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Bifunctional Avidin with Covalently Modifiable Ligand Binding Site
- Source :
- PLoS ONE, PLoS ONE, Vol 6, Iss 1, p e16576 (2011)
- Publication Year :
- 2011
-
Abstract
- The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (strept)avidin to improve the existing applications. Even so, (strept)avidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces. Public Library of Science
- Subjects :
- Streptavidin
Macromolecular Assemblies
Proteomics
Biophysics
lcsh:Medicine
Biotin
Protein Engineering
Ligands
Biochemistry
Protein Chemistry
Biokemia, solu- ja molekyylibiologia - Biochemistry, cell and molecular biology
03 medical and health sciences
chemistry.chemical_compound
Chemical Biology
Point Mutation
Sulfhydryl Compounds
Binding site
Biomacromolecule-Ligand Interactions
lcsh:Science
Bifunctional
Maleimide
Biology
030304 developmental biology
0303 health sciences
Multidisciplinary
Binding Sites
biology
lcsh:R
030302 biochemistry & molecular biology
Proteins
Ligand (biochemistry)
Avidin
Combinatorial chemistry
Recombinant Proteins
Cross-Linking Reagents
chemistry
Small Molecules
Biotinylation
Bionanotechnology
biology.protein
lcsh:Q
Genetic Engineering
Research Article
Biotechnology
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- PLoS ONE, PLoS ONE, Vol 6, Iss 1, p e16576 (2011)
- Accession number :
- edsair.doi.dedup.....8c58c3ca63a18b6b3e4bb0160bd96e91