272 ¹ 2003 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 1439-4227/03/04/04 $ 20.00+.50/0 ChemBioChem 2003, 4, 272±276 A Powerful Combinatorial Screen to Identify High-Affinity Terbium(III)-Binding Peptides Mark Nitz, Katherine J. Franz, Rebecca L. Maglathlin, and Barbara Imperiali* [a] Lanthanide-binding tags (LBTs) are protein fusion partners con- sisting 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. Or- thogonally 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 macro- cyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2-nM apparent dissociation constant for Tb 3 ions. KEYWORDS: combinatorial chemistry ¥ fluorescent probes ¥ lanthanides ¥ peptides Introduction There is an ever-increasing need for chemical and physical probes that can be conveniently integrated into proteins to facilitate the analysis of structure and function. Lanthanide- binding tags (LBTs) are protein fusion partners of minimal dimensions capable of harnessing a vast array of lanthanide- dependent biophysical techniques, which include luminescence analyses with Tb 3 and Eu 3 species, [1, 2] NMR spectroscopy (through use of the paramagnetic lanthanides), [3] and X-ray crystallography, which utilizes the strong scattering power of the lanthanide ions. [4] Herein we demonstrate that a series of focused combinatorial libraries, coupled with a novel lumines- cent screening technique can be successfully implemented to identify peptide loops with low-nanomolar dissociation con- stants for Tb 3 ions. These sequences provide the next generation of LBT fusion partners for a broad range of biophysical applications. [5] Incorporation of lanthanide ion binding sites into proteins has previously been accomplished through either chemical labeling of amino acid side chains with lanthanide chelators, [6] or by protein engineering to introduce calcium binding loops, such as the EF-hand motif. [7, 3h] Both of these approaches have limita- tions. Chemical labeling necessitates the presence of a uniquely reactive residue within the protein of interest, as well as the optimization of the labeling chemistry. Generation of a fusion protein with an EF-hand motif is an easier approach, however once the motif is removed from the context of the calcium- binding protein, the lanthanide affinity drops precipitously (10 8 ±10 9 M to 10 5 ±10 6 M). The loss of lanthanide affinity can lead to complications with nonspecific lanthanide bind- ing, competing ligands, and lanthanide-induced protein aggre- gation. [7a, 8] In order to extend the utility of protein-bound lanthanide ions, we sought to optimize the Tb 3 -affinity and luminescent properties of short oligopeptides (including only the encoded amino acids) for use as LBT fusion partners. Two classes of peptide sequence were envisaged as useful for the intended eventual applications: disulfide-constrained peptide loops with maximal lanthanide affinity [1] and cysteine-free peptides for redox-sensitive applications. The studies described herein have resulted in rapid access to unconstrained peptides with 140-fold improvement in Tb 3 affinity over previously synthesized Tb 3 -binding loops, and a constrained peptide with an increase in affinity for terbium ions of over three orders of magnitude relative to the native peptide sequences. Screening Methodology The luminescence of Tb 3 peptide chelates in the presence of a sensitizing tyrosine or tryptophan residue provides a convenient handle for screening lanthanide-binding peptides. However, preliminary experiments indicated that screening of peptides covalently attached to the solid support yielded false-positive luminescent signals caused by interference from the polyethy- lene glycol resin matrix. In order to circumvent this problem, a [a] Prof. B. Imperiali, Dr. M. Nitz, Dr. K. J. Franz, R. L. Maglathlin Department of Chemistry Massachusetts Institute of Technology Cambridge, MA 02139 (USA) Fax: ( 1)617-452-2419 E-mail: imper@mit.edu Supporting information for this article is available on the WWW under http:// www.chemphyschem.org or from the author.