Intracellular Protein-Labeling Probes for Multicolor Single-Molecule Imaging of Immune Receptor-Adaptor Molecular Dynamics

Ryota Sato, Jun Kozuka, Masahiro Ueda, Reiko Mishima, Yutaro Kumagai, Akimasa Yoshimura, Masafumi Minoshima, Shin Mizukami, Kazuya Kikuchi

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

Single-molecule imaging (SMI) has been widely utilized to investigate biomolecular dynamics and protein-protein interactions in living cells. However, multicolor SMI of intracellular proteins is challenging because of high background signals and other limitations of current fluorescence labeling approaches. To achieve reproducible intracellular SMI, a labeling probe ensuring both efficient membrane permeability and minimal non-specific binding to cell components is essential. We developed near-infrared fluorescent probes for protein labeling that specifically bind to a mutant β-lactamase tag. By structural fine-tuning of cell permeability and minimized non-specific binding, SiRcB4 enabled multicolor SMI in combination with a HaloTag-based red-fluorescent probe. Upon addition of both chemical probes at sub-nanomolar concentrations, single-molecule imaging revealed the dynamics of TLR4 and its adaptor protein, TIRAP, which are involved in the innate immune system. Statistical analysis of the quantitative properties and time-lapse changes in dynamics revealed a protein-protein interaction in response to ligand stimulation.

Original languageEnglish
Pages (from-to)17397-17404
Number of pages8
JournalJournal of the American Chemical Society
Volume139
Issue number48
DOIs
Publication statusPublished - 2017 Dec 6

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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