Designed, functionalized helix-loop-helix motifs that bind human carbonic anhydrase II: a new class of synthetic receptor molecules.
Polypeptides designed to fold into helix-loop-helix motifs and to dimerize to form four-helix bundles were functionalized by the introduction of a sulfonamide derivative known to bind human carbonic anhydrase II (HCAII) and one or both of the dansyl- and methoxycoumarin fluorescent probes. The 42-residue sequence DC that carries all three substituents in solvent-exposed positions was found to bind HCAII with a dissociation constant of 5 nM in aqueous solution at pH 7. At 2 muM concentration, DC was mainly dimeric in aqueous solution but bound HCAII as a monomer. Upon addition of a large excess of a helix-loop-helix motif without a high-affinity ligand, KE2-Q, a ternary complex was formed between HCAII, DC, and KE2-Q. Hydrophobic interactions between DC and HCAII and coordination of the sulfonamide group to the zinc ion of HCAII contributed cooperatively to binding in a demonstration of the usefulness of folded polypeptide-small organic molecule chimera as novel protein receptors. The DC homodimer was found to be a very sensitive biosensor component due to intermolecular quenching of its fluorescence that was inhibited upon binding to HCAII.
Enander
Karin
K
Department of Chemistry-IFM, Linköping University, 581 83 Linköping, Sweden.
Dolphin
Gunnar T
GT
Baltzer
Lars
L
D000595:Amino Acid Sequence; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D010455:Peptides; D024402:Carbonic Anhydrase II