Executive Summary : | Modern bioconjugation approaches have been successful due to the development of chemoselective organic reactions that form stable covalent linkages under physiological conditions. Protein bioconjugation was traditionally performed in vitro on pure proteins, with the resulting bioconjugates used for both in vitro and in vivo applications. However, there is a lack of facile approaches for in cellulo bioconjugation, which could open doors to powerful applications, such as studying protein dynamics and activity in cells or animals with unprecedented spatiotemporal control. The proposed bioconjugation strategy enables facile protein modification in both in vitro settings on pure proteins and in cellulo on endogenous proteins. The approach involves recombinantly producing proteins appended with N-terminal cysteine residues and labeling them with fluorescent tags using 1,2-aminothiol-triggered chemistry. This approach can be used for labeling proteins such as eGFP, MPB, streptavidin, and human carbonic anhydrase. The proposed approach involves the installation of a 1,2-aminothiol moiety on proteins of interest (POI) without compromising protein activity. A probe consisting of a ligand of the POI, a cleavable thiazolidine moiety, and an electrophile is designed to achieve this goal. The probe-POI complex is generated by a ligand-driven selective recruitment of the probe to the POI, followed by a proximity-driven nucleophilic attack of neighboring amino acid side chains of the POI on the electrophilic moiety. The pendant 1,2-aminothiol moiety is then utilized for fluorescent labelling. |