Cysteine cross-linking defines part of the dimer and B/C domain interface of the Escherichia coli mannitol permease
| Autor: | Berend Poolman, Ria H. Duurkens, R. Mensen, B.A. van Montfort, Gea K. Schuurman-Wolters, G.T. Robillard |
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| Přispěvatelé: | Enzymologie, Faculty of Science and Engineering, Groningen Biomolecular Sciences and Biotechnology |
| Jazyk: | Dutch; Flemish |
| Rok vydání: | 2001 |
| Předmět: |
Monosaccharide Transport Proteins
Stereochemistry Protein subunit Dimer B-DOMAIN C-DOMAIN SUBSTRATE-BINDING medicine.disease_cause Biochemistry Maleimides chemistry.chemical_compound Escherichia coli medicine Mannitol ENZYME-IIMTL Cysteine Disulfides Phosphorylation Binding site PHOSPHORYLATION SITE MUTANTS Phosphoenolpyruvate Sugar Phosphotransferase System Molecular Biology GeneralLiterature_REFERENCE(e.g. dictionaries encyclopedias glossaries) Gel electrophoresis chemistry.chemical_classification Binding Sites TRANSPORT PROTEIN Escherichia coli Proteins Cell Biology Recombinant Proteins STATE Transport protein Cross-Linking Reagents Enzyme Amino Acid Substitution chemistry DEPENDENT PHOSPHOTRANSFERASE SYSTEM PHOSPHOENOLPYRUVATE Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Mutagenesis Site-Directed ComputingMethodologies_DOCUMENTANDTEXTPROCESSING MEMBRANE Dimerization Oxidation-Reduction Copper Phenanthrolines |
| Zdroj: | The Journal of Biological Chemistry, 276(16), 12756-12763. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC |
| ISSN: | 0021-9258 |
| Popis: | Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain. The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis. Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit. The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124). The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124). The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length. The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain. |
| Databáze: | OpenAIRE |
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