Genetically Encoded Click Chemistry for Single-Molecule FRET of Proteins

Publication type:
Chapter
Metadata:
Autoren
Autoren-URL
https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000318445900009&DestLinkType=FullRecord&DestApp=WOS_CPL
DOI
10.1016/B978-0-12-407239-8.00009-4
ISBN-13
978-0-12-407239-8
Paginierung
169 - 187
Buchtitel
LABORATORY METHODS IN CELL BIOLOGY: IMAGING
Datum der Veröffentlichung
2012
Status
Published
Titel
Genetically Encoded Click Chemistry for Single-Molecule FRET of Proteins
Ausgabe der Zeitschrift
113

Data source: Web of Science (Lite)

Other metadata sources:
Autoren
DOI
10.1016/b978-0-12-407239-8.00009-4
Paginierung
169 - 187
Buchtitel
Laboratory Methods in Cell Biology - Imaging
Datum der Veröffentlichung
2013
Status
Published
Herausgeber
Elsevier
Herausgeber URL
http://dx.doi.org/10.1016/b978-0-12-407239-8.00009-4
Datum der Datenerfassung
2018
Titel
Genetically Encoded Click Chemistry for Single-Molecule FRET of Proteins

Data source: Crossref

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.
Addresses
  • EMBL, Structural and Computational Biology Unit, Heidelberg, Germany.
Autoren
DOI
10.1016/b978-0-12-407239-8.00009-4
Open access
false
Schlüsselwörter
  • Escherichia coli
  • Maleimides
  • Muramidase
  • Lysine
  • Proteins
  • Recombinant Proteins
  • Viral Proteins
  • Fluorescent Dyes
  • Fluorescence Resonance Energy Transfer
  • Staining and Labeling
  • Amino Acid Substitution
  • Mutagenesis, Site-Directed
  • Genetic Vectors
  • Fenofibrate
  • Click Chemistry
Medium
Print
Paginierung
169 - 187
Datum der Veröffentlichung
2013
Status
Published
Datum der Datenerfassung
2013
Titel
Genetically encoded click chemistry for single-molecule FRET of proteins.
Ausgabe der Zeitschrift
113

Data source: Europe PubMed Central

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.
Autoren
Autoren-URL
https://www.ncbi.nlm.nih.gov/pubmed/23317903
DOI
10.1016/B978-0-12-407239-8.00009-4
Schlüsselwörter
  • Amino Acid Substitution
  • Click Chemistry
  • Escherichia coli
  • Fenofibrate
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes
  • Genetic Vectors
  • Lysine
  • Maleimides
  • Muramidase
  • Mutagenesis, Site-Directed
  • Proteins
  • Recombinant Proteins
  • Staining and Labeling
  • Viral Proteins
Paginierung
169 - 187
Datum der Veröffentlichung
2013
Status
Published
Datum, an dem der Datensatz öffentlich gemacht wurde
2013
Titel
Genetically encoded click chemistry for single-molecule FRET of proteins.
Ausgabe der Zeitschrift
113

Data source: PubMed

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