TEC IT OR LEAVE IT: SITE-SPECIFIC FLUORESCENT LABELLING OF ALKENES

Abstract

The ability to selectively label proteins with fluorescent tags is essential for studying biological processes in real time. This project explored thiol–ene click (TEC) chemistry as a site-specific, bioorthogonal protein labelling strategy using genetically encoded terminal alkene groups. Four alkene-containing noncanonical amino acids (ncAAs) were screened for incorporation via amber codon suppression, with S-allyl-cysteine (SAC) and S-2-amino-hept-6-enoic acid (SahA) selected for further study. These ncAAs were incorporated into two protein scaffolds, LmrR and PFE, at solvent-exposed positions, followed by labelling using a novel thiol-containing fluorophore, AbzSH. The TEC reaction was optimized using LmrR (V15SAC), with 0.5 mM AbzSH and 10 minutes of UV exposure identified as optimal conditions. Labelling was highly specific and efficient, confirmed via SDS-PAGE, in-gel fluorescence, and LC-MS. Importantly, no offtarget labelling was observed in wild-type proteins or in E. coli lysates, demonstrating excellent chemoselectivity, even in complex mixtures. Although PFE variants failed to show reactivity, likely due to buried alkene groups, these results validate the TEC platform for precise protein labelling and pave the way for future applications in live-cell imaging.