23 research outputs found
Ligand-directed dibromophenyl benzoate chemistry for rapid and selective acylation of intracellular natural proteins
A rapid and selective ligand-directed chemical reaction was developed for the acylation of proteins in living cells on the basis of ligand-directed chemistry. By fine tuning the reactivity and stability of the phenyl ester derivatives, we successfully identified ortho-dibromophenyl benzoate as the optimal reactive motif. It was sufficiently stable in an aqueous buffer, hydrolyzing less than 10% after 13 h of incubation, but reactive enough for efficient and selective protein labeling in living mammalian cells, as well as in vitro (referred to as ligand-directed dibromophenyl benzoate (LDBB) chemistry). Using this chemistry, various fluorophores can be tethered to the target protein directly, which allows fluorescence visualization of the labeled protein in live cells using different colored fluorophore groups (including coumarin, fluorescein and rhodamine). Furthermore, this labeling is applicable to not only an overexpressed protein (E. coli dihydrofolate reductase) but also endogenous human carbonic anhydrase II and XII under living cell conditions. LDBB chemistry is a new entry of ligand-directed protein labeling methods, and should be particularly useful for the imaging of natural proteins in living cells
Specific Cell Surface Protein Imaging by Extended Self-Assembling Fluorescent Turn-on Nanoprobes
Visualization of tumor-specific protein biomarkers on
cell membranes
has the potential to contribute greatly to basic biological research
and therapeutic applications. We recently reported a unique supramolecular
strategy for specific protein detection using self-assembling fluorescent
nanoprobes consisting of a hydrophilic protein ligand and a hydrophobic
BODIPY fluorophore in test tube settings. This method is based on
recognition-driven disassembly of the nanoprobes, which induces a
clear turn-on fluorescent signal. In the present study, we have successfully
extended the range of applicable fluorophores to the more hydrophilic
ones such as fluorescein or rhodamine by introducing a hydrophobic
module near the fluorophore. Increasing the range of available fluorophores
allowed selective imaging of membrane-bound proteins under live cell
conditions. That is, overexpressed folate receptor (FR) or hypoxia-inducible
membrane-bound carbonic anhydrases (CA) on live cell surfaces as cancer-specific
biomarkers were fluorescently visualized using the designed supramolecular
nanoprobes in the turn-on manner. Moreover, a cell-based inhibitor-assay
platform for CA on a live cell surface was constructed, highlighting
the potential applicability of the self-assembling turn-on probes
Intracellular Protein-Responsive Supramolecules: Protein Sensing and In-Cell Construction of Inhibitor Assay System
Supramolecular nanomaterials responsive
to specific intracellular
proteins should be greatly promising for protein sensing and imaging,
controlled drug release or dynamic regulation of cellular processes.
However, valid design strategies to create useful probes are poorly
developed, particularly for proteins inside living cells as targets.
We recently reported a unique supramolecular strategy for specific
protein detection using self-assembling fluorescent probes consisting
of a protein ligand and a fluorophore on the live cell surface, as
well as in test tube settings. Herein, we discovered that our self-assembled
supramolecular probes having a rhodamine derivative (tetramethylrhodamine
or rhodamine-green) can incorporate and stay as less-fluorescent aggregates
inside the living cells, so as to sense the protein activity in a
reversible manner. Using the overexpressed model protein (dihydrofolate
reductase), we demonstrated that this turn-on/off mode is controlled
by selective ligand–protein recognition inside the live cells.
Not only such a model protein, but also endogenous human carbonic
anhydrase and heat shock protein 90 were specifically visualized in
living mammalian cells, by use of the similar ligand-tethered supramolecular
probes. Furthermore, such reversibility allowed us to intracellularly
construct a unique system to evaluate the inhibitors affinity toward
specific endogenous proteins in live cells, highlighting the potential
of dynamic supramolecules as novel intelligent biomaterials
Analysis of Cell-Surface Receptor Dynamics through Covalent Labeling by Catalyst-Tethered Antibody
A general technique for introducing
biophysical probes into selected
receptors in their native environment is valuable for the study of
their structure, dynamics, function, and molecular interactions. A
number of such techniques rely on genetic engineering, which is not
applicable for the study of endogenous proteins, and such approaches
often suffer from artifacts due to the overexpression and bulky size
of the probes/protein tags used. Here we designed novel catalyst-antibody
conjugates capable of introducing small chemical probes into receptor
proteins such as epidermal growth factor receptor (EGFR) and human
epidermal growth factor receptor 2 (HER2) in a selective manner on
the surface of living cells. Because of the selectivity and efficiency
of this labeling technique, we were able to monitor the cellular dynamics
and lifetime of HER2 endogenously expressed on cancer cells. More
significantly, the current labeling technique comprises a stable covalent
bond, which combined with a peptide mass fingerprinting analysis allowed
epitope mapping of antibodies on living cells and identification of
potential binding sites of anti-EGFR affibody. Although as yet unreported
in the literature, the binding sites predicted by our labeling method
were consistently supported by the subsequent mutation and binding
assay experiments. In addition, this covalent labeling method provided
experimental evidence that HER2 exhibits a more dynamic structure
than expected on the basis of crystallographic analysis alone. Our
novel catalyst-antibody conjugates are expected to provide a general
tool for investigating the protein trafficking, fluctuation, and molecular
interactions of an important class of cell-surface receptors on live
cell surfaces