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Maurice Hofnung - One of the best experts on this subject based on the ideXlab platform.
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Degradation versus aggregation of misfolded Maltose-Binding protein in the periplasm of Escherichia coli.
The Journal of biological chemistry, 1998Co-Authors: Jean Michel Betton, Nathalie Sassoon, Maurice Hofnung, Michel LaurentAbstract:The periplasmic fates of misfolded MalE31, a defective folding mutant of the Maltose-Binding protein, were determined by manipulating two cellular activities affecting the protein folding pathway in host cells: (i) the malEp promoter activity, which is controlled by the transcriptional activator MalT, and (ii) the DegP and Protease III periplasmic proteolytic activity. At a low level of expression, the degradation of misfolded MalE31 was partially impaired in cells lacking DegP or Protease III. At a high level of expression, misfolded MalE31 rapidly formed periplasmic inclusion bodies and thus escaped degradation. However, the manipulated host cell activities did not enhance the production of periplasmic, soluble MalE31. A kinetic competition between folding, aggregation, and degradation is proposed as a general model for the biogenesis of periplasmic proteins.
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Folding of a Mutant Maltose-Binding Protein of Escherichia coli Which Forms Inclusion Bodies
The Journal of biological chemistry, 1996Co-Authors: Jean Michel Betton, Maurice HofnungAbstract:Abstract The Maltose-Binding protein (MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. We have examined the characteristics of a Mal mutant of malE corresponding to the double substitution Gly Asp/Ile Pro, MalE31, previously obtained by random mutagenesis. In vivo, the MalE31 precursor is efficiently processed, but the mature protein forms inclusion bodies in the periplasm. Furthermore, the accumulation of insoluble MalE31 is independent of its cellular localization; MalE31 lacking its signal sequence forms inclusion bodies in the cytoplasm. The native MalE31 protein can be purified by affinity chromatography from inclusion bodies after denaturation by 8 M urea. The renatured protein exhibits full Maltose Binding affinity (K= 9 × 10M), suggesting that its folded structure is similar to that of the wild-type protein. Unfolding/refolding experiments show that MalE31 is less stable (−5.5 kcal/mol) than the wild-type protein (−9.5 kcal/mol) and that folding intermediates have a high tendency to form aggregates. In conclusion, the observed phenotype of cells expressing malE31 can be explained by a defective folding pathway of the protein.
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location of tolerated insertions deletions in the structure of the Maltose Binding protein
FEBS Letters, 1993Co-Authors: Jean Michel Betton, William Saurin, Pierre Martineau, Maurice HofnungAbstract:In a previous study [(1987) J. Mol. Biol. 194, 663-673], we isolated ten insertion/deletion mutants (indels) of the Maltose Binding protein for which the Maltose Binding constant was only a little or not at all affected. In this paper, we have localized these mutations in the recently solved three-dimensional structure. Contrary to the general expectation, most of the insertion/deletion modifications occurred within elements of secondary structure. An analysis of the inserted residues for three indels found within α helices allowed an interpretation regarding protein structure accommodation to such modifications.
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Location of tolerated insertions/deletions in the structure of the Maltose Binding protein
FEBS Letters, 1993Co-Authors: Jean Michel Betton, William Saurin, Pierre Martineau, Maurice HofnungAbstract:In a previous study [(1987) J. Mol. Biol. 194, 663-673], we isolated ten insertion/deletion mutants (indels) of the Maltose Binding protein for which the Maltose Binding constant was only a little or not at all affected. In this paper, we have localized these mutations in the recently solved three-dimensional structure. Contrary to the general expectation, most of the insertion/deletion modifications occurred within elements of secondary structure. An analysis of the inserted residues for three indels found within α helices allowed an interpretation regarding protein structure accommodation to such modifications. © 1993.
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Immune responses to hybrid Maltose-Binding proteins
Vaccine, 1993Co-Authors: David O'callaghan, Pierre Martineau, Catherine Fayolle, Claude Leclerc, Jean-gérard Guillet, A. Charbit, Maurice HofnungAbstract:The Escherichia coli Maltose-Binding proteins is a highly versatile carrier protein allowing the construction of genetically engineered hybrid proteins. It accepts large fusions to both C- and N-termini as well as the insertion of shorter peptides at permissive sites within the continuity of the protein. We have genetically inserted immunogenic peptides corresponding to defined viral B- and T-cell epitopes into two permissive sites: one at amino acid site 133, the other at site 303
Jean Michel Betton - One of the best experts on this subject based on the ideXlab platform.
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Folding and aggregation of export-defective mutants of the Maltose-Binding protein.
Research in microbiology, 2002Co-Authors: Jean Michel Betton, Denis Phichith, Sabine HunkeAbstract:We previously characterized a defective-folding variant of the periplasmic Maltose-Binding protein, MalE31. To examine the alternative folding pathways open to the MalE31 precursor, we have analyzed the cellular fates of this aggregation-prone protein carrying altered signal sequences. Our results are most easily interpreted by a kinetic competition between exportation, folding, and degradation.
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Degradation versus aggregation of misfolded Maltose-Binding protein in the periplasm of Escherichia coli.
The Journal of biological chemistry, 1998Co-Authors: Jean Michel Betton, Nathalie Sassoon, Maurice Hofnung, Michel LaurentAbstract:The periplasmic fates of misfolded MalE31, a defective folding mutant of the Maltose-Binding protein, were determined by manipulating two cellular activities affecting the protein folding pathway in host cells: (i) the malEp promoter activity, which is controlled by the transcriptional activator MalT, and (ii) the DegP and Protease III periplasmic proteolytic activity. At a low level of expression, the degradation of misfolded MalE31 was partially impaired in cells lacking DegP or Protease III. At a high level of expression, misfolded MalE31 rapidly formed periplasmic inclusion bodies and thus escaped degradation. However, the manipulated host cell activities did not enhance the production of periplasmic, soluble MalE31. A kinetic competition between folding, aggregation, and degradation is proposed as a general model for the biogenesis of periplasmic proteins.
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Folding of a Mutant Maltose-Binding Protein of Escherichia coli Which Forms Inclusion Bodies
The Journal of biological chemistry, 1996Co-Authors: Jean Michel Betton, Maurice HofnungAbstract:Abstract The Maltose-Binding protein (MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. We have examined the characteristics of a Mal mutant of malE corresponding to the double substitution Gly Asp/Ile Pro, MalE31, previously obtained by random mutagenesis. In vivo, the MalE31 precursor is efficiently processed, but the mature protein forms inclusion bodies in the periplasm. Furthermore, the accumulation of insoluble MalE31 is independent of its cellular localization; MalE31 lacking its signal sequence forms inclusion bodies in the cytoplasm. The native MalE31 protein can be purified by affinity chromatography from inclusion bodies after denaturation by 8 M urea. The renatured protein exhibits full Maltose Binding affinity (K= 9 × 10M), suggesting that its folded structure is similar to that of the wild-type protein. Unfolding/refolding experiments show that MalE31 is less stable (−5.5 kcal/mol) than the wild-type protein (−9.5 kcal/mol) and that folding intermediates have a high tendency to form aggregates. In conclusion, the observed phenotype of cells expressing malE31 can be explained by a defective folding pathway of the protein.
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location of tolerated insertions deletions in the structure of the Maltose Binding protein
FEBS Letters, 1993Co-Authors: Jean Michel Betton, William Saurin, Pierre Martineau, Maurice HofnungAbstract:In a previous study [(1987) J. Mol. Biol. 194, 663-673], we isolated ten insertion/deletion mutants (indels) of the Maltose Binding protein for which the Maltose Binding constant was only a little or not at all affected. In this paper, we have localized these mutations in the recently solved three-dimensional structure. Contrary to the general expectation, most of the insertion/deletion modifications occurred within elements of secondary structure. An analysis of the inserted residues for three indels found within α helices allowed an interpretation regarding protein structure accommodation to such modifications.
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Location of tolerated insertions/deletions in the structure of the Maltose Binding protein
FEBS Letters, 1993Co-Authors: Jean Michel Betton, William Saurin, Pierre Martineau, Maurice HofnungAbstract:In a previous study [(1987) J. Mol. Biol. 194, 663-673], we isolated ten insertion/deletion mutants (indels) of the Maltose Binding protein for which the Maltose Binding constant was only a little or not at all affected. In this paper, we have localized these mutations in the recently solved three-dimensional structure. Contrary to the general expectation, most of the insertion/deletion modifications occurred within elements of secondary structure. An analysis of the inserted residues for three indels found within α helices allowed an interpretation regarding protein structure accommodation to such modifications. © 1993.
Wolfram Welte - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of a liganded trehalose Maltose Binding protein from the hyperthermophilic archaeon thermococcus litoralis at 1 85 a
Journal of Molecular Biology, 2001Co-Authors: Joachim Diez, Winfried Boos, Kay Diederichs, Gerhard Greller, Reinhold Horlacher, Wolfram WelteAbstract:Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/Maltose-Binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/Maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same Binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-Binding protein and is structurally related to the Maltose-Binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to Maltose Binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.
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The crystal structure of a liganded trehalose/Maltose-Binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A.
Journal of molecular biology, 2001Co-Authors: Joachim Diez, Winfried Boos, Kay Diederichs, Gerhard Greller, Reinhold Horlacher, Wolfram WelteAbstract:Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/Maltose-Binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/Maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same Binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-Binding protein and is structurally related to the Maltose-Binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to Maltose Binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.
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the crystal structure of a liganded trehalose Maltose Binding protein from the hyperthermophilic archaeon thermococcus litoralis at 1 85 a
Journal of Molecular Biology, 2001Co-Authors: Joachim Diez, Winfried Boos, Kay Diederichs, Gerhard Greller, Reinhold Horlacher, Wolfram WelteAbstract:Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/Maltose-Binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/Maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same Binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-Binding protein and is structurally related to the Maltose-Binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to Maltose Binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.
Pierre Martineau - One of the best experts on this subject based on the ideXlab platform.
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location of tolerated insertions deletions in the structure of the Maltose Binding protein
FEBS Letters, 1993Co-Authors: Jean Michel Betton, William Saurin, Pierre Martineau, Maurice HofnungAbstract:In a previous study [(1987) J. Mol. Biol. 194, 663-673], we isolated ten insertion/deletion mutants (indels) of the Maltose Binding protein for which the Maltose Binding constant was only a little or not at all affected. In this paper, we have localized these mutations in the recently solved three-dimensional structure. Contrary to the general expectation, most of the insertion/deletion modifications occurred within elements of secondary structure. An analysis of the inserted residues for three indels found within α helices allowed an interpretation regarding protein structure accommodation to such modifications.
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Location of tolerated insertions/deletions in the structure of the Maltose Binding protein
FEBS Letters, 1993Co-Authors: Jean Michel Betton, William Saurin, Pierre Martineau, Maurice HofnungAbstract:In a previous study [(1987) J. Mol. Biol. 194, 663-673], we isolated ten insertion/deletion mutants (indels) of the Maltose Binding protein for which the Maltose Binding constant was only a little or not at all affected. In this paper, we have localized these mutations in the recently solved three-dimensional structure. Contrary to the general expectation, most of the insertion/deletion modifications occurred within elements of secondary structure. An analysis of the inserted residues for three indels found within α helices allowed an interpretation regarding protein structure accommodation to such modifications. © 1993.
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Evaluation of several affinity chromatographic supports for the purification of Maltose-Binding protein from Escherichia coli
Journal of Chromatography A, 1993Co-Authors: Yolande Kroviarski, Sylvie Cochet, Pierre Martineau, Jean-pierre Cartron, Olivier BertrandAbstract:To obtain affinity adsorbents with good mechanical resistance, suitable for the purification of Maltose-Binding protein (MBP) from Escherichia coli and genetically engineered proteins fused to MBP, a series of supports were prepared by grafting amylose on to agarose by different chemistries. Their capacities for MBP and their abilities to be used at relatively high flow-rates were examined. Efficient supports were most conveniently prepared by coupling amylose to epoxy-activated agarose in an aqueous-organic mixture. © 1993.
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Immune responses to hybrid Maltose-Binding proteins
Vaccine, 1993Co-Authors: David O'callaghan, Pierre Martineau, Catherine Fayolle, Claude Leclerc, Jean-gérard Guillet, A. Charbit, Maurice HofnungAbstract:The Escherichia coli Maltose-Binding proteins is a highly versatile carrier protein allowing the construction of genetically engineered hybrid proteins. It accepts large fusions to both C- and N-termini as well as the insertion of shorter peptides at permissive sites within the continuity of the protein. We have genetically inserted immunogenic peptides corresponding to defined viral B- and T-cell epitopes into two permissive sites: one at amino acid site 133, the other at site 303
Joachim Diez - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of a liganded trehalose Maltose Binding protein from the hyperthermophilic archaeon thermococcus litoralis at 1 85 a
Journal of Molecular Biology, 2001Co-Authors: Joachim Diez, Winfried Boos, Kay Diederichs, Gerhard Greller, Reinhold Horlacher, Wolfram WelteAbstract:Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/Maltose-Binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/Maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same Binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-Binding protein and is structurally related to the Maltose-Binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to Maltose Binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.
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The crystal structure of a liganded trehalose/Maltose-Binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A.
Journal of molecular biology, 2001Co-Authors: Joachim Diez, Winfried Boos, Kay Diederichs, Gerhard Greller, Reinhold Horlacher, Wolfram WelteAbstract:Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/Maltose-Binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/Maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same Binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-Binding protein and is structurally related to the Maltose-Binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to Maltose Binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.
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the crystal structure of a liganded trehalose Maltose Binding protein from the hyperthermophilic archaeon thermococcus litoralis at 1 85 a
Journal of Molecular Biology, 2001Co-Authors: Joachim Diez, Winfried Boos, Kay Diederichs, Gerhard Greller, Reinhold Horlacher, Wolfram WelteAbstract:Abstract We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/Maltose-Binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/Maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo , this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same Binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-Binding protein and is structurally related to the Maltose-Binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel β-sheet flanked by α-helices on both sides. Both are connected by a hinge region consisting of two antiparallel β-strands and an α-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to Maltose Binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.