title: A Unique Genetically Encoded FRET Pair in Mammalian Cells.
abstract: Förster resonance energy transfer (FRET) between two suitable fluorophores is a powerful tool to monitor dynamic changes in protein structure in vitro and in vivo. The ability to genetically encode a FRET pair represents a convenient "labeling-free" strategy to incorporate them into target protein(s). Currently, the only genetically encoded FRET pairs available for use in mammalian cells use fluorescent proteins. However, their large size can lead to unfavorable perturbations, particularly when two are used at the same time. Additionally, fluorescent proteins are largely restricted to a terminal attachment to the target, which might not be optimal. Here, we report the development of an alternative genetically encoded FRET pair in mammalian cells that circumvents these challenges by taking advantage of a small genetically encoded fluorescent unnatural amino acid as the donor and enhanced green fluorescent protein (EGFP) as the acceptor. The small size of Anap relative to fluorescent proteins, and the ability to co-translationally incorporate it into internal sites on the target protein, endows this novel FRET pair with improved versatility over its counterparts that rely upon two fluorescent proteins.
authors.affiliation: Department of Chemistry, Boston College, 2609 Beacon Street, 246B Merkert Chemistry Center, Chestnut Hill, MA, 02467, USA.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Mitchell
authors.affiliation: Department of Chemistry, Boston College, 2609 Beacon Street, 246B Merkert Chemistry Center, Chestnut Hill, MA, 02467, USA.
authors.forename: Partha Sarathi
authors.identifier: 0000-0002-0516-7307
authors.initials: PS
authors.lastname: Addy
authors.affiliation: Department of Chemistry, Boston College, 2609 Beacon Street, 246B Merkert Chemistry Center, Chestnut Hill, MA, 02467, USA.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Chin
authors.affiliation: Department of Chemistry, Boston College, 2609 Beacon Street, 246B Merkert Chemistry Center, Chestnut Hill, MA, 02467, USA.
authors.forename: Abhishek
authors.identifier: 0000-0002-6231-5302
authors.initials: A
authors.lastname: Chatterjee
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D003584:Cytological Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Anap; EGFP; FRET; genetic code expansion; unnatural amino acid