1887

Abstract

The ferric uptake regulator (Fur) of was expressed in in its native form and as a fusion to the maltose-binding protein (MBP). Fur from the MBP fusion bound to MBP after proteolytic cleavage, and the two could only be separated by partial unfolding. The refolded protein was in the same conformation as native protein (as judged by circular dichroism and fluorescence spectroscopies) and was fully active in DNA-binding assays. As-prepared native Fur contained small amounts of Zn that were easily removed by treatment with EDTA, and apo-protein could be reconstituted with approximately one Zn ion per monomer. Thus, the Fur can probably accommodate a single Zn ion bound to the metal-sensing site. The single cysteine residue of Fur aligns with a cysteine in other members of the Fur family that is essential for activity of the protein, and is believed to provide one of the ligands to a structural Zn ion. This cysteine residue was shown to be dispensable for the activity of Fur, which is consistent with the suggestion that the protein does not contain a structural Zn ion. Members of the Fur family contain a highly conserved His-His-Asp-His motif. Alanine substitutions of residues in this motif showed His-87 and His-89 of Fur to be essential for activity, whilst His-86 and Asp-88 are partially dispensable.

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2002-08-01
2019-10-16
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