Difference between revisions of "PMID:9045630"
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| + | !align=left align='left' bgcolor='#CCCCFF' |Citation | ||
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| + | '''Sone, M, Kishigami, S, Yoshihisa, T and Ito, K''' (1997) Roles of disulfide bonds in bacterial alkaline phosphatase. ''J. Biol. Chem.'' '''272''':6174-8 | ||
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| + | !align=left align='left' bgcolor='#CCCCFF' |Abstract | ||
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| + | Alkaline phosphatase of Escherichia coli (a homodimeric protein found in the periplasmic space) contains two intramolecular disulfide bonds (Cys-168-Cys-178 and Cys-286-Cys-336) that are formed after export to the periplasmic space. The location-specific folding character of this enzyme allowed its wide usage as a reporter of protein localization in prokaryotic cells. To study the roles of disulfide bonds in alkaline phosphatase, we eliminated each of them by Cys to Ser mutations. Intracellular stability of alkaline phosphatase decreased in the absence of either one or both of the disulfide bonds. The mutant proteins were stabilized in a DegP protease-deficient strain, allowing accumulation at significant levels and subsequent characterization. A mutant protein that lacked the N-terminally located disulfide bond (Cys-168-Cys-178) was found to have Cys-286 and Cys-336 residues disulfide-bonded, to have a dimeric structure, and to have almost full enzymatic activity. Nevertheless, the mutant protein lost the trypsin-resistant conformation that is characteristically observed for the wild-type enzyme. In contrast, mutants lacking Cys-286 and Cys-336 were monomeric and inactive. These results indicate that the Cys-286-Cys-336 disulfide bond is required and is sufficient for correctly positioning the active site region of this enzyme, but such an active conformation is still insufficient for the conformational stability of the enzyme. Thus, a fully active state of this enzyme can be formed without full protein stability, and the two disulfide bonds differentially contribute to these properties. | ||
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| + | !align=left align='left' bgcolor='#CCCCFF' |Links | ||
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| + | [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9045630 PubMed] | ||
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| + | !align=left align='left' bgcolor='#CCCCFF' |Keywords | ||
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| + | Alkaline Phosphatase/chemistry; Bacterial Proteins/chemistry; Binding Sites; Cysteine/chemistry; Disulfides/chemistry; Mutagenesis, Site-Directed; Protein Conformation; Protein Denaturation; Protein Folding; Serine/chemistry; Structure-Activity Relationship; Trypsin/pharmacology | ||
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| + | <!--box uid=d41d8cd98f00b204e9800998ecf8427e.32382.T5c7e6c24d0118--> | ||
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| + | ==Main Points of the Paper == | ||
| + | {{LitSignificance}} | ||
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| + | == Materials and Methods Used == | ||
| + | {{LitMaterials}} | ||
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| + | ==Phenotype Annotations== | ||
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| + | !|Phenotype of!!Taxon Information!!Genotype Information (if known)!!Condition Information!!OMP ID!!OMP Term Name!!ECO ID!!ECO Term Name!!Notes!!Status | ||
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| + | ==Notes== | ||
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| + | ==References== | ||
| + | {{RefHelp}} | ||
| + | <references/> | ||
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| + | [[Category:Publication]] | ||
Latest revision as of 07:31, 5 March 2019
| Citation |
Sone, M, Kishigami, S, Yoshihisa, T and Ito, K (1997) Roles of disulfide bonds in bacterial alkaline phosphatase. J. Biol. Chem. 272:6174-8 |
|---|---|
| Abstract |
Alkaline phosphatase of Escherichia coli (a homodimeric protein found in the periplasmic space) contains two intramolecular disulfide bonds (Cys-168-Cys-178 and Cys-286-Cys-336) that are formed after export to the periplasmic space. The location-specific folding character of this enzyme allowed its wide usage as a reporter of protein localization in prokaryotic cells. To study the roles of disulfide bonds in alkaline phosphatase, we eliminated each of them by Cys to Ser mutations. Intracellular stability of alkaline phosphatase decreased in the absence of either one or both of the disulfide bonds. The mutant proteins were stabilized in a DegP protease-deficient strain, allowing accumulation at significant levels and subsequent characterization. A mutant protein that lacked the N-terminally located disulfide bond (Cys-168-Cys-178) was found to have Cys-286 and Cys-336 residues disulfide-bonded, to have a dimeric structure, and to have almost full enzymatic activity. Nevertheless, the mutant protein lost the trypsin-resistant conformation that is characteristically observed for the wild-type enzyme. In contrast, mutants lacking Cys-286 and Cys-336 were monomeric and inactive. These results indicate that the Cys-286-Cys-336 disulfide bond is required and is sufficient for correctly positioning the active site region of this enzyme, but such an active conformation is still insufficient for the conformational stability of the enzyme. Thus, a fully active state of this enzyme can be formed without full protein stability, and the two disulfide bonds differentially contribute to these properties. |
| Links | |
| Keywords |
Alkaline Phosphatase/chemistry; Bacterial Proteins/chemistry; Binding Sites; Cysteine/chemistry; Disulfides/chemistry; Mutagenesis, Site-Directed; Protein Conformation; Protein Denaturation; Protein Folding; Serine/chemistry; Structure-Activity Relationship; Trypsin/pharmacology |
Main Points of the Paper
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Materials and Methods Used
Please list the materials and methods used in this paper (strains, plasmids, antibodies, etc).
Phenotype Annotations
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| Phenotype of | Taxon Information | Genotype Information (if known) | Condition Information | OMP ID | OMP Term Name | ECO ID | ECO Term Name | Notes | Status |
|---|---|---|---|---|---|---|---|---|---|
Notes
References
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