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Genetically encoded click chemistry for single-molecule FRET of proteins.
- Source :
-
Methods in cell biology [Methods Cell Biol] 2013; Vol. 113, pp. 169-87. - Publication Year :
- 2013
-
Abstract
- Single molecule Fluorescence Resonance Energy Transfer (FRET) has been widely applied to study structure, function and dynamics of complex biological systems. Labeling of proteins at specific positions with fluorescent dyes is a challenging and key step for any single molecule FRET measurement. Genetic code expansion has facilitated site specific incorporation of unnatural amino acids into proteins. These unnatural amino acid bears bioorthognal functional groups that provide opportunity to install a unique chemical handle into proteins. Propargyllysine is an unnatural amino acid which, when incorporated into a protein, can be exploited to attach commercially available fluorescent azide dyes through copper-catalyzed alkyne-azide cycloaddition click reaction (also known as click reaction). We describe here an optimized strategy to combine synthesis of propargyllysine, its genetic incorporation in the protein and click reaction to site-specifically label the protein with azide derivative of Alexa® 488. Later the protein is labeled at unique cysteine residue via maleimide coupling chemistry with acceptor Alexa® 594 dye to yield double labeled protein as required for any single molecule FRET experiments.<br /> (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Subjects :
- Amino Acid Substitution
Escherichia coli
Fenofibrate
Fluorescent Dyes chemistry
Genetic Vectors
Lysine chemistry
Maleimides chemistry
Muramidase biosynthesis
Muramidase chemistry
Muramidase genetics
Mutagenesis, Site-Directed
Recombinant Proteins biosynthesis
Recombinant Proteins chemistry
Recombinant Proteins genetics
Staining and Labeling
Viral Proteins biosynthesis
Viral Proteins chemistry
Viral Proteins genetics
Click Chemistry
Fluorescence Resonance Energy Transfer
Lysine analogs & derivatives
Lysine chemical synthesis
Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0091-679X
- Volume :
- 113
- Database :
- MEDLINE
- Journal :
- Methods in cell biology
- Publication Type :
- Academic Journal
- Accession number :
- 23317903
- Full Text :
- https://doi.org/10.1016/B978-0-12-407239-8.00009-4