Advancing biophysical methods to study protein conformational changes requires overcoming challenges related to site-specific labeling. Transition-metal ion FRET (tmFRET) provides exceptionally high-resolution information on ligand dependent protein structural changes and dynamics associated with those changes. To address challenges associated with labeling for tmFRET, we employed genetic code expansion (GCE) to incorporate a noncanonical metal-chelating amino acid—3-(2,2'-Bipyridin-5-Yl)-L-Alanine (bpyA) into maltose-binding protein (MBP). BpyA is a versatile metal-binding group with sub-micromolar affinity for transition metal acceptors including Cu(II) and Ni(II). We validated the site-specific incorporation of bpyA into MBP-K295bpyA expressed in E. coli using western blot analysis and mass spectrometry (LC-MS/MS). We then labeled a single cysteine at site 237 with BODIPY-MTS as a FRET donor (MBP-T237C-BODIPY-K295bpyA). As expected, the addition of free Cu(II) decreased the steady-state fluorescence of BODIPY. This indicates bpyA binds Cu(II) forming Cu(bpyA) 2+ , an effective tmFRET acceptor. We then conducted time-resolved tmFRET experiments using time correlated single photon counting (TCSPC) to measure the lifetime of BODIPY in the presence and absence of Cu(II). We extracted donor-acceptor distance distributions by fitting fluorescence lifetime data in both the apo and maltose-bound (holo) states of MBP. The fitting revealed average distances of 18Å and 12Å in the absence and presence of maltose, respectively, with narrow distribution widths. These data are consistent with known structures of MBP. These results suggest metal-binding bpyA is a powerful tool for introducing a metal acceptor site for tmFRET. In the future, donor fluorophores could also be incorporated as noncanonical amino acids, providing cysteine-independent tmFRET labeling.
DeFreest et al. (Sun,) studied this question.