These data show that the tri-histidine mutant provides the most robust and sensitive metal binding site which modulates the fluorescence of mEmerald. The specific Kd of the engineered 3H site was determined with a two-site binding model used in previous tmFRET experiments. The first site accounts for the low affinity metal quenching seen in the FP controls and the second site accounts for the specific engineered site. Figure 1C shows the emission spectra of the six FP variants we used in this study: EBFP2, mCerulean3, mEmerald, mVenus, mApple, and mKate2. Each is relatively bright, well folded and monomeric, and their emission spectra overlap the absorbance spectrum of colored transition metal ion FRET acceptors including copper, cobalt, and nickel. For each FP variant we made surface-exposed tri-histidine mutants at similar positions to the iq-mEmerald construct. Aside from the mutant based on mKate2, the excitation, emission, quantum yield, and relative brightness values of these engineered proteins were similar to their parent protein counterparts. In the nickel-bound structure, nickel is coordinated by the two engineered histidines and an aspartate from a neighboring iq-mEmerald in the crystal lattice. These three residues form an imperfect tetrahedron with the metal at its center. The D117 metal contact is a crystal-packing artifact because iq-mEmerald is a monomer in solution in the presence or absence of metals as indicated by analytical ultracentrifugation data. The third engineered histidine points away from the metal and is blocked from interacting with the ion by the D117 residue from the adjacent FP. The increased affinity in the mEmerald-3H over the mEmerald-2H mutant in our fluorescence data, however, indicates that H147 does interact with the metal outside the context of the crystal. The nickel is spaced 2 A ˚ from the un-protonated nitrogens of the histidine imidazoles and 2.5 A ˚ from the oxygen of D117. The metal ion position in the zinc-bound structure is similar. In both metalbound structures the two histidines rotate towards each other to bind to the metal ion compared with the metal-free structure. The metal ions are positioned to the closest and farthest non-hydrogen atom in the chromophore. Aside from the position of the histidines, the only other difference in the structures was the orientation of Y145. This tyrosine rotates 180u away from the chromophore and is surface exposed in the zinc-bound structure compared to the nickel and apo structures. Because there is no substantial fluorescence change upon zinc binding, the functional relevance of this zinc-specific structural feature is unknown. While single iq-FPs can respond to metal ion concentrations in titration experiments it is difficult to quantitatively measure steadystate levels of ions without a reference marker.