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Studies on the biotin-binding sites of avidin and streptavidin. A chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues.

ABSTRACT: We applied the protein photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) method to explore the conformation of the side chains of tyrosine, tryptophan and histidine residues in three biotin-binding proteins. The c.i.d.n.p. spectra of avidin, streptavidin and 'core' streptavidin were compared with those of their complexes with biotin and its derivatives. The data indicate that the single tyrosine residue (Tyr-33) of avidin is clearly inaccessible to the triplet flavin photo-c.i.d.n.p. probe. The same holds for all tryptophan and histidine side chains. Although the analogous Tyr-43 residue of streptavidin is also buried, at least three of the other tyrosine residues of this protein are exposed. The same conclusions apply to the truncated form of the protein, core streptavidin. As judged by the photo-c.i.d.n.p. results, complexing of avidin and streptavidin with biotin, N-epsilon-biotinyl-L-lysine (biocytin) or biotinyltyrosine has little or no effect on tyrosine accessibility in these proteins. Biotinyltyrosine can be used to probe the depth of the corresponding binding site. The accessibility of the tyrosine side chain of biotinyltyrosine in the complex demonstrates the exquisite fit of the biotin-binding cleft of avidin: only the biotin moiety appears to be accommodated, leaving the tyrosine side chain exposed.

SUBMITTER: Gitlin G

PROVIDER: S-EPMC1138535 | biostudies-other | 1989 Apr

REPOSITORIES: biostudies-other

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Project description:Affinity interactions between the small molecule biotin and the protein avidin have been used extensively to functionalize biomaterials. More recently, researchers have leveraged the changes in biotin-avidin affinity that occur upon biotin conjugation to larger molecules to control the release of biotinylated drugs and proteins. However, the effects of biotin-avidin interactions on hydrogel properties have not been thoroughly investigated. The objective of this study was to evaluate the effect of increasing biotin and avidin concentrations on hydrogel swelling properties, as an indicator of crosslinking. Gelatin, selected as a model hydrogel material, was biotinylated at increasing fold molar excesses of biotin with a PEG linker using N-hydroxysuccinimide chemistry. Afterwards, biotinylated gelatin was formed into hydrogels and stabilized with glutaraldehyde. Swelling properties of the biotinylated hydrogels were investigated by conducting swelling studies in different avidin solutions. Increasing the degree of biotinylation caused significant decreases in swelling ratios of the hydrogels in a dose-dependent manner, suggesting increases in crosslinking of the hydrogels. However, increasing avidin concentrations in excess of biotin content did not significantly affect swelling ratios. Moving hydrogels to phosphate-buffered saline following avidin incorporation resulted in increased swelling ratios for hydrogels prepared with a lower concentration of biotin. However, hydrogels prepared with the highest concentration of biotin did not experience increased swelling ratios, implying that the stability of biotin-avidin-mediated crosslinking depends on the number of biotin molecules available for binding. Collectively, these results demonstrate that biotin-avidin interactions control hydrogel swelling properties, and that the magnitude and stability of the effects depend on the biotin concentration. These results have important implications for affinity-based controlled release of biotinylated drugs or proteins from biotin-avidin-crosslinked hydrogels.

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Studies on the biotin-binding sites of avidin and streptavidin. A chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues.

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