PMID:12475238
| Citation |
Rimon, A, Tzubery, T, Galili, L and Padan, E (2002) Proximity of cytoplasmic and periplasmic loops in NhaA Na+/H+ antiporter of Escherichia coli as determined by site-directed thiol cross-linking. Biochemistry 41:14897-905 |
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| Abstract |
The unique trypsin cleavable site of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, was exploited to detect a change in mobility of cross-linked products of NhaA by polyacrylamide gel electrophoresis. Double-Cys replacements were introduced into loops, one on each side of the trypsin cleavage site (Lys 249). The proximity of paired Cys residues was assessed by disulfide cross-linking of the two tryptic fragments, using three homobifunctional cross-linking agents: 1,6-bis(maleimido)hexane (BMH), N,N'-o-phenylenedimaleimide (o-PDM), and N,N'-p-phenylenedimaleimide (p-PDM). The interloop cross-linking was found to be very specific, indicating that the loops are not merely random coils that interact randomly. In the periplasmic side of NhaA, two patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently between the double-Cys replacements A118C/S286C, N177C/S352C, and H225C/S352C; (b) only BMH cross-links the double-Cys replacements A118C/S352C, N177C/S286C, and H225C/S286C. In the cytoplasmic side of NhaA, three patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently the pairs of Cys replacements L4C/E252C, S146C/L316C, S146C/R383C, and E241C/E252C; (b) BMH and p-PDM cross-link efficiently the pairs of Cys replacements S87C/E252C, S87C/L316C, and S146C/E252C; (c) none of the reagents cross-links the double-Cys replacements L4C/L316C, L4C/R383C, S87C/R383C, A202C/E252C, A202C/L316C, A202C/R383C, E241C/L316C, and E241C/R383C. The data reveal that the N-terminus and loop VIII-IX that have previously been shown to change conformation with pH are in close proximity within the NhaA protein. The data also suggest close proximity between N-terminal and C-terminal helices at both the cytoplasmic and the periplasmic face of NhaA. |
| Links | |
| Keywords |
Amino Acid Sequence; Amino Acid Substitution/genetics; Animals; Cricetinae; Cross-Linking Reagents/metabolism; Cysteine/genetics; Cytoplasm/chemistry; Cytoplasm/genetics; Cytoplasm/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Fluoresceins/metabolism; Fluorescent Dyes/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments/chemistry; Peptide Fragments/genetics; Peptide Fragments/metabolism; Periplasm/chemistry; Periplasm/genetics; Periplasm/metabolism; Protein Structure, Secondary/genetics; Protein Structure, Tertiary/genetics; Sodium-Hydrogen Antiporter/chemistry; Sodium-Hydrogen Antiporter/genetics; Sodium-Hydrogen Antiporter/metabolism; Sulfhydryl Compounds/metabolism; Tumor Cells, Cultured |
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