The HaloTag as a general scaffold for far-red tunable chemigenetic indicators

Nat Chem Biol. 2021 Jun;17(6):718-723. doi: 10.1038/s41589-021-00775-w. Epub 2021 Apr 1.

Abstract

Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Action Potentials / drug effects
  • Animals
  • Bioengineering
  • Calcium / chemistry
  • Cells, Cultured
  • Crystallography, X-Ray
  • Electrophysiological Phenomena
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • Hydrolases / chemical synthesis
  • Hydrolases / chemistry*
  • Kinetics
  • Molecular Conformation
  • Molecular Structure
  • Neurons / drug effects
  • Primary Cell Culture
  • Proteins / chemistry
  • Rats
  • Rhodamines

Substances

  • Fluorescent Dyes
  • Proteins
  • Rhodamines
  • Hydrolases
  • haloalkane dehalogenase
  • Calcium