Immunoglobulin Mutant Library Genetically Screened for Folding Stability Exploiting Bacterial Signal Transduction

Kolmar, Harald; Frisch, Christian; Götze, K.; Fritz, Hans-Joachim

doi:10.1006/jmbi.1995.0448

Cited by 18 publications

(17 citation statements)

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“…In agreement with the dimerization model are also the results obtained with other ToxR fusion proteins in which two domains with different tendencies to dimerize, PhoA, GCN4 leucine zipper domain and monomeric maltose binding protein have been fused to ToxR at the periplasmic level [6]. As a further development of this system, the ToxR activator has been used as an indicator for the association [7]and the folding stability [8]of dimers of immunoglobulin V L domains and for the analysis of residues critical for the dimerization of the transmembrane segment of glycophorin A [9]. In contrast, another study has shown that hybrid proteins in which ToxR periplasmic domain was replaced with either PhoA, GCN4 leucine zipper domain or β‐lactamase (Bla), all activate transcription from the ctx promoter at high level.…”

Section: Introductionsupporting

confidence: 58%

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Changes in the periplasmic linker and in the expression level affect the activity of ToxR and λ‐ToxR fusion proteins in Escherichia coli

Jappelli

Brenner

1998

FEBS Letters

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In order to assess the potentiality of Vibrio cholerae ToxR protein and of bacteriophage V V repressor as indicators of the dimerization of periplasmic proteins in Escherichia coli, we have constructed a series of plasmids encoding transmembrane fusion proteins. The amino-terminal part, containing the DNA binding domain of either ToxR or V V repressor, is located in the cytoplasm and acts as reporter for dimerization. As models of periplasmic proteins we have used alkaline phosphatase (a dimer) and L L-lactamase (a monomer). Both the expression level and the distance between the transmembrane segment and the periplasmic protein substantially affect the activity of the reporter domains.z 1998 Federation of European Biochemical Societies.

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Section: Introductionsupporting

confidence: 58%

“…The DNA binding domains of ToxR and λ repressors have been applied as genetic indicators of the dimerization of both periplasmic and membrane proteins [5–9]. It is important to validate the applicability of these reporter systems to different experimental conditions.…”

Section: Discussionmentioning

confidence: 99%

Changes in the periplasmic linker and in the expression level affect the activity of ToxR and λ‐ToxR fusion proteins in Escherichia coli

Jappelli

Brenner

1998

FEBS Letters

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“…As mentioned above, a fusion protein consisting of E. coli ␤-lactamase and the variable domain of the Bence-Jones protein REI (REI v ) is likely to be degraded by DegP in vivo (11,13). Figure 3 shows that the melting temperature of this protein is approximately 45ЊC.…”

Section: Resultsmentioning

confidence: 99%

“…By contrast, neither protease cleaved native bovine serum albumin, ovalbumin, carbonic anhydrase, or an N-terminal fusion of ␤-lactamase to the immunoglobulin domain REI v (12). The last result was somewhat surprising, as this chimeric protein is expressed at much higher levels in strains lacking functional DegP, suggesting that it is normally degraded by DegP in vivo (11,13). As we show later, this protein becomes susceptible to degradation by either DegP or DegQ if its disulfide bonds are reduced and alkylated.…”

Section: Resultsmentioning

confidence: 99%

The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation

1996

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DegP and DegQ are homologous endoproteases found in the periplasmic compartment of Escherichia coli.The studies presented here suggest that DegP and DegQ have very similar substrate specificities and cleave substrates which are transiently or globally denatured. Model substrates were cleaved at discrete Val/Xaa or Ile/Xaa sites, suggesting that aliphatic, ␤-branched residues, which are typically buried in the hydrophobic core of most proteins, are important determinants of cleavage specificity. Indeed, the peptide bonds cleaved in the model substrates are generally inaccessible in the native three-dimensional structures. In addition, a chimeric fusion protein, which is a DegP substrate in vivo, is degraded in vitro only after reduction of its intramolecular disulfide bonds. Taken together, these findings suggest that DegP and DegQ may degrade transiently denatured proteins, unfolded proteins which accumulate in the periplasm following heat shock or other stress conditions, and/or newly secreted proteins prior to folding and disulfide bond formation. Cross-linking studies indicate that both DegP and DegQ form dodecamers in solution and thus are similar to many other intracellular proteases which form large oligomeric complexes.Intracellular proteases play important regulatory roles and also serve essential housekeeping functions by removing damaged or misfolded proteins (18). Escherichia coli contains at least two cytoplasmic proteases, Lon and Clp, which function to degrade abnormal proteins (5). The biochemical properties of these ATP-dependent proteases have been studied extensively (4). Relatively little, however, is known about the biochemical or structural properties of proteases which may be responsible for degradation of misfolded or abnormal proteins in the periplasmic compartment of bacteria. Such a function has been attributed to the periplasmic protease DegP, which is also known as HtrA or protease Do (15,28,30,31). DegP is required for survival of E. coli at elevated temperatures, and mutations in the degP gene result in decreased degradation of chimeric membrane and periplasmic proteins (14,30,31). The temperature sensitivity of degP mutants is reduced in strains which release periplasmic proteins into the medium because of outer-membrane defects, as expected if DegP is required for the removal of misfolded proteins which may accumulate at high temperatures (16,30).Several groups recently identified another periplasmic protease of E. coli, DegQ (HhoA), which is homologous to DegP (2, 33). The DegQ and DegP proteins are of similar size (455 and 474 residues, respectively) and display approximately 60% sequence identity. Overproduction of DegQ suppresses the temperature-sensitive defect of a DegP Ϫ strain, suggesting that the two enzymes must be capable of degrading similar substrates (33). On the basis of sequence conservation, inhibition by diisopropyl fluorophosphate, and mutagenesis experiments, both enzymes seem to contain the Ser-His-Asp catalytic triad found in traditional serine proteases (29,...

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“…Additionally, the plasmid-free reporter strain FHK12 (without plasmid) was used as a control for strains without ToxR function, and E. coli strain FHK12 expressing pHKToxR′REI-T39K served as control (PC), which is noted for a strong increase in dimerization. 44 lacZ reporter gene. Comparisons of the Gcn4 mutant and wild-type leucine zipper revealed an approximately fivefold decrease in dimerization ability in the mutant Gcn4 L267S compared to that of wild-type (Fig.…”

Section: Dimerization Of Gcn4 L267s Is Reduced Compared To That Of Wimentioning

confidence: 99%

A Feedback Circuit between Transcriptional Activation and Self-Destruction of Gcn4 Separates Its Metabolic and Morphogenic Response in Diploid Yeasts

Herzog

Streckfuß‐Bömeke

Braus

2011

Journal of Molecular Biology

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Immunoglobulin Mutant Library Genetically Screened for Folding Stability Exploiting Bacterial Signal Transduction

Cited by 18 publications

References 0 publications

Changes in the periplasmic linker and in the expression level affect the activity of ToxR and λ‐ToxR fusion proteins in Escherichia coli

Changes in the periplasmic linker and in the expression level affect the activity of ToxR and λ‐ToxR fusion proteins in Escherichia coli

The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation

A Feedback Circuit between Transcriptional Activation and Self-Destruction of Gcn4 Separates Its Metabolic and Morphogenic Response in Diploid Yeasts

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