Dataset Information

Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies.

ABSTRACT: The streptavidin-based enrichment of biotin-tagged molecules is a common methodology that is routinely used across multiple disciplines in biomedical research. Numerous and varied formats of immobilized streptavidin and related proteins are available, but predicting which product is most apt for a given application is complicated by the fact that there are numerous technical considerations and no universal reporting standards for describing the binding capacity of the beads. Here, we define criteria that should be considered when performing a fit-for-purpose evaluation of streptavidin beads. We also describe a colorimetric competitive displacement assay, the streptAVIdin binDing capacITY (AVIDITY) assay, a fast, easy, and inexpensive absorbance-based method to measure the binding capacity of streptavidin beads, which can be used to compare different products and evaluate variation among many of the same product. We expect that the fit-for-purpose criteria and the AVIDITY assay will benefit users across disciplines to make informed decisions regarding the most apt streptavidin bead products for their own experiments.

SUBMITTER: Berg Luecke L

PROVIDER: S-EPMC8116117 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies.

Berg Luecke Linda L Gundry Rebekah L RL

Journal of proteome research 20201203 2

The streptavidin-based enrichment of biotin-tagged molecules is a common methodology that is routinely used across multiple disciplines in biomedical research. Numerous and varied formats of immobilized streptavidin and related proteins are available, but predicting which product is most apt for a given application is complicated by the fact that there are numerous technical considerations and no universal reporting standards for describing the binding capacity of the beads. Here, we define crit ...[more]

PMID: 33270449

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Project description:The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.

Dataset Information

Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies.

Publications

Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies.

Similar Datasets

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