title: A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein.
abstract: Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California, USA.
authors.forename: Erik A
authors.initials: EA
authors.lastname: Rodriguez
authors.affiliation: School of Medicine, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Geraldine N
authors.initials: GN
authors.lastname: Tran
authors.affiliation: Howard Hughes Medical Institute, La Jolla, California, USA.
authors.forename: Larry A
authors.identifier: 0000-0001-7759-0430
authors.initials: LA
authors.lastname: Gross
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Crisp
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA.|Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Shu
authors.affiliation: School of Medicine, University of Tasmania, Hobart, Tasmania, Australia.
authors.forename: John Y
authors.initials: JY
authors.lastname: Lin
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California, USA.|Howard Hughes Medical Institute, La Jolla, California, USA.
authors.forename: Roger Y
authors.initials: RY
authors.lastname: Tsien
mesh_terms: D001664:Biliverdine / Q000737:chemistry; D015374:Biosensing Techniques; D002453:Cell Cycle / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000528:radiation effects; D009154:Mutation; D010798:Phycocyanin / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D021122:Protein Subunits; D000072359:Trichodesmium / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D001664:Biliverdine; D008164:Luminescent Proteins; D010798:Phycocyanin; D021122:Protein Subunits; D000097573:Red Fluorescent Protein; C002497:allophycocyanin