Measuring protein conformational changes with genetically encoded bipyridylalanine and transition-metal ion FRET

Transition-metal ion Förster resonance energy transfer (tmFRET) measures short-range distances and conformational changes in proteins, but its distance sensitivity depends on the donor– acceptor pair. Here, we establish the metal-chelating noncanonical amino acid bipyridylalanine (bpyA) as a flexible, genetically encoded acceptor platform whose Förster distance (R ₀ ) can be tuned without changing protein labeling. Cu²⁺, Cu²⁺ complexed with nitrilotriacetic acid (NTA), and Fe²⁺ form distinct acceptors at the same bpyA site, allowing distance sensitivity to be adjusted by changing the solution conditions. We incorporated bpyA at two sites in maltose binding protein (MBP) and labeled nearby engineered cysteines with the donor fluorophore BODIPY. The resulting [Cu(bpyA)]²⁺, [CuNTA(bpyA)]²⁺, and [Fe(bpyA)]²⁺ acceptors provided R ₀ values ranging from 13.9 to 21.2 Å. Analysis of time-resolved tmFRET data recovered donor–acceptor distance distributions and maltose-dependent changes consistent with structural predictions. At the 237/295 site pair, both copper and iron acceptors resolved the maltose-dependent decrease in distance. At the 309/322 site pair, [Cu(bpyA)]²⁺ did not appreciably resolve the distance increase, whereas adding NTA shifted R ₀ toward shorter distances and enabled detection of an increase from 12.1 to 14.4 Å. These results demonstrate that a single bpyA-labeled protein preparation supports multiple tmFRET acceptors, providing a cysteine-independent acceptor-labeling strategy with adjustable distance sensitivity.