A powerful combinatorial screen to identify high-affinity terbium(III)-binding peptides.
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.
Nitz
Mark
M
Department of Chemistry Massachusetts Institute of Technology Cambridge, MA 02139, USA.
Franz
Katherine J
KJ
Maglathlin
Rebecca L
RL
Imperiali
Barbara
B
D000595:Amino Acid Sequence; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D016384:Consensus Sequence; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D013725:Terbium / Q000737:chemistry*
D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013725:Terbium