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Derek Marsh - One of the best experts on this subject based on the ideXlab platform.
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Association of Spin-Labeled Lipids with â-Barrel Proteins from the Outer Membrane of Escherichia coli†
2016Co-Authors: Muthu Ramakrishnan, Cosmin L Pocanschi, Jörg H. Kleinschmidt, Derek MarshAbstract:ABSTRACT: The interaction of spin-labeled lipids with â-barrel transmembrane proteins has been studied by the electron spin resonance (ESR) methods developed for R-helical integral proteins. The outer membrane protein OmpA and the ferrichrome-iron receptor FhuA from the outer membrane of Escherichia coli were reconstituted in bilayers of Dimyristoylphosphatidylglycerol. The ESR spectra from phosphatidyl-glycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the â-barrel, in addition to that from the fluid bilayer lipids. The stoichiometry of motionally restricted lipids, 11 and 32 lipids/monomer for OmpA and FhuA, respectively, is constant irrespective of the total lipid/protein ratio. It is proportional to the number of transmembrane â-strands, eight for OmpA and 22 for FhuA, and correlates reasonably well with the intramembranous perimeter of the protein. Spin-labeled lipids with different polar headgroups display a differential selectivity of interaction with the two proteins. The more pronounced pattern of lipid selectivity for FhuA than for OmpA correlates with the preponderance of positively charged residues facing the lipids in the extensions of the â-sheet and shorter interconnecting loops on the extracellular side of FhuA. Because of the favorable time scale of electron spin resonance (ESR)1 spectroscopy, ESR spectra of spin-labele
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association of spin labeled lipids with beta barrel proteins from the outer membrane of escherichia coli
Biochemistry, 2004Co-Authors: Muthu Ramakrishnan, Cosmin L Pocanschi, Jorg H Kleinschmidt, Derek MarshAbstract:The interaction of spin-labeled lipids with ‚-barrel transmembrane proteins has been studied by the electron spin resonance (ESR) methods developed for R-helical integral proteins. The outer membrane protein OmpA and the ferrichrome-iron receptor FhuA from the outer membrane of Escherichia coli were reconstituted in bilayers of Dimyristoylphosphatidylglycerol. The ESR spectra from phosphatidyl- glycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the ‚-barrel, in addition to that from the fluid bilayer lipids. The stoichiometry of motionally restricted lipids, 11 and 32 lipids/monomer for OmpA and FhuA, respectively, is constant irrespective of the total lipid/protein ratio. It is proportional to the number of transmembrane ‚-strands, eight for OmpA and 22 for FhuA, and correlates reasonably well with the intramembranous perimeter of the protein. Spin-labeled lipids with different polar headgroups display a differential selectivity of interaction with the two proteins. The more pronounced pattern of lipid selectivity for FhuA than for OmpA correlates with the preponderance of positively charged residues facing the lipids in the extensions of the ‚-sheet and shorter interconnecting loops on the extracellular side of FhuA.
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Articles Association of Spin-Labeled Lipids with â-Barrel Proteins from the Outer Membrane of Escherichia coli†
2004Co-Authors: Muthu Ramakrishnan, Cosmin L Pocanschi, Jörg H. Kleinschmidt, Derek MarshAbstract:ABSTRACT: The interaction of spin-labeled lipids with â-barrel transmembrane proteins has been studied by the electron spin resonance (ESR) methods developed for R-helical integral proteins. The outer membrane protein OmpA and the ferrichrome-iron receptor FhuA from the outer membrane of Escherichia coli were reconstituted in bilayers of Dimyristoylphosphatidylglycerol. The ESR spectra from phosphatidyl-glycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the â-barrel, in addition to that from the fluid bilayer lipids. The stoichiometry of motionally restricted lipids, 11 and 32 lipids/monomer for OmpA and FhuA, respectively, is constant irrespective of the total lipid/protein ratio. It is proportional to the number of transmembrane â-strands, eight for OmpA and 22 for FhuA, and correlates reasonably well with the intramembranous perimeter of the protein. Spin-labeled lipids with different polar headgroups display a differential selectivity of interaction with the two proteins. The more pronounced pattern of lipid selectivity for FhuA than for OmpA correlates with the preponderance of positively charged residues facing the lipids in the extensions of the â-sheet and shorter interconnecting loops on the extracellular side of FhuA. Because of the favorable time scale of electron spin resonance (ESR)1 spectroscopy, ESR spectra of spin-labele
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Non-linear electron paramagnetic resonance studies of the interaction of cytochrome c oxidase with spin-labelled lipids in gel-phase membranes. Biochemistry 39
2000Co-Authors: Jörg H. Kleinschmidt, Gary L. Powell, Derek MarshAbstract:ABSTRACT: The interaction of lipids, spin-labeled at different positions in the sn-2 chain, with cytochrome c oxidase reconstituted in gel-phase membranes of Dimyristoylphosphatidylglycerol has been studied by electron paramagnetic resonance (EPR) spectroscopy. Nonlinear EPR methods, both saturation transfer EPR and progressive saturation EPR, were used. Interaction with the protein largely removes the flexibility gradient of the lipid chains in gel-phase membranes. The rotational mobility of the chain segments is reduced, relative to that for gel-phase lipids, by the intramembranous interaction with cytochrome c oxidase. This holds for all positions of chain labeling, but the relative effect is greater for chain segments closer to the terminal methyl ends. Modification of the paramagnetic metal-ion centers in the protein by binding azide has a pronounced effect on the spin-lattice relaxation of the lipid spin labels. This demonstrates that the centers modified are sufficiently close to the first-shell lipids to give appreciable dipolar interactions and that their vertical location in the membrane is closer to the 5-position than to the 14-position of the lipid chains. Cytochrome c oxidase is the terminal member of the mitochondrial respiratory chain that effects the reduction of molecular oxygen to water by means of electrons donate
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lipid protein and protein protein interactions in double recombinants of myelin proteolipid apoprotein and myelin basic protein with Dimyristoylphosphatidylglycerol
Biochemistry, 1991Co-Authors: M B Sankaram, Peter J Brophy, Derek MarshAbstract:The integral proteolipid apoprotein (PLP) from bovine spinal cord has been reconstituted in Dimyristoylphosphatidylglycerol (DMPG) bilayers, and the mutual interactions on binding the peripheral myelin basic protein (MBP) have been studied. Quantitation of protein and lipid contents in the MBP-PLP-DMPG double recombinants at different PLP:DMPG ratios led to the conclusion that MBP binds only to the DMPG lipid headgroups and is hindered from interaction with the first shell of lipids surrounding the PLP. No specific PLP-MBP association could be detected. Electron spin resonance (ESR) spectra of phosphatidylglycerol spin-labeled at position n = 5 in the sn-2 chain showed that complexation of MBP with the PLP-DMPG recombinants leads to a decrease in lipid chain mobility to an extent which correlates with the degree of MBP binding. At low DMPG:PLP ratios, the perturbations of lipid mobility by both proteins are mutually enhanced. In single recombinants of PLP with DMPG, the ESR spectra of phosphatidylglycerol spin-labeled at position n = 14 in the sn-2 chain indicated that approximately 10 lipids/protein are motionally restricted by direct contact with the intramembranous surface of the protein. This number is in agreement with earlier results for reconstitutions of PLP in dimyristoylphosphatidylcholine (DMPC) [Brophy, P. J., Horvath, L. I., & Marsh, D. (1984) Biochemistry 23, 860-865] and is consistent with a hexameric arrangement of the PLP molecules in DMPG bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)
Michele Auger - One of the best experts on this subject based on the ideXlab platform.
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interaction between β purothionin and Dimyristoylphosphatidylglycerol a 31p nmr and infrared spectroscopic study
Biophysical Journal, 2002Co-Authors: Julieandree Richard, Isabelle Kelly, Didier Marion, Michel Pezolet, Michele AugerAbstract:Abstract The interaction of β -purothionin, a small basic and antimicrobial protein from the endosperm of wheat seeds, with multilamellar vesicles of Dimyristoylphosphatidylglycerol (DMPG) was investigated by 31 P solid-state NMR and infrared spectroscopy. NMR was used to study the organization and dynamics of DMPG in the absence and presence of β -purothionin. The results indicate that β -purothionin does not induce the formation of nonlamellar phases in DMPG. Two-dimensional exchange spectroscopy shows that β -purothionin decreases the lateral diffusion of DMPG in the fluid phase. Infrared spectroscopy was used to investigate the perturbations, induced by β -purothionin, of the polar and nonpolar regions of the phospholipid bilayers. At low concentration of β -purothionin, the temperature of the gel-to-fluid phase transition of DMPG increases from 24°C to ∼33°C, in agreement with the formation of electrostatic interactions between the cationic protein and the anionic phospholipid. At higher protein concentration, the lipid transition is slightly shifted toward lower temperature and a second transition is observed below 20°C, suggesting an insertion of the protein in the hydrophobic core of the lipid bilayer. The results also suggest that the presence of β -purothionin significantly modifies the lipid packing at the surface of the bilayer to increase the accessibility of water molecules in the interfacial region. Finally, orientation measurements indicate that the α -helices and the β -sheet of β -purothionin have tilt angles of ∼60° and 30°, respectively, relative to the normal of the ATR crystal.
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two dimensional infrared correlation spectroscopy study of the aggregation of cytochrome c in the presence of Dimyristoylphosphatidylglycerol
Biophysical Journal, 2001Co-Authors: Mariejosee Paquet, Michel Pezolet, Mario Laviolette, Michele AugerAbstract:Two-dimensional infrared correlation spectroscopy (2D-IR) was used in this study to investigate the aggregation of cytochrome c in the presence of Dimyristoylphosphatidylglycerol. The influence of temperature on the aggregation has been evaluated by monitoring the intensity of a band at 1616 cm(-1), which is characteristic of aggregated proteins, and the 2D-IR analysis has been used to determine the various secondary structure components of cytochrome c involved before and during its aggregation. The 2D-IR correlation analysis clearly reveals for the first time that aggregation starts to occur between nearly native proteins, which then unfold, yielding to further aggregation of the protein. Later in the aggregation process, the formation of intermolecular bonds and unfolding of the alpha-helices appear to be simultaneous. These results lead us to propose a two-step aggregation process. Finally, the results obtained during the heating period clearly indicate that before the protein starts to aggregate, there is a loosening of the tertiary structure of cytochrome c, resulting in a decrease of the beta-sheet content and an increase of the amount of beta-turns. This study clearly demonstrates the potential of 2D-IR spectroscopy to investigate the aggregation of proteins and this technique could therefore be applied to other proteins such as those involved in fibrilogenesis.
Auger Michèle - One of the best experts on this subject based on the ideXlab platform.
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Determining the Mode of Action Involved in the Antimicrobial Activity of Synthetic Peptides: A Solid-State NMR and FTIR Study
Biophysical Society. Published by Elsevier Inc., 2012Co-Authors: Lorin Aurélien, Noël Mathieu, Provencher Marie-Ève, Turcotte Vanessa, Cardinal Sébastien, Lagüe Patrick, Voyer Normand, Auger MichèleAbstract:AbstractWe have previously shown that leucine to lysine substitution(s) in neutral synthetic crown ether containing 14-mer peptide affect the peptide structure and its ability to permeabilize bilayers. Depending on the substitution position, the peptides adopt mainly either a α-helical structure able to permeabilize dimyristoylphosphatidylcholine (DMPC) and Dimyristoylphosphatidylglycerol (DMPG) vesicles (nonselective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). In this study, we have used a combination of solid-state NMR and Fourier transform infrared spectroscopy to investigate the effects of nonselective α-helical and selective intermolecular β-sheet peptides on both types of bilayers. 31P NMR results indicate that both types of peptides interact with the headgroups of DMPC and DMPG bilayers. 2H NMR and Fourier transform infrared results reveal an ordering of the hydrophobic core of bilayers when leakage is noted, i.e., for DMPG vesicles in the presence of both types of peptides and DMPC vesicles in the presence of nonselective peptides. However, selective peptides have no significant effect on the ordering of DMPC acyl chains. The ability of these 14-mer peptides to permeabilize lipid vesicles therefore appears to be related to their ability to increase the order of the bilayer hydrophobic core
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Insights on the Interactions of Synthetic Amphipathic Peptides with Model Membranes as Revealed by 31P and 2H Solid-State NMR and Infrared Spectroscopies
The Biophysical Society. Published by Elsevier Inc., 2006Co-Authors: Ouellet Marise, Voyer Normand, Bernard Geneviève, Auger MichèleAbstract:AbstractWe studied the interaction between synthetic amphipathic peptides and model membranes by solid-state NMR and infrared spectroscopies. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers were synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure. To shed light on their membrane interaction, 31P and 2H solid-state NMR experiments were performed on both peptides in interaction with dimyristoylphosphatidylcholine vesicles in the absence and presence of cholesterol, Dimyristoylphosphatidylglycerol vesicles, and oriented bicelles. 31P NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are weakly yet markedly affected by the presence of the peptides, whereas 31P NMR experiments on bicelles indicate no significant changes in the morphology and orientation of the bicelles. On the other hand, 2H NMR experiments on vesicles reveal that the acyl chain order is affected differently depending on the membrane lipidic composition and on the peptide hydrophobic length. Finally, infrared spectroscopy was used to study the interfacial region of the bilayer. Based on these studies, mechanisms of membrane perturbation are proposed for the 14-mer and 21-mer peptides in interaction with model membranes depending on the bilayer composition and peptide length
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Insights on the Interactions of Synthetic Amphipathic Peptides with Model Membranes as Revealed by (31)P and (2)H Solid-State NMR and Infrared Spectroscopies
Biophysical Society, 2006Co-Authors: Ouellet Marise, Voyer Normand, Bernard Geneviève, Auger MichèleAbstract:We studied the interaction between synthetic amphipathic peptides and model membranes by solid-state NMR and infrared spectroscopies. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers were synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure. To shed light on their membrane interaction, (31)P and (2)H solid-state NMR experiments were performed on both peptides in interaction with dimyristoylphosphatidylcholine vesicles in the absence and presence of cholesterol, Dimyristoylphosphatidylglycerol vesicles, and oriented bicelles. (31)P NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are weakly yet markedly affected by the presence of the peptides, whereas (31)P NMR experiments on bicelles indicate no significant changes in the morphology and orientation of the bicelles. On the other hand, (2)H NMR experiments on vesicles reveal that the acyl chain order is affected differently depending on the membrane lipidic composition and on the peptide hydrophobic length. Finally, infrared spectroscopy was used to study the interfacial region of the bilayer. Based on these studies, mechanisms of membrane perturbation are proposed for the 14-mer and 21-mer peptides in interaction with model membranes depending on the bilayer composition and peptide length
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Interaction between beta-Purothionin and Dimyristoylphosphatidylglycerol: a (31)P-NMR and infrared spectroscopic study.
2002Co-Authors: Richard Julie-andrée, Kelly Isabelle, Marion Didier, Pézolet Michel, Auger MichèleAbstract:The interaction of beta-purothionin, a small basic and antimicrobial protein from the endosperm of wheat seeds, with multilamellar vesicles of Dimyristoylphosphatidylglycerol (DMPG) was investigated by (31)P solid-state NMR and infrared spectroscopy. NMR was used to study the organization and dynamics of DMPG in the absence and presence of beta-purothionin. The results indicate that beta-purothionin does not induce the formation of nonlamellar phases in DMPG. Two-dimensional exchange spectroscopy shows that beta-purothionin decreases the lateral diffusion of DMPG in the fluid phase. Infrared spectroscopy was used to investigate the perturbations, induced by beta-purothionin, of the polar and nonpolar regions of the phospholipid bilayers. At low concentration of beta-purothionin, the temperature of the gel-to-fluid phase transition of DMPG increases from 24 degrees C to ~33 degrees C, in agreement with the formation of electrostatic interactions between the cationic protein and the anionic phospholipid. At higher protein concentration, the lipid transition is slightly shifted toward lower temperature and a second transition is observed below 20 degrees C, suggesting an insertion of the protein in the hydrophobic core of the lipid bilayer. The results also suggest that the presence of beta-purothionin significantly modifies the lipid packing at the surface of the bilayer to increase the accessibility of water molecules in the interfacial region. Finally, orientation measurements indicate that the alpha-helices and the beta-sheet of beta-purothionin have tilt angles of ~60 degrees and 30 degrees, respectively, relative to the normal of the ATR crystal
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Two-Dimensional Infrared Correlation Spectroscopy Study of the Aggregation of Cytochrome c in the Presence of Dimyristoylphosphatidylglycerol
The Biophysical Society. Published by Elsevier Inc., 2001Co-Authors: Paquet Marie-josée, Pézolet Michel, Laviolette Mario, Auger MichèleAbstract:AbstractTwo-dimensional infrared correlation spectroscopy (2D-IR) was used in this study to investigate the aggregation of cytochrome c in the presence of Dimyristoylphosphatidylglycerol. The influence of temperature on the aggregation has been evaluated by monitoring the intensity of a band at 1616cm−1, which is characteristic of aggregated proteins, and the 2D-IR analysis has been used to determine the various secondary structure components of cytochrome c involved before and during its aggregation. The 2D-IR correlation analysis clearly reveals for the first time that aggregation starts to occur between nearly native proteins, which then unfold, yielding to further aggregation of the protein. Later in the aggregation process, the formation of intermolecular bonds and unfolding of the α-helices appear to be simultaneous. These results lead us to propose a two-step aggregation process. Finally, the results obtained during the heating period clearly indicate that before the protein starts to aggregate, there is a loosening of the tertiary structure of cytochrome c, resulting in a decrease of the β-sheet content and an increase of the amount of β-turns. This study clearly demonstrates the potential of 2D-IR spectroscopy to investigate the aggregation of proteins and this technique could therefore be applied to other proteins such as those involved in fibrilogenesis
Michel Pezolet - One of the best experts on this subject based on the ideXlab platform.
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interaction between β purothionin and Dimyristoylphosphatidylglycerol a 31p nmr and infrared spectroscopic study
Biophysical Journal, 2002Co-Authors: Julieandree Richard, Isabelle Kelly, Didier Marion, Michel Pezolet, Michele AugerAbstract:Abstract The interaction of β -purothionin, a small basic and antimicrobial protein from the endosperm of wheat seeds, with multilamellar vesicles of Dimyristoylphosphatidylglycerol (DMPG) was investigated by 31 P solid-state NMR and infrared spectroscopy. NMR was used to study the organization and dynamics of DMPG in the absence and presence of β -purothionin. The results indicate that β -purothionin does not induce the formation of nonlamellar phases in DMPG. Two-dimensional exchange spectroscopy shows that β -purothionin decreases the lateral diffusion of DMPG in the fluid phase. Infrared spectroscopy was used to investigate the perturbations, induced by β -purothionin, of the polar and nonpolar regions of the phospholipid bilayers. At low concentration of β -purothionin, the temperature of the gel-to-fluid phase transition of DMPG increases from 24°C to ∼33°C, in agreement with the formation of electrostatic interactions between the cationic protein and the anionic phospholipid. At higher protein concentration, the lipid transition is slightly shifted toward lower temperature and a second transition is observed below 20°C, suggesting an insertion of the protein in the hydrophobic core of the lipid bilayer. The results also suggest that the presence of β -purothionin significantly modifies the lipid packing at the surface of the bilayer to increase the accessibility of water molecules in the interfacial region. Finally, orientation measurements indicate that the α -helices and the β -sheet of β -purothionin have tilt angles of ∼60° and 30°, respectively, relative to the normal of the ATR crystal.
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two dimensional infrared correlation spectroscopy study of the aggregation of cytochrome c in the presence of Dimyristoylphosphatidylglycerol
Biophysical Journal, 2001Co-Authors: Mariejosee Paquet, Michel Pezolet, Mario Laviolette, Michele AugerAbstract:Two-dimensional infrared correlation spectroscopy (2D-IR) was used in this study to investigate the aggregation of cytochrome c in the presence of Dimyristoylphosphatidylglycerol. The influence of temperature on the aggregation has been evaluated by monitoring the intensity of a band at 1616 cm(-1), which is characteristic of aggregated proteins, and the 2D-IR analysis has been used to determine the various secondary structure components of cytochrome c involved before and during its aggregation. The 2D-IR correlation analysis clearly reveals for the first time that aggregation starts to occur between nearly native proteins, which then unfold, yielding to further aggregation of the protein. Later in the aggregation process, the formation of intermolecular bonds and unfolding of the alpha-helices appear to be simultaneous. These results lead us to propose a two-step aggregation process. Finally, the results obtained during the heating period clearly indicate that before the protein starts to aggregate, there is a loosening of the tertiary structure of cytochrome c, resulting in a decrease of the beta-sheet content and an increase of the amount of beta-turns. This study clearly demonstrates the potential of 2D-IR spectroscopy to investigate the aggregation of proteins and this technique could therefore be applied to other proteins such as those involved in fibrilogenesis.
Frances Separovic - One of the best experts on this subject based on the ideXlab platform.
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Structural effects of the antimicrobial peptide maculatin 1.1 on supported lipid bilayers
European Biophysics Journal, 2013Co-Authors: David I. Fernandez, Anton P. Brun, Tzong-hsien Lee, Paramjit Bansal, Marie-isabel Aguilar, Michael James, Frances SeparovicAbstract:The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH_2) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC–Dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC–DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides.