title: Fluorophore targeting to cellular proteins via enzyme-mediated azide ligation and strain-promoted cycloaddition.
abstract: Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue. Cambridge, Massachusetts 02139, USA.
authors.forename: Jennifer Z
authors.initials: JZ
authors.lastname: Yao
authors.forename: Chayasith
authors.initials: C
authors.lastname: Uttamapinant
authors.forename: Andrei
authors.initials: A
authors.lastname: Poloukhtine
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Baskin
authors.forename: Julian A
authors.initials: JA
authors.lastname: Codelli
authors.forename: Ellen M
authors.initials: EM
authors.lastname: Sletten
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Popik
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Ting
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003500:Cyclization; D034242:Cyclooctanes / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation
chemical_list: D001386:Azides; D034242:Cyclooctanes; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C490345:lplA protein, E coli; D008025:Ligases