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Valerie Belle - One of the best experts on this subject based on the ideXlab platform.
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Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins
Journal of Physical Chemistry Letters, 2017Co-Authors: Christoph Gmeiner, Elisabetta Mileo, Sylvain R. A. Marque, Georg Dorn, Frédéric H.-t. Allain, Bruno Guigliarelli, Valerie Belle, Daniel Klose, Gunnar Jeschke, Maxim YulikovAbstract:Site-Directed Spin Labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional Spin Labeling of cysteine residues. Double electron–electron resonance (DEER) EPR measurements were performed for both the nitroxide–nitroxide and Gd(III)–nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial Spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.
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Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins
Journal of Physical Chemistry Letters, 2017Co-Authors: Christoph Gmeiner, Elisabetta Mileo, Sylvain R. A. Marque, Georg Dorn, Frédéric H.-t. Allain, Bruno Guigliarelli, Valerie Belle, Daniel Klose, Gunnar Jeschke, Maxim YulikovAbstract:Abstract Image Site-Directed Spin Labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional Spin Labeling of cysteine residues. Double electron–electron resonance (DEER) EPR measurements were performed for both the nitroxide–nitroxide and Gd(III)–nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial Spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.
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exploring intrinsically disordered proteins using site directed Spin Labeling electron paramagnetic resonance spectroscopy
Frontiers in molecular biosciences, 2015Co-Authors: Nolwenn Le Breton, Marlène Martinho, Elisabetta Mileo, Emilien Etienne, Bruno Guigliarelli, Guillaume Gerbaud, Valerie BelleAbstract:Proteins are highly variable biological systems, not only in their structures but also in their dynamics. The most extreme example of dynamics is encountered within the family of Intrinsically Disordered Proteins (IDPs), which are proteins lacking a well-defined 3D structure under physiological conditions. Among the biophysical techniques well-suited to study such highly flexible proteins, Site Directed Spin Labelling combined with EPR spectroscopy (SDSL-EPR) is one of the most powerful, being able to reveal, at the residue level, structural transitions such as folding events. SDSL-EPR is based on selective grafting of a paramagnetic label on the protein under study and is limited neither by the size nor by the complexity of the system. The objective of this mini-review is to describe the basic strategy of SDSL-EPR and to illustrate how it can be successfully applied to characterize the structural behaviour of IDPs. Recent developments aimed at enlarging the panoply of SDSL-EPR approaches are presented in particular newly synthesized Spin labels that allow the limitations of the classical ones to be overcome. The potentialities of these new Spin labels will be demonstrated on different examples of IDPs.
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Enlarging the Panoply of Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR): Sensitive and Selective Spin-Labeling of Tyrosine Using an Isoindoline-Based Nitroxide
Bioconjugate Chemistry, 2013Co-Authors: Elisabetta Mileo, Régine Lebrun, Valérie Roubaud, Marlène Martinho, Paul Tordo, Sylvain R. A. Marque, Brigitte Gontero, Emilien Etienne, Bruno Guigliarelli, Valerie BelleAbstract:Site-Directed Spin Labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical Spin labels are functionalized to be grafted on natural or Site-Directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable ( when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 with its partner protein, we showed that the isoindoline-based nitroxide is a good reporter to reveal changes in its local environment contrary to the previous study where the label was poorly sensitive to probe structural changes. The successful targeting of tyrosine residues with the isoindoline-based riitroxide thus offers a highly promising approach, complementary to the classical cysteine-SDSL one, which significantly enlarges the field of applications of the technique for probing protein dynamics.
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Enlarging the Panoply of Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR): Sensitive and Selective Spin-Labeling of Tyrosine Using an Isoindoline-Based Nitroxide
Bioconjugate Chemistry, 2013Co-Authors: Elisabetta Mileo, Régine Lebrun, Valérie Roubaud, Marlène Martinho, Paul Tordo, Sylvain R. A. Marque, Brigitte Gontero, Emilien Etienne, Bruno Guigliarelli, Valerie BelleAbstract:Site-Directed Spin Labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical Spin labels are functionalized to be grafted on natural or Site-Directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 wi...
Bruno Guigliarelli - One of the best experts on this subject based on the ideXlab platform.
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Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins
Journal of Physical Chemistry Letters, 2017Co-Authors: Christoph Gmeiner, Elisabetta Mileo, Sylvain R. A. Marque, Georg Dorn, Frédéric H.-t. Allain, Bruno Guigliarelli, Valerie Belle, Daniel Klose, Gunnar Jeschke, Maxim YulikovAbstract:Site-Directed Spin Labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional Spin Labeling of cysteine residues. Double electron–electron resonance (DEER) EPR measurements were performed for both the nitroxide–nitroxide and Gd(III)–nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial Spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.
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Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins
Journal of Physical Chemistry Letters, 2017Co-Authors: Christoph Gmeiner, Elisabetta Mileo, Sylvain R. A. Marque, Georg Dorn, Frédéric H.-t. Allain, Bruno Guigliarelli, Valerie Belle, Daniel Klose, Gunnar Jeschke, Maxim YulikovAbstract:Abstract Image Site-Directed Spin Labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional Spin Labeling of cysteine residues. Double electron–electron resonance (DEER) EPR measurements were performed for both the nitroxide–nitroxide and Gd(III)–nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial Spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.
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exploring intrinsically disordered proteins using site directed Spin Labeling electron paramagnetic resonance spectroscopy
Frontiers in molecular biosciences, 2015Co-Authors: Nolwenn Le Breton, Marlène Martinho, Elisabetta Mileo, Emilien Etienne, Bruno Guigliarelli, Guillaume Gerbaud, Valerie BelleAbstract:Proteins are highly variable biological systems, not only in their structures but also in their dynamics. The most extreme example of dynamics is encountered within the family of Intrinsically Disordered Proteins (IDPs), which are proteins lacking a well-defined 3D structure under physiological conditions. Among the biophysical techniques well-suited to study such highly flexible proteins, Site Directed Spin Labelling combined with EPR spectroscopy (SDSL-EPR) is one of the most powerful, being able to reveal, at the residue level, structural transitions such as folding events. SDSL-EPR is based on selective grafting of a paramagnetic label on the protein under study and is limited neither by the size nor by the complexity of the system. The objective of this mini-review is to describe the basic strategy of SDSL-EPR and to illustrate how it can be successfully applied to characterize the structural behaviour of IDPs. Recent developments aimed at enlarging the panoply of SDSL-EPR approaches are presented in particular newly synthesized Spin labels that allow the limitations of the classical ones to be overcome. The potentialities of these new Spin labels will be demonstrated on different examples of IDPs.
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Enlarging the Panoply of Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR): Sensitive and Selective Spin-Labeling of Tyrosine Using an Isoindoline-Based Nitroxide
Bioconjugate Chemistry, 2013Co-Authors: Elisabetta Mileo, Régine Lebrun, Valérie Roubaud, Marlène Martinho, Paul Tordo, Sylvain R. A. Marque, Brigitte Gontero, Emilien Etienne, Bruno Guigliarelli, Valerie BelleAbstract:Site-Directed Spin Labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical Spin labels are functionalized to be grafted on natural or Site-Directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable ( when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 with its partner protein, we showed that the isoindoline-based nitroxide is a good reporter to reveal changes in its local environment contrary to the previous study where the label was poorly sensitive to probe structural changes. The successful targeting of tyrosine residues with the isoindoline-based riitroxide thus offers a highly promising approach, complementary to the classical cysteine-SDSL one, which significantly enlarges the field of applications of the technique for probing protein dynamics.
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Enlarging the Panoply of Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR): Sensitive and Selective Spin-Labeling of Tyrosine Using an Isoindoline-Based Nitroxide
Bioconjugate Chemistry, 2013Co-Authors: Elisabetta Mileo, Régine Lebrun, Valérie Roubaud, Marlène Martinho, Paul Tordo, Sylvain R. A. Marque, Brigitte Gontero, Emilien Etienne, Bruno Guigliarelli, Valerie BelleAbstract:Site-Directed Spin Labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical Spin labels are functionalized to be grafted on natural or Site-Directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 wi...
Elisabetta Mileo - One of the best experts on this subject based on the ideXlab platform.
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Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins
Journal of Physical Chemistry Letters, 2017Co-Authors: Christoph Gmeiner, Elisabetta Mileo, Sylvain R. A. Marque, Georg Dorn, Frédéric H.-t. Allain, Bruno Guigliarelli, Valerie Belle, Daniel Klose, Gunnar Jeschke, Maxim YulikovAbstract:Site-Directed Spin Labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional Spin Labeling of cysteine residues. Double electron–electron resonance (DEER) EPR measurements were performed for both the nitroxide–nitroxide and Gd(III)–nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial Spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.
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Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins
Journal of Physical Chemistry Letters, 2017Co-Authors: Christoph Gmeiner, Elisabetta Mileo, Sylvain R. A. Marque, Georg Dorn, Frédéric H.-t. Allain, Bruno Guigliarelli, Valerie Belle, Daniel Klose, Gunnar Jeschke, Maxim YulikovAbstract:Abstract Image Site-Directed Spin Labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional Spin Labeling of cysteine residues. Double electron–electron resonance (DEER) EPR measurements were performed for both the nitroxide–nitroxide and Gd(III)–nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial Spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.
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exploring intrinsically disordered proteins using site directed Spin Labeling electron paramagnetic resonance spectroscopy
Frontiers in molecular biosciences, 2015Co-Authors: Nolwenn Le Breton, Marlène Martinho, Elisabetta Mileo, Emilien Etienne, Bruno Guigliarelli, Guillaume Gerbaud, Valerie BelleAbstract:Proteins are highly variable biological systems, not only in their structures but also in their dynamics. The most extreme example of dynamics is encountered within the family of Intrinsically Disordered Proteins (IDPs), which are proteins lacking a well-defined 3D structure under physiological conditions. Among the biophysical techniques well-suited to study such highly flexible proteins, Site Directed Spin Labelling combined with EPR spectroscopy (SDSL-EPR) is one of the most powerful, being able to reveal, at the residue level, structural transitions such as folding events. SDSL-EPR is based on selective grafting of a paramagnetic label on the protein under study and is limited neither by the size nor by the complexity of the system. The objective of this mini-review is to describe the basic strategy of SDSL-EPR and to illustrate how it can be successfully applied to characterize the structural behaviour of IDPs. Recent developments aimed at enlarging the panoply of SDSL-EPR approaches are presented in particular newly synthesized Spin labels that allow the limitations of the classical ones to be overcome. The potentialities of these new Spin labels will be demonstrated on different examples of IDPs.
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Enlarging the Panoply of Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR): Sensitive and Selective Spin-Labeling of Tyrosine Using an Isoindoline-Based Nitroxide
Bioconjugate Chemistry, 2013Co-Authors: Elisabetta Mileo, Régine Lebrun, Valérie Roubaud, Marlène Martinho, Paul Tordo, Sylvain R. A. Marque, Brigitte Gontero, Emilien Etienne, Bruno Guigliarelli, Valerie BelleAbstract:Site-Directed Spin Labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical Spin labels are functionalized to be grafted on natural or Site-Directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable ( when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 with its partner protein, we showed that the isoindoline-based nitroxide is a good reporter to reveal changes in its local environment contrary to the previous study where the label was poorly sensitive to probe structural changes. The successful targeting of tyrosine residues with the isoindoline-based riitroxide thus offers a highly promising approach, complementary to the classical cysteine-SDSL one, which significantly enlarges the field of applications of the technique for probing protein dynamics.
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Enlarging the Panoply of Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR): Sensitive and Selective Spin-Labeling of Tyrosine Using an Isoindoline-Based Nitroxide
Bioconjugate Chemistry, 2013Co-Authors: Elisabetta Mileo, Régine Lebrun, Valérie Roubaud, Marlène Martinho, Paul Tordo, Sylvain R. A. Marque, Brigitte Gontero, Emilien Etienne, Bruno Guigliarelli, Valerie BelleAbstract:Site-Directed Spin Labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical Spin labels are functionalized to be grafted on natural or Site-Directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 wi...
Marlène Martinho - One of the best experts on this subject based on the ideXlab platform.
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exploring intrinsically disordered proteins using site directed Spin Labeling electron paramagnetic resonance spectroscopy
Frontiers in molecular biosciences, 2015Co-Authors: Nolwenn Le Breton, Marlène Martinho, Elisabetta Mileo, Emilien Etienne, Bruno Guigliarelli, Guillaume Gerbaud, Valerie BelleAbstract:Proteins are highly variable biological systems, not only in their structures but also in their dynamics. The most extreme example of dynamics is encountered within the family of Intrinsically Disordered Proteins (IDPs), which are proteins lacking a well-defined 3D structure under physiological conditions. Among the biophysical techniques well-suited to study such highly flexible proteins, Site Directed Spin Labelling combined with EPR spectroscopy (SDSL-EPR) is one of the most powerful, being able to reveal, at the residue level, structural transitions such as folding events. SDSL-EPR is based on selective grafting of a paramagnetic label on the protein under study and is limited neither by the size nor by the complexity of the system. The objective of this mini-review is to describe the basic strategy of SDSL-EPR and to illustrate how it can be successfully applied to characterize the structural behaviour of IDPs. Recent developments aimed at enlarging the panoply of SDSL-EPR approaches are presented in particular newly synthesized Spin labels that allow the limitations of the classical ones to be overcome. The potentialities of these new Spin labels will be demonstrated on different examples of IDPs.
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Enlarging the Panoply of Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR): Sensitive and Selective Spin-Labeling of Tyrosine Using an Isoindoline-Based Nitroxide
Bioconjugate Chemistry, 2013Co-Authors: Elisabetta Mileo, Régine Lebrun, Valérie Roubaud, Marlène Martinho, Paul Tordo, Sylvain R. A. Marque, Brigitte Gontero, Emilien Etienne, Bruno Guigliarelli, Valerie BelleAbstract:Site-Directed Spin Labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical Spin labels are functionalized to be grafted on natural or Site-Directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable ( when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 with its partner protein, we showed that the isoindoline-based nitroxide is a good reporter to reveal changes in its local environment contrary to the previous study where the label was poorly sensitive to probe structural changes. The successful targeting of tyrosine residues with the isoindoline-based riitroxide thus offers a highly promising approach, complementary to the classical cysteine-SDSL one, which significantly enlarges the field of applications of the technique for probing protein dynamics.
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Enlarging the Panoply of Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR): Sensitive and Selective Spin-Labeling of Tyrosine Using an Isoindoline-Based Nitroxide
Bioconjugate Chemistry, 2013Co-Authors: Elisabetta Mileo, Régine Lebrun, Valérie Roubaud, Marlène Martinho, Paul Tordo, Sylvain R. A. Marque, Brigitte Gontero, Emilien Etienne, Bruno Guigliarelli, Valerie BelleAbstract:Site-Directed Spin Labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical Spin labels are functionalized to be grafted on natural or Site-Directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 wi...
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Monitoring structural transitions in IDPs by Site-Directed Spin Labeling EPR spectroscopy.
Methods of Molecular Biology, 2012Co-Authors: Johnny Habchi, Marlène Martinho, Bruno Guigliarelli, Antoine Gruet, Sonia Longhi, Valerie BelleAbstract:Electron paramagnetic resonance (EPR) spectroscopy is a technique that specifically detects unpaired electrons. EPR sensitive reporter groups (Spin labels or Spin probes) can be introduced into biological systems via Site-Directed Spin Labeling (SDSL). This is usually accomplished by cysteine-substitution mutagenesis followed by covalent modification of the unique sulfhydryl group with a selective nitroxide reagent. SDSL EPR spectroscopy has been shown to be a sensitive and powerful method to study structural transitions within intrinsically disordered proteins (IDPs). In this chapter, we provide a detailed experimental protocol for this approach and present a few examples of EPR spectral shapes illustrative of various mobility regimes of the Spin probe, reflecting different protein topologies.
David S Cafiso - One of the best experts on this subject based on the ideXlab platform.
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Peptide-membrane interactions determined using Site-Directed Spin Labeling
Current Topics in Membranes, 2020Co-Authors: David S CafisoAbstract:Abstract The study of peptide-membrane interactions is important for understanding a wide range of fundamental molecular mechanisms, including the action of antibiotic peptides, the association of proteins involved in cell signaling, and membrane fusion. This review discusses the use of Site-Directed Spin Labeling to investigate peptide-membrane interactions. Site-Directed Spin Labeling involves the incorporation of a Spin label in a site-specific fashion into a macromolecule, such as a protein or peptide, either through synthesis or Site-Directed mutagenesis. Electron paramagnetic resonance (EPR) spectroscopy is then used to determine molecular structure and dynamics. A number of examples discussed in this review demonstrate how this methodology can be used to determine the conformation of membrane-associated peptides, their position along the bilayer normal, and their state of aggregation. Recent studies have shown that this approach is not highly perturbing, and with new advances in EPR resonator design, it is a highly sensitive technique.
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Identifying and quantitating conformational exchange in membrane proteins using Site-Directed Spin Labeling.
Accounts of Chemical Research, 2014Co-Authors: David S CafisoAbstract:ConspectusProtein structures are not static but sample different conformations over a range of amplitudes and time scales. These fluctuations may involve relatively small changes in bond angles or quite large rearrangements in secondary structure and tertiary fold. The equilibrium between discrete structural substates on the microsecond to millisecond time scale is sometimes termed conformational exchange. Protein dynamics and conformational exchange are believed to provide the basis for many important activities, such as protein–protein and protein–ligand interactions, enzymatic activity and protein allostery; however, for many proteins, the dynamics and conformational exchange that lead to function are poorly defined.Spectroscopic methods, such as NMR, are among the most important methods to explore protein dynamics and conformational exchange; however, they are difficult to implement in some systems and with some types of exchange events. Site-Directed Spin Labeling (SDSL) is an EPR based approach that...
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Probing the conformation of the resting state of a bacterial multidrug ABC transporter, BmrA, by a Site-Directed Spin Labeling approach.
Protein Science, 2009Co-Authors: Serge Crouzy, David S Cafiso, Michel Becchi, Attilio Di Pietro, Jean-michel JaultAbstract:Previously published 3-D structures of a prototypic ATP-binding cassette (ABC) transporter, MsbA, have been recently corrected revealing large rigid-body motions possibly linked to its catalytic cycle. Here, a closely related multidrug bacterial ABC transporter, BmrA, was studied using Site-Directed Spin Labeling by focusing on a region connecting the transmembrane domain and the nucleotide-binding domain (NBD). Electron paramagnetic resonance (EPR) spectra of single Spin-labeled cysteine mutants suggests that, in the resting state, this sub-domain essentially adopts a partially extended conformation, which is consistent with the crystal structures of MsbA and Sav1866. Interestingly, one of the single point mutants (Q333C) yielded an immobilized EPR spectrum that could arise from a direct interaction with a vicinal tyrosine residue. Inspection of different BmrA models pointed to Y408, within the NBD, as the putative interacting partner, and its mutation to a Phe residue indeed dramatically modified the EPR spectra of the Spin labeled Q333C. Moreover, unlike the Y408F mutation, the Y408A mutation abolished both ATPase activity and drug transport of BmrA, suggesting that a nonpolar bulky residue is required at this position. The spatial proximity of Q333 and Y408 was also confirmed by formation of a disulfide bond when both Q333 and T407 (or S409) were replaced jointly by a cysteine residue. Overall, these results indicate that the two regions surrounding Q333 and Y408 are close together in the 3-D structure of BmrA and that residues within these two sub-domains are essential for proper functioning of this transporter.
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Conformation and Membrane Position of the Region Linking the two C2 Domains in Synaptotagmin 1 by Site-Directed Spin Labeling
Biochemistry, 2008Co-Authors: Hao Huang, David S CafisoAbstract:Synaptotagmin 1 (syt1) is an integral membrane protein localized on the synaptic vesicle that acts as the Ca2+ sensor for neuronal exocytosis. Synaptotagmin 1 contains two C2 domains, C2A and C2B, which bind Ca2+ ions, membranes, and SNAREs. Here, Site-Directed Spin Labeling (SDSL) was used to determine the position and dynamics of the region that links the two C2 domains in a water soluble construct encompassing the two C2 domains (syt1C2AB). An analysis of the EPR line shapes from this region indicates that the linker is flexible and unstructured when syt1 is in solution or bound to lipid bilayers. The nanosecond dynamics of the linker does not change, in the presence or absence of Ca2+, suggesting that there is no Ca2+-dependent intramolecular association between the two domains. When syt1C2AB is membrane-bound, the position of the linker relative to the membrane interface was determined by measuring parameters for the collision of the Spin-labeled syt1C2AB mutants with both soluble and membrane-bound ...
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Recent advances and applications of Site-Directed Spin Labeling
Current Opinion in Structural Biology, 2006Co-Authors: Gail E. Fanucci, David S CafisoAbstract:Site-Directed Spin Labeling has become a popular biophysical tool for the characterization of protein structure, dynamics and conformational change. This method is well suited and widely used to study small soluble proteins, membrane proteins and large protein complexes. Recent advances in Site-Directed Spin Labeling methodology have occurred in two areas. The first involves an understanding of the conformations and local dynamics of the Spin-labeled sidechain, including the features of proteins that influence electron paramagnetic resonance lineshape. The second advance is the application of pulse techniques to determine long-range distances and distance distributions in proteins. During the past two years, these technical developments have been used to address several important problems concerning the molecular function of proteins.