A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein

Nat Methods. 2016 Sep;13(9):763-9. doi: 10.1038/nmeth.3935. Epub 2016 Aug 1.

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.

MeSH terms

  • Biliverdine / chemistry
  • Biosensing Techniques*
  • Cell Cycle / physiology
  • Escherichia coli / genetics
  • HEK293 Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / isolation & purification*
  • Luminescent Proteins / radiation effects
  • Mutation
  • Phycocyanin / chemistry*
  • Phycocyanin / metabolism
  • Protein Conformation
  • Protein Stability
  • Protein Subunits
  • Red Fluorescent Protein
  • Trichodesmium / metabolism*

Substances

  • Luminescent Proteins
  • Protein Subunits
  • allophycocyanin
  • Phycocyanin
  • Biliverdine