conditions, provides a short linker, and allows for selective and efficient Spin Labelling via cysteines. Beyond establishing synthetic access to Ox-SLIM, its suitability as a Spin Label is illustrated and ultimately, highly sensitive PDS measurements are presented down to protein concentrations as low as 45 nm resolving interSpin distances of up to 5.5 nm.
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determination of nitroxide Spin Label conformations via peldor and x ray crystallography
Physical Chemistry Chemical Physics, 2016Co-Authors: Dinar Abdullin, Gregor Hagelueken, Olav SchiemannAbstract:Pulsed electron–electron double resonance (PELDOR or DEER) in combination with site-directed Spin Labelling has emerged as an important method for measuring nanometer distance constraints that are used to obtain coarse-grained structures of biomolecules or to follow their conformational changes. Translating measured Spin–Spin distances between Spin Labels into structural information requires taking the conformational flexibility of Spin Label side chains into account. Here, we present an analysis of orientation selective PELDOR data recorded on six singly MTSSL-Labelled azurin mutants. The analysis yielded conformational MTSSL ensembles, which are considerably narrower than those predicted using in silico Spin Labeling methods but match well with Spin Label conformations found in the corresponding crystal structures. The possible reasons and consequences for predicting Spin Label conformers in the fold of biomolecules are discussed.
