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Site-Directed Incorporation of Fluorescent Nonnatural Amino Acids into Streptavidin for Highly Sensitive Detection of Biotin
- Source :
- Biomacromolecules. 1:118-125
- Publication Year :
- 2000
- Publisher :
- American Chemical Society (ACS), 2000.
-
Abstract
- Fluorescent nonnatural amino acids were incorporated into specific positions of streptavidin. The positions of the nonnatural amino acids were directed by a CGGG/CCCG four-base codon/anticodon pair. The nonnatural mutants with a single 2-anthrylalanine at the 22nd, 43rd, 54th, and 120th positions, respectively, were found to bind biotin, indicating that the mutants retained active conformation. The fluorescence intensities of the anthryl groups were relatively insensitive to the positions and the biotin binding when excited at 265 nm. When the anthryl group at the 120th position was excited through energy transfer from tryptophan units, the fluorescence intensity markedly decreased with biotin binding, because of a suppression of the energy transfer. Amino acids carrying 7-methoxycoumarine fluorophore were also incorporated at the 120th position. Their fluorescence quantum yields were very sensitive to the biotin binding. The high sensitivity of the coumarine-labeled streptavidin exemplifies potential applications of fluorescent nonnatural mutants for detecting specific molecules at very low concentrations.
- Subjects :
- Streptavidin
Biotin binding
Fluorophore
Polymers and Plastics
Stereochemistry
Biotin
Bioengineering
Biomaterials
chemistry.chemical_compound
RNA, Transfer
Coumarins
Materials Chemistry
Amino Acids
Fluorescent Dyes
chemistry.chemical_classification
Chemistry
Tryptophan
Fluorescence
Recombinant Proteins
Amino acid
Spectrometry, Fluorescence
Mutation
Transfer RNA
Indicators and Reagents
Subjects
Details
- ISSN :
- 15264602 and 15257797
- Volume :
- 1
- Database :
- OpenAIRE
- Journal :
- Biomacromolecules
- Accession number :
- edsair.doi.dedup.....37e9acf6831f81cfe426274f67aac8a7