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Lanthanide-binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodology for the identification of new LBT sequences with high affinity for Tb(3+) ions and intense luminescence properties. This methodology utilizes solid-phase split-and-pool combinatorial peptide synthesis. Orthogonally cleavable linkers allow an efficient two-step screening procedure. The initial screen avoids the interference caused by on-bead screening by photochemically releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined aqueous solution. Employment of this methodology on a series of focused combinatorial libraries yielded a linear peptide sequence of 17 encoded amino acids that demonstrated a 140-fold increase in affinity (57 nM dissociation constant, K(D)) over previously reported lanthanide-binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2-nM apparent dissociation constant for Tb(3+) ions.Supporting information for this article is available on the WWW under http://www.chemphyschem.org or from the author.

The cover picture shows the chemistry and biology used to develop a new protein labeling strategy based on lanthanide-binding peptides. Focused peptide libraries are screened to identify oligopeptide motifs of dual function: the peptides bind TbIII with remarkably high affinity and promote intense TbIII luminescence. The peptide sequences are then genetically encoded to create recombinant fusion proteins that possess a site-specific and minimally invasive fluorophore. Further information can be found in the two articles by Imperiali and co-workers on pp. 265–271 and pp. 272–276.