78025-74-8Relevant academic research and scientific papers
Site-Selective Synthetic Acylation of a Target Protein in Living Cells Promoted by a Chemical Catalyst/Donor System
Hamajima, Wataru,Fujimura, Akiko,Fujiwara, Yusuke,Yamatsugu, Kenzo,Kawashima, Shigehiro A.,Kanai, Motomu
, p. 1102 - 1109 (2019)
Cell biology is tightly regulated by post-translational modifications of proteins. Methods to modulate post-translational modifications in living cells without relying on enzymes or genetic manipulation are, however, largely underexplored. We previously reported that a chemical catalyst (DSH) conjugated with a nucleosome-binding ligand can activate an acyl-CoA and promote site-selective lysine acylation of histones in test tubes. In-cell acylation by this catalyst system is challenging, however, mainly due to the low cell permeability of acyl-CoA and the propensity of DSH to form inactive disulfide. Here, we report a new catalyst system effective for in-cell acylation, comprising a cell-permeable acyl donor and pro-drugged DSH. Using E. coli dihydrofolate reductase and trimethoprim as a model protein and ligand pair, the catalyst system enabled site-selective acylation of the target protein in living cells. The findings will lead to the development of useful chemical biology tools and new therapeutic strategies capable of synthetically modulating post-translational modifications.
A General Strategy for the Semisynthesis of Ratiometric Fluorescent Sensor Proteins with Increased Dynamic Range
Xue, Lin,Prifti, Efthymia,Johnsson, Kai
supporting information, p. 5258 - 5261 (2016/05/19)
We demonstrate how a combination of self-labeling protein tags and unnatural amino acid technology permits the semisynthesis of ratiometric fluorescent sensor proteins with unprecedented dynamic range in vitro and on live cells. To generate such a sensor, a binding protein is labeled with a fluorescent competitor of the analyte using SNAP-tag in conjugation with a second fluorophore that is positioned in vicinity of the binding site of the binding protein using unnatural amino acid technology. Binding of the analyte by the sensor displaces the tethered fluorescent competitor from the binding protein and disrupts fluorescence resonance energy transfer between the two fluorophores. Using this design principle, we generate a ratiometric fluorescent sensor protein for methotrexate that exhibits large dynamic ranges both in vitro (ratio changes up to 32) and on cell surfaces (ratio change of 13). The performance of these semisynthetic sensor proteins makes them attractive for applications in basic research and diagnostics.
