title: Genetically encoded click chemistry for single-molecule FRET of proteins.
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.
authors: EMBL, Structural and Computational Biology Unit, Heidelberg, Germany.
authors: Swati
authors: S
authors: Tyagi
authors: Edward A
authors: EA
authors: Lemke
mesh_terms: D019943:Amino Acid Substitution; D057930:Click Chemistry; D004926:Escherichia coli; D011345:Fenofibrate; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D005822:Genetic Vectors; D008239:Lysine / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013194:Staining and Labeling; D014764:Viral Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D008301:Maleimides; D011506:Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C043592:maleimide; D009113:Muramidase; D008239:Lysine; D011345:Fenofibrate