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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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Probing the Structural Role of an αβ Loop of Maltose-Binding Protein by Mutagenesis: Heat-shock Induction by Loop Variants of the Maltose-Binding Protein that Form Periplasmic Inclusion Bodies
Journal of molecular biology, 1996Co-Authors: Jean Michel Betton, Didier Boscus, Dominique Missiakas, Satish Raina, Maurice HofnungAbstract:The Maltose-Binding Protein (MBP) of Escherichia coli is the periplasmic receptor of the Maltose transport system. Previous studies have identified amino acid substitutions in an alpha/beta loop of the structure of MBP that are critical for the in vivo folding. To probe genetically the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 of malE were mutagenized. The Maltose phenotype, which correlates with a biologically active structure of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correct in vivo folding pathway of the Protein. By the same genetic screens, we characterized loop-variant MBPs associated with a defective periplasmic folding pathway and aggregated into inclusion bodies. Heat-shock induction with production of misfolded loop variants was examined using both lon-lacZ and htrA-lacZ fusions. We found that the extent of formation of inclusion bodies in the periplasm of E. coli, from misfolded loop variant MBPs, correlated with the level of heat-shock response regulated by the alternate heat-shock sigma factor, sigma 24.
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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.
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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Probing the Structural Role of an αβ Loop of Maltose-Binding Protein by Mutagenesis: Heat-shock Induction by Loop Variants of the Maltose-Binding Protein that Form Periplasmic Inclusion Bodies
Journal of molecular biology, 1996Co-Authors: Jean Michel Betton, Didier Boscus, Dominique Missiakas, Satish Raina, Maurice HofnungAbstract:The Maltose-Binding Protein (MBP) of Escherichia coli is the periplasmic receptor of the Maltose transport system. Previous studies have identified amino acid substitutions in an alpha/beta loop of the structure of MBP that are critical for the in vivo folding. To probe genetically the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 of malE were mutagenized. The Maltose phenotype, which correlates with a biologically active structure of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correct in vivo folding pathway of the Protein. By the same genetic screens, we characterized loop-variant MBPs associated with a defective periplasmic folding pathway and aggregated into inclusion bodies. Heat-shock induction with production of misfolded loop variants was examined using both lon-lacZ and htrA-lacZ fusions. We found that the extent of formation of inclusion bodies in the periplasm of E. coli, from misfolded loop variant MBPs, correlated with the level of heat-shock response regulated by the alternate heat-shock sigma factor, sigma 24.
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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, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, 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, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, 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, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, 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.
Linda L. Randall - One of the best experts on this subject based on the ideXlab platform.
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Novel Intermediates in the Synthesis of Maltose‐Binding Protein in Escherichia coli
European journal of biochemistry, 2005Co-Authors: Linda L. Randall, Lars-göran Josefsson, Simon J. S. HardyAbstract:Nascent intermediates in the synthesis of Maltose-Binding Protein, a periplasmic Protein in Escherichia coli, were demonstrated both in vivo and in vitro. They are likely to result from a drastic reduction in the rate of elongation at specific sites on the mRNA leading to detectable accumulation of distinct species of incomplete polypeptides. In order to reach its final destination in the periplasmic space, Maltose-Binding Protein is transferred across the cytoplasmic membrane as it is elongated. It is possible that variations in the rate of elongation are involved in this export process.
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Demonstration in vivo that interaction of Maltose-Binding Protein with SecB is determined by a kinetic partitioning.
Journal of bacteriology, 1995Co-Authors: V J Khisty, Linda L. RandallAbstract:An early step in the export of Maltose-Binding Protein to the periplasm is interaction with the molecular chaperone SecB. We demonstrate that Binding to SecB in vivo is determined by a kinetic partitioning between the folding of Maltose-Binding Protein to its native state and its association with SecB. A complex of SecB and a species of Maltose-Binding Protein that folds slowly is shown to be longer-lived than a complex of the wild-type Maltose-Binding Protein and SecB. In addition, we show that incomplete nascent chains, which are unable to fold, remain complexed with SecB.
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Interaction of SecB with intermediates along the folding pathway of Maltose-Binding Protein.
Protein science : a publication of the Protein Society, 1995Co-Authors: Deborah L. Diamond, S. Strobel, Sang-yearn Chun, Linda L. RandallAbstract:SecB, a molecular chaperone involved in Protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative. The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB. SecB has no affinity for native, stably folded polypeptides but interacts tightly with polypeptides that are nonnative. In order to better understand the nature of the Binding, we have examined the interaction of SecB with intermediates along the folding pathway of Maltose-Binding Protein. Taking advantage of forms of Maltose-Binding Protein that are altered in their folding properties, we show that the first intermediate in folding, represented by the collapsed state, binds to SecB, and that the polypeptide remains active as a ligand until it crosses the final energy barrier to attain the native state.
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Folding of Maltose-Binding Protein. Evidence for the identity of the rate-determining step in vivo and in vitro.
The Journal of biological chemistry, 1993Co-Authors: Sang-yearn Chun, S. Strobel, P. Bassford, Linda L. RandallAbstract:The folding of Maltose-Binding Protein, a periplasmic Protein in Escherichia coli, was shown to proceed through the same rate-limiting step whether folding occurred in the cell under physiological conditions or in vitro in the absence of other Proteins. Four species of Maltose-Binding Protein containing aminoacyl substitutions identified as decreasing the rate of folding of the Protein in vivo were purified, and their denaturant-induced folding transitions were analyzed by monitoring the intrinsic fluorescence of tryptophan. In all four cases the rate of folding in vitro was slower than that of the wild-type Maltose-Binding Protein; thus the same step determines the rate of folding in vivo and in vitro. Furthermore, examination of the three-dimensional structure of Maltose-Binding Protein as determined by x-ray crystallography (F. Quiocho, personal communication; Spurlino, J. C., Lu, G.-Y., and Quiocho, F. A. (1991) J. Biol. Chem. 266, 5202-5219) indicates that all 4 of the residues identified as crucial to folding lie in one structural element of the native Protein. We conclude that the rate-limiting step both in vivo and in vitro involves formation of this element of structure.
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, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, 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, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, 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, Reinhold Horlacher, Winfried Boos, Kay Diederichs, Gerhard Greller, 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.