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

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.

Optical Manipulation of Subcellular Protein Translocation Using a Photoactivatable Covalent Labeling System

“…We have previously developed acovalent protein-labeling system, named the BL-tag system, which utilizes am utant (E166N) TEM-1 b-lactamase (BL-tag) and b-lactam ligands. [24][25][26][27] One of the characteristics of the BL-tag system is that various b-lactam antibiotics,s uch as penicillin and cephalosporin, can be used as specific ligands.T he common structure of the ligands contains ac arboxylate residue,a nd the esterification of the carboxylate leads to areduction in the protein-labeling rate. [28] In addition, the crystal structure of the complex of the mutant E166N b-lactamase and a b-lactam antibiotic suggests that the carboxylate residue plays an important role in complex formation, including the formation of hydrogen bonds.…”

“…We have previously developed a covalent protein‐labeling system, named the BL‐tag system, which utilizes a mutant (E166N) TEM‐1 β‐lactamase (BL‐tag) and β‐lactam ligands [24–27] . One of the characteristics of the BL‐tag system is that various β‐lactam antibiotics, such as penicillin and cephalosporin, can be used as specific ligands.…”

Covalently Tethered Rhodamine Voltage Reporters for High Speed Functional Imaging in Brain Tissue

“…We present the design, synthesis, and application of genetically targetable tetramethylrhodamine voltage indicators (RhoVR-Halos) that combine the specificity of self-labeling proteins [45][46][47][48][49][50][51] with the favorable photophysics of small-molecule Rhodamine Voltage Reporters. RhoVR-Halos label HaloTag expressing cells and neurons with exceptional selectivity (>30-fold selectivity over untransfected neurons in culture) and record membrane potential changes with high sensitivities (up to 34% ± 2% ΔF/F per 100 mV in HEK cells, 6.7% ± 0.2% ΔF/F per spike in cultured neurons, and 4.3% ± 0.3% per spike for neurons in slice).…”

Near-infrared fluorescent probes: a next-generation tool for protein-labeling applications

“…35 Our group developed SiR-based NIR uorescent probes for intracellular protein labeling using BL-tag as a protein tag and applied them for multicolor single-molecule imaging inside living cells. 60 We developed a series of membrane-permeable probes SiRcB(n) (n ¼ 2, 4, 6) ( Fig. 7B), where SiR attached as a NIR uorophore, is conjugated to bacampicillin, a prodrug of penicillin, as a BL-tag ligand.…”