Biochemical characterization of WrbA, founding member of a new family of multimeric flavodoxin-like proteins.
The protein WrbA had been identified as an Escherichia coli stationary-phase protein that copurified and coimmunoprecipitated with the tryptophan repressor. Sequences homologous to WrbA have been reported in several species of yeast and plants. We previously showed that this new family of proteins displays low but structurally significant sequence similarity with flavodoxins and that its members are predicted to share the alpha/beta core of the flavodoxin fold but with a short conserved insertion unique to the new family, which could account for reports that some family members may be dimeric in solution. The general sequence similarity to flavodoxins suggests that the members of the new family might bind FMN, but their wide evolutionary distribution indicates that, unlike the flavodoxins, these proteins may be ubiquitous. In this paper, we report the purification and biochemical characterization of WrbA, demonstrating that the protein binds FMN specifically and is a multimer in solution. The FMN binding constant is weaker than for many flavodoxins, being approximately 2 microM at 25 degreesC in 0. 1 mM sodium phosphate, pH 7.2. The protein participates in a dimer-tetramer equilibrium over a wide range of solution conditions, with a midpoint at approximately 1.4 microM. One FMN binds per monomer and has no apparent effect on the multimerization equilibrium. WrbA has no effect on the affinity or mode of DNA binding by the tryptophan repressor; thus, its physiological role remains unclear. Although many proteins with flavodoxin-like domains are known to be multimers, WrbA is apparently the first characterized case in which multimerization is associated directly with the flavodoxin-like domain itself.
Department of Chemistry, Princeton University, Princeton, New Jersey 08544-1009, USA.
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Grandori
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Khalifah
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Boice
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Fairman
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Giovanielli
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Carey
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005486:Flavin Mononucleotide / Q000378:metabolism; D005418:Flavodoxin / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D012097:Repressor Proteins
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005418:Flavodoxin; D012097:Repressor Proteins; C081759:WrbA protein, E coli; D005486:Flavin Mononucleotide