Mutations that Allow Disulfide Bond Formation in the Cytoplasm of
            <i>Escherichia coli</i>

Derman, Alan I.; Prinz, William A.; Belin, Dominique; Beckwith, Jon

doi:10.1126/science.8259521

Cited by 411 publications

(322 citation statements)

References 35 publications

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“…This construct produces Dom1PI with an NH 2 -terminal Escherichia coli thioredoxin fusion (Trx tag, Novagen, Madison, WI) connected by a 43-amino acid residue linker containing six histidine residues and a factor Xa cleavage site. To ensure accurate disulfide formation, an E. coli trxB -/gor522 -double mutant [E. coli Origami (DE3), Novagen] with an oxidative cytoplasm was used (21,22). This strategy has been shown to result in the correct LEKTI domain 1 disulfide pattern (20).…”

Section: Methodsmentioning

confidence: 99%

The Solution Structure of a Chimeric LEKTI Domain Reveals a Chameleon Sequence

et al. 2004

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The conversion of an R-helical to a -strand conformation and the presence of chameleon sequences are fascinating from the perspective that such structural features are implicated in the induction of amyloid-related fatal diseases. In this study, we have determined the solution structure of a chimeric domain (Dom1PI) from the multidomain Kazal-type serine proteinase inhibitor LEKTI using multidimensional NMR spectroscopy. This chimeric protein was constructed to investigate the reasons for differences in the folds of the homologous LEKTI domains 1 and 6 [Lauber, T., et al. (2003) J. Mol. Biol. 328, 205-219]. In Dom1PI, two adjacent phenylalanine residues (F28 and F29) of domain 1 were substituted with proline and isoleucine, respectively, as found in the corresponding P4′ and P5′ positions of domain 6. The three-dimensional structure of Dom1PI is significantly different from the structure of domain 1 and closely resembles the structure of domain 6, despite the sequence being identical to that of domain 1 except for the two substituted phenylalanine residues and being only 31% identical to the sequence of domain 6. The mutation converted a short 3 10 -helix into an extended loop conformation and parts of the long COOH-terminal R-helix of domain 1 into a -hairpin structure. The latter conformational change occurs in a sequence stretch distinct from the region containing the substituted residues. Therefore, this switch from an R-helical structure to a -hairpin structure indicates a chameleon sequence of seven residues. We conclude that the secondary structure of Dom1PI is determined not only by the local protein sequence but also by nonlocal interactions.

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Section: Methodsmentioning

confidence: 99%

The Solution Structure of a Chimeric LEKTI Domain Reveals a Chameleon Sequence

et al. 2004

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“…There may be other substrates but they remain to be identified [4]. Thioredoxin reductase has diverse functions in the cell.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

Cloning and sequencing of a human thioredoxin reductase

et al. 1995

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E. coli thioredoxin reductase has been cloned and sequenced [21,22] and its biochemical and physical properties extensively studied [23,24]. Eukaryotic thioredoxin reductases have so far been only cloned from Penicillium chrysogenum [25], Saccharomyces cerevisiae [26], and Arabidopsis thaliana [27] and they show 44-50% sequence identity to the bacterial enzyme. We now report the cloning and sequencing of a putative thioredoxin reductase from human placenta.

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“…Bacterial strains, phage, plasmids and growth conditions E. coli DH5␣ [supE44 hsd R17 recA1 endA1 ⌬lac U169 ( 80 lacZ⌬M15 )] and E. coli A237 (trxB36 ) and A304 (trxB::kan) have been used as hosts for genetic transformation (Sambrook et al, 1989;Derman et al, 1993). E. coli HB101 (pNH101) (Díaz et al, 1992a) and E. coli HB101 (pGL80) (Díaz et al, 1991) were used as sources of LytA101 and LytA respectively.…”

Section: Methodsmentioning

confidence: 99%

The lytic enzyme of the pneumococcal phage Dp‐1: a chimeric lysin of intergeneric origin

Sheehan

Garcı́a

López

et al. 1997

Molecular Microbiology

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SummaryWe have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp-1. The lytic enzyme of this phage (Pal), previously identified as an N-acetyl-muramoyl-L-alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram-positive families has shown that the interchange of functional domains switches enzyme specificity. Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N-terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5-T that infects Lactococcus lactis, whereas the C-terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate. Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate. These peculiar characteristics have been ascribed to a modified C-terminal domain. The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.

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Mutations that Allow Disulfide Bond Formation in the Cytoplasm of Escherichia coli

Cited by 411 publications

References 35 publications

The Solution Structure of a Chimeric LEKTI Domain Reveals a Chameleon Sequence

The Solution Structure of a Chimeric LEKTI Domain Reveals a Chameleon Sequence

Cloning and sequencing of a human thioredoxin reductase

The lytic enzyme of the pneumococcal phage Dp‐1: a chimeric lysin of intergeneric origin

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