FIG 1.

GCE and click labeling of HIV-1 CA. (a) Experimental scheme for GCE and click-labeling. The system used here requires the introduction of an amber stop codon (UAG) at a specific site into the CA coding sequence. A genetically engineered bio-orthogonal tRNA/aminoacyl-tRNA synthetase (PylRS) pair mediates incorporation of a noncanonical amino acid (ncAA) at the chosen position. In a second step, a highly reactive group of the ncAA is covalently linked to a fluorophore carrying a cognate reactive group (e.g., a tetrazine group reacting with a cyclopropene group at the ncAA via strain-promoted inverse electron-demand Diels-Alder cycloaddition [SPIEDAC]). Image created with BioRender.com. (b) The tetrazine-derivative of silicon rhodamine (Tet-SiR) reacts with the strained alkene of the ncAA cyclopropene-l-lysine (CpK) via SPIEDAC. The open SiR conformation results in fluorescence (highlighted in red).