ABSTRACT
Affinity labeling of a target protein is a powerful method for chemical biology studies. However, it is still difficult to label intracellular proteins efficiently in living cells. We propose the novel design strategy of a reactive group-embedded affinity labeling reagent for efficient protein labeling. With FKBP12 as the model target protein, the ligand binding pocket-oriented labeling reagent could label intracellular protein, whereas protein surface-oriented reagent was ineffective for labeling in living cells, partially because of the intracellular protein fluctuation under the macromolecular crowding effects. These results provide new insight for efficient intracellular protein labeling.
Subject(s)
Affinity Labels/chemistry , Tacrolimus Binding Protein 1A/analysis , Dose-Response Relationship, Drug , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
In plant biology, calcium ions are involved in a variety of intriguing biological phenomena as a secondary messenger. However, most conventional calcium indicators are not applicable for plant cells because of the difficulty with their localization control in plant cells. We here introduce a method to monitor spatiotemporal Ca(2+) dynamics in living plant cells based on linking the synthetic calcium indicator Calcium Green-1 to a natural product-based protein ligand. In a proof-of-concept study using cultured BY-2 cells overexpressing the target protein for the ligand, the ligand-tethered probe accumulated in the cytosol and nucleus, and enabled real-time monitoring of the cytosolic and nucleus Ca(2+) dynamics under the physiological condition. The present strategy using ligand-tethered fluorescent sensors may be successfully applied to reveal the spatiotemporal dynamics of calcium ions in living plant cells.
Subject(s)
Calcium/analysis , Calcium/metabolism , Fluorescent Dyes/analysis , Nicotiana/metabolism , Spatio-Temporal Analysis , Cells, Cultured , Cytosol/chemistry , Cytosol/metabolism , Fluorescent Dyes/chemistry , Ligands , Microscopy, Fluorescence , Molecular Structure , Organic Chemicals/analysis , Organic Chemicals/chemical synthesis , Organic Chemicals/chemistry , Nicotiana/cytologyABSTRACT
12-O-ß-D-Glucopyranosyljasmonic acid (JAG, 1) induces nyctinastic leaf-folding of Samanea saman. The SAR studies of 1 revealed the unique role of its glycone moiety. Biological activity and the target affinity of 1 were affected by the stereochemistry of the glycone moiety. JAG belongs to a unique class of ligands in which the structure of the glycone moiety is involved in the molecular recognition by the target protein.
