Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals

Chem Biol. 2009 Nov 25;16(11):1169-79. doi: 10.1016/j.chembiol.2009.10.009.

Abstract

Fluorescent proteins have become valuable tools for biomedical research as protein tags, reporters of gene expression, biosensor components, and cell lineage tracers. However, applications of fluorescent proteins for deep tissue imaging in whole mammals have been constrained by the opacity of tissues to excitation light below 600 nm, because of absorbance by hemoglobin. Fluorescent proteins that excite efficiently in the "optical window" above 600 nm are therefore highly desirable. We report here the evolution of far-red fluorescent proteins with peak excitation at 600 nm or above. The brightest one of these, Neptune, performs well in imaging deep tissues in living mice. The crystal structure of Neptune reveals a novel mechanism for red-shifting involving the acquisition of a new hydrogen bond with the acylimine region of the chromophore.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Crystallography, X-Ray
  • Fluorescent Dyes / chemistry*
  • Hydrogen Bonding
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • neptune protein
  • red fluorescent protein

Associated data

  • PDB/3IP2
  • PDB/FN565568
  • PDB/FN565569
  • PDB/GU189532
  • PDB/GU189533