Design of lanthanide fingers: Compact lanthanide-binding metalloproteins

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide-binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp₂Glu₂ metal-coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide-binding peptide incorporated the following key elements: 1) residues with high α-helix and β-sheet propensities in the respective secondary structures; 2) an optimized big box a-helix N-cap; 3) a Schellman α-helix C-cap motif; and 4) an optional D-Pro-Ser type II′ β-turn in the β-hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25-residue peptide that was a general lanthanide-binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er³⁺. CD spectra of the peptide-lanthanide complexes are similar to those of zinc fingers and other bba proteins. Metal binding involves residues from the N-terminal β-hairpin and the C terminal α-helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D-Pro-Ser type II′ β-turn motif could be replaced by Thr-Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF-hand peptide.