Abstract
Bradavidin is a homotetrameric biotin-binding protein from Bradyrhizobium japonicum, a nitrogen fixing and root nodule-forming symbiotic bacterium of the soybean. Wild-type (wt) bradavidin has 138 amino acid residues, whereas the C-terminally truncated core-bradavidin has only 118 residues. We have solved the X-ray structure of wt bradavidin and found that the C-terminal amino acids of each subunit were uniquely bound to the biotin-binding pocket of an adjacent subunit. The biotin-binding pocket occupying peptide (SEKLSNTK) was named “Brad-tag” and it serves as an intrinsic stabilizing ligand in wt bradavidin. The binding of Brad-tag to core-bradavidin was analysed by isothermal titration calorimetry and a binding affinity of ∼25 µM was measured. In order to study the potential of Brad-tag, a green fluorescent protein tagged with Brad-tag was prepared and successfully concentrated from a bacterial cell lysate using core-bradavidin-functionalized Sepharose resin.
Highlights
Chicken egg-white avidin and its eukaryotic and prokaryotic homologs, known collectively as avidins, are proteins that have extreme affinity towards D-biotin (Kd,10–15 M for chicken avidin) [1,2,3]
Eukaryotic avidins exist in the eggs of e.g. birds, reptilia and amphibians [4,5,6,7], whereas prokaryotic avidins have been isolated from a few bacterial species: Streptomyces avidinii [8], Bradyrhizobium japonicum [9,10], Rhizobium etli [11] and Burkholderia pseudomallei [12]
We tried to crystallize wt bradavidin in the absence and presence of biotin, and in the presence of an azo dye HABA (4-hydroxyazobenzene-2-carboxylic acid, called 2(49-hydroxybenzene)azobenzoic acid) in the hope that the dye would bind to the biotin-binding site
Summary
Chicken egg-white avidin and its eukaryotic and prokaryotic homologs, known collectively as avidins, are proteins that have extreme affinity towards D-biotin (Kd ,10–15 M for chicken avidin) [1,2,3]. The best-studied avidins are the mature forms of chicken avidin and streptavidin. In the chicken egg-white, only the 28 amino acid signal peptide is removed from the full-length polypeptide chain to form the mature avidin protein (residues 1–128) [1], whereas several different cleavage products have been detected for streptavidin [16]. The most stable truncated form of streptavidin is the so-called core streptavidin, which contains residues 13–139 but still retains extremely high affinity towards D-biotin Kd ,10–15 M [16]. C-terminal residues 150–153 (Asn150Gly151-Asn152-Pro153) of wt streptavidin fold back into the biotin-binding site in each monomer [19], thereby competing with the binding of at least low-affinity biotinylated macromolecules [16,20]
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