New Orange Ligand-Dependent Fluorescent Reporter for Anaerobic Imaging.
Hannah E ChiaKarl J KoebkeAathmaja A RangarajanNicole M KoropatkinE Neil G MarshJulie Suzanne BiteenPublished in: ACS chemical biology (2021)
Bilin-binding fluorescent proteins like UnaG-bilirubin are noncovalent ligand-dependent reporters for oxygen-free microscopy but are restricted to blue and far-red fluorescence. Here we describe a high-throughput screening approach to provide a new UnaG-ligand pair that can be excited in the 532 nm green excitation microscopy channel. We identified a novel orange UnaG-ligand pair that maximally emits at 581 nm. Whereas the benzothiazole-based ligand itself is nominally fluorescent, the compound binds UnaG with high affinity (Kd = 3 nM) to induce a 2.5-fold fluorescence intensity enhancement and a 10 nm red shift. We demonstrated this pair in the anaerobic fluorescence microscopy of the prevalent gut bacterium Bacteroides thetaiotaomicron and in Escherichia coli. This UnaG-ligand pair can also be coupled to IFP2.0-biliverdin to differentiate cells in mixed-species two-color imaging. Our results demonstrate the versatility of the UnaG ligand-binding pocket and extend the ability to image cells at longer wavelengths in anoxic environments.
Keyphrases
- single molecule
- high resolution
- escherichia coli
- label free
- living cells
- energy transfer
- induced apoptosis
- photodynamic therapy
- quantum dots
- microbial community
- wastewater treatment
- cell cycle arrest
- high speed
- high throughput
- optical coherence tomography
- deep learning
- oxidative stress
- cystic fibrosis
- mass spectrometry
- signaling pathway
- multidrug resistant
- heavy metals
- binding protein
- pi k akt
- klebsiella pneumoniae