Intracellular Protein-Labeling Probes for Multicolor Single-Molecule Imaging of Immune Receptor–Adaptor Molecular Dynamics
| Autor: | Yutaro Kumagai, Shin Mizukami, Jun Kozuka, Kazuya Kikuchi, Ryota Sato, Reiko Mishima, Masafumi Minoshima, Akimasa Yoshimura, Masahiro Ueda |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
TIRAP Membrane permeability Cell Color Immune receptor Molecular Dynamics Simulation Ligands 010402 general chemistry 01 natural sciences Biochemistry beta-Lactamases Catalysis 03 medical and health sciences Colloid and Surface Chemistry medicine Fluorescent Dyes Membrane Glycoproteins Staining and Labeling Chemistry Proteins Receptors Interleukin-1 Signal transducing adaptor protein General Chemistry Fluorescence Single Molecule Imaging Molecular biology 0104 chemical sciences Toll-Like Receptor 4 030104 developmental biology medicine.anatomical_structure Molecular Probes Biophysics Intracellular Protein Binding |
| Zdroj: | Journal of the American Chemical Society. 139:17397-17404 |
| ISSN: | 1520-5126 0002-7863 |
| DOI: | 10.1021/jacs.7b08262 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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