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Salvador Casares - One of the best experts on this subject based on the ideXlab platform.
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The effect of a Proline Residue on the rate of growth and the space group of α-spectrin SH3-domain crystals
Acta Crystallographica Section D Biological Crystallography, 2009Co-Authors: Ana Cámara-artigas, Monserrat Andújar-sánchez, Emilia Ortiz-salmerón, Celia Cuadri, Salvador CasaresAbstract:α-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a Proline Residue that participates in a crystal contact mimicking the binding of Proline-rich sequences to SH3 domains. This Residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the Residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical Proline Residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P41212, with unit-cell parameters a = b = 42.231, c = 93.655 A, and that diffracts to 1.45 A resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2 M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 Residue plays a key role in the rate of crystal growth.
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The effect of a Proline Residue on the rate of growth and the space group of alpha-spectrin SH3-domain crystals.
Acta crystallographica. Section D Biological crystallography, 2009Co-Authors: Ana Cámara-artigas, Monserrat Andújar-sánchez, Emilia Ortiz-salmerón, Celia Cuadri, Salvador CasaresAbstract:alpha-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a Proline Residue that participates in a crystal contact mimicking the binding of Proline-rich sequences to SH3 domains. This Residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the Residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical Proline Residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 42.231, c = 93.655 A, and that diffracts to 1.45 A resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2 M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 Residue plays a key role in the rate of crystal growth.
Stefan Gründer - One of the best experts on this subject based on the ideXlab platform.
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A single conserved Proline Residue determines the membrane topology of stomatin.
The Biochemical journal, 2009Co-Authors: Ivan Kadurin, Stephan M. Huber, Stefan GründerAbstract:Stomatin is an integral membrane protein which is widely expressed in many cell types. It is accepted that stomatin has a unique hairpin-loop topology: it is anchored to the membrane with an N-terminal hydrophobic domain and the N- and C-termini are cytoplasmically localized. Stomatin is a prototype for a family of related proteins, containing among others MEC-2 (mechanosensory protein 2) from Caenorhabditis elegans, SLP (stomatin-like protein)-3 and podocin, all of which interact with ion channels to regulate their activity. Members of the stomatin family partly localize in DRMs (detergent-resistant membrane domains) enriched in cholesterol and sphingolipids. It has been proposed that a highly conserved Proline Residue in the middle of the hydrophobic domain directly binds cholesterol and that cholesterol binding is necessary for the regulation of ion channels. In the present study we show that a small part of the stomatin pool exists as a single-pass transmembrane protein rather than a hairpin-loop protein. The highly conserved Proline Residue is crucial for adopting the hairpin-loop topology: substitution of this Proline Residue by serine transfers the whole stomatin pool to the single-pass transmembrane form, which no longer localizes to DRMs. These results suggest that formation of the hairpin loop is inefficient and that the conserved Proline Residue is indispensable for formation of the hairpin loop. The single-pass transmembrane form exists also for SLP-3 and it should be considered that it mediates part of the physiological functions of stomatin and related proteins.
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A single conserved Proline Residue determines the membrane topology of stomatin
Biochemical Journal, 2009Co-Authors: Ivan Kadurin, Stephan Huber, Stefan GründerAbstract:Stomatin is an integral membrane protein which is widely expressed in many cell types. It is accepted that stomatin has a unique hairpin loop topology: it is anchored to the membrane with an N-terminal domain and N- and C-termini are cytoplasmically localized. Stomatin is a prototype for a family of related proteins, containing among others MEC-2 from C. elegans, stomatin-like protein (SLP) 3, and podocin, all of which interact with ion channels to regulate their activity. Members of the stomatin family partly localize in detergent resistant membrane domains (DRMs) enriched in cholesterol and sphingolipids. It has been proposed that a highly conserved Proline Residue in the middle of the hydrophobic domain directly binds cholesterol and that cholesterol binding is necessary for the regulation of ion channels. Here we show that a small part of the stomatin pool exists as a single pass transmembrane protein rather than a hairpin loop protein. The highly conserved Proline is crucial for adopting the hairpin loop topology: substitution of this Proline by serine transfers the whole stomatin pool to the single pass transmembrane form, which no longer localizes to DRMs. These results suggest that formation of the hairpin loop is inefficient and that the conserved Proline is indispensable for formation of the hairpin loop. The single pass transmembrane form exists also for SLP-3 and it should be considered that it mediates part of the physiological functions of stomatin and related proteins.
Edwin De Pauw - One of the best experts on this subject based on the ideXlab platform.
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Influences of Proline and Cysteine Residues on Fragment Yield in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mass Spectrometry
Journal of The American Society for Mass Spectrometry, 2014Co-Authors: Daiki Asakawa, Nicolas Smargiasso, Loic Quinton, Edwin De PauwAbstract:Matrix-assisted laser desorption/ionization in-source decay produces highly informative fragments for the sequencing of peptides/proteins. Among amino acids, cysteine and Proline Residues were found to specifically influence the fragment yield. As they are both frequently found in small peptide structures for which de novo sequencing is mandatory, the understanding of their specific behaviors would allow useful fragmentation rules to be established. In the case of cysteine, a c •/ w fragment pair originating from Xxx–Cys is formed by side-chain loss from the cysteine Residue. The presence of a Proline Residue contributes to an increased yield of ISD fragments originating from N–C_α bond cleavage at Xxx_1–Xxx_2Pro, which is attributable to the cyclic structure of the Proline Residue. Our results suggest that the aminoketyl radical formed by MALDI-ISD generally induces the homolytic N–C_α bond cleavage located on the C–terminal side of the radical site. In contrast, N–C_α bond cleavage at Xxx–Pro produces no fragments and the N–C_α bond at the Xxx_1–Xxx_2Pro bond is alternatively cleaved via a heterolytic cleavage pathway.
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influences of Proline and cysteine Residues on fragment yield in matrix assisted laser desorption ionization in source decay mass spectrometry
Journal of the American Society for Mass Spectrometry, 2014Co-Authors: Daiki Asakawa, Nicolas Smargiasso, Loic Quinton, Edwin De PauwAbstract:Matrix-assisted laser desorption/ionization in-source decay produces highly informative fragments for the sequencing of peptides/proteins. Among amino acids, cysteine and Proline Residues were found to specifically influence the fragment yield. As they are both frequently found in small peptide structures for which de novo sequencing is mandatory, the understanding of their specific behaviors would allow useful fragmentation rules to be established. In the case of cysteine, a c•/w fragment pair originating from Xxx–Cys is formed by side-chain loss from the cysteine Residue. The presence of a Proline Residue contributes to an increased yield of ISD fragments originating from N–Cα bond cleavage at Xxx1–Xxx2Pro, which is attributable to the cyclic structure of the Proline Residue. Our results suggest that the aminoketyl radical formed by MALDI-ISD generally induces the homolytic N–Cα bond cleavage located on the C–terminal side of the radical site. In contrast, N–Cα bond cleavage at Xxx–Pro produces no fragments and the N–Cα bond at the Xxx1–Xxx2Pro bond is alternatively cleaved via a heterolytic cleavage pathway.
Ana Cámara-artigas - One of the best experts on this subject based on the ideXlab platform.
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The effect of a Proline Residue on the rate of growth and the space group of α-spectrin SH3-domain crystals
Acta Crystallographica Section D Biological Crystallography, 2009Co-Authors: Ana Cámara-artigas, Monserrat Andújar-sánchez, Emilia Ortiz-salmerón, Celia Cuadri, Salvador CasaresAbstract:α-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a Proline Residue that participates in a crystal contact mimicking the binding of Proline-rich sequences to SH3 domains. This Residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the Residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical Proline Residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P41212, with unit-cell parameters a = b = 42.231, c = 93.655 A, and that diffracts to 1.45 A resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2 M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 Residue plays a key role in the rate of crystal growth.
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The effect of a Proline Residue on the rate of growth and the space group of alpha-spectrin SH3-domain crystals.
Acta crystallographica. Section D Biological crystallography, 2009Co-Authors: Ana Cámara-artigas, Monserrat Andújar-sánchez, Emilia Ortiz-salmerón, Celia Cuadri, Salvador CasaresAbstract:alpha-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a Proline Residue that participates in a crystal contact mimicking the binding of Proline-rich sequences to SH3 domains. This Residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the Residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical Proline Residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 42.231, c = 93.655 A, and that diffracts to 1.45 A resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2 M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 Residue plays a key role in the rate of crystal growth.
Ivan Kadurin - One of the best experts on this subject based on the ideXlab platform.
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A single conserved Proline Residue determines the membrane topology of stomatin.
The Biochemical journal, 2009Co-Authors: Ivan Kadurin, Stephan M. Huber, Stefan GründerAbstract:Stomatin is an integral membrane protein which is widely expressed in many cell types. It is accepted that stomatin has a unique hairpin-loop topology: it is anchored to the membrane with an N-terminal hydrophobic domain and the N- and C-termini are cytoplasmically localized. Stomatin is a prototype for a family of related proteins, containing among others MEC-2 (mechanosensory protein 2) from Caenorhabditis elegans, SLP (stomatin-like protein)-3 and podocin, all of which interact with ion channels to regulate their activity. Members of the stomatin family partly localize in DRMs (detergent-resistant membrane domains) enriched in cholesterol and sphingolipids. It has been proposed that a highly conserved Proline Residue in the middle of the hydrophobic domain directly binds cholesterol and that cholesterol binding is necessary for the regulation of ion channels. In the present study we show that a small part of the stomatin pool exists as a single-pass transmembrane protein rather than a hairpin-loop protein. The highly conserved Proline Residue is crucial for adopting the hairpin-loop topology: substitution of this Proline Residue by serine transfers the whole stomatin pool to the single-pass transmembrane form, which no longer localizes to DRMs. These results suggest that formation of the hairpin loop is inefficient and that the conserved Proline Residue is indispensable for formation of the hairpin loop. The single-pass transmembrane form exists also for SLP-3 and it should be considered that it mediates part of the physiological functions of stomatin and related proteins.
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A single conserved Proline Residue determines the membrane topology of stomatin
Biochemical Journal, 2009Co-Authors: Ivan Kadurin, Stephan Huber, Stefan GründerAbstract:Stomatin is an integral membrane protein which is widely expressed in many cell types. It is accepted that stomatin has a unique hairpin loop topology: it is anchored to the membrane with an N-terminal domain and N- and C-termini are cytoplasmically localized. Stomatin is a prototype for a family of related proteins, containing among others MEC-2 from C. elegans, stomatin-like protein (SLP) 3, and podocin, all of which interact with ion channels to regulate their activity. Members of the stomatin family partly localize in detergent resistant membrane domains (DRMs) enriched in cholesterol and sphingolipids. It has been proposed that a highly conserved Proline Residue in the middle of the hydrophobic domain directly binds cholesterol and that cholesterol binding is necessary for the regulation of ion channels. Here we show that a small part of the stomatin pool exists as a single pass transmembrane protein rather than a hairpin loop protein. The highly conserved Proline is crucial for adopting the hairpin loop topology: substitution of this Proline by serine transfers the whole stomatin pool to the single pass transmembrane form, which no longer localizes to DRMs. These results suggest that formation of the hairpin loop is inefficient and that the conserved Proline is indispensable for formation of the hairpin loop. The single pass transmembrane form exists also for SLP-3 and it should be considered that it mediates part of the physiological functions of stomatin and related proteins.
