BPS2026 – Genetic code expansion to incorporate the metal-binding noncanonical amino acid bipyridyl alanine for transition metal ion FRET

Genetic code expansion is a powerful method to introduce new functionalities to amino acid side chains. It is particularly useful to introduce site-specific labels with very high efficiency. We used genetic code expansion to label maltose binding protein (MBP) with a chelating amino acid side chain, bipyridyl alanine (bpyA), for binding transition metals for transition metal ion FRET (tmFRET). The bpyA side chain is especially useful because it can bind different transition metals, including Cu(II), Co(II), and Ni(II), to give a range of Förster radii when paired with a FRET donor. We used amber codon suppression with a previously published evolved aminoacyl tRNA synthetase/tRNA pair to incorporate bpyA into two different sites in MBP expressed in E. coli. Western blot analysis and mass spectrometry demonstrated that purified MBP-bpyA protein was highly pure, without detectable off-target incorporation or near-cognate suppression. Each MBP-bpyA also included a single cysteine for labeling with the fluorophore BODIPY. Using steady-state and time-resolved fluorescence, we measured FRET between the BODIPY and metal-bound bpyA. We compared the data with predictions based on rotameric ensembles of the donor/acceptor labels and found excellent agreement with known MBP structures in the absence and presence of maltose. This approach is an improvement over binding of transition metals to other motifs because (1) it does not require a cysteine for incorporation of the metal-ion acceptor; (2) it exhibits high labeling efficiency; and (3) the bpyA has sub-micromolar affinity for transition metals.