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Designed, functionalized helix-loop-helix motifs that bind human carbonic anhydrase II: a new class of synthetic receptor molecules
Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
2004 (Engelska)Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, nr 14, s. 4464-4465Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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 μM 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.

Ort, förlag, år, upplaga, sidor
2004. Vol. 126, nr 14, s. 4464-4465
Nationell ämneskategori
Teknik och teknologier
Identifikatorer
URN: urn:nbn:se:liu:diva-45770DOI: 10.1021/ja038799cOAI: oai:DiVA.org:liu-45770DiVA, id: diva2:266666
Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2017-12-13
Ingår i avhandling
1. Folded polypeptide scaffolds for biosensor and biochip applications: design, synthesis, functionalisation and characterisation
Öppna denna publikation i ny flik eller fönster >>Folded polypeptide scaffolds for biosensor and biochip applications: design, synthesis, functionalisation and characterisation
2003 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

This thesis describes the design, synthesis and evaluation of functional molecular units intended for use in biosensor and microarray applications. A flexible, synthetic helix-loop-helix polypeptide that dimerises to form four-helix bundles was used as a scaffold and was modified with affinity ligands and fluorescent probes to specifically bind a target biomolecule and report on this event in an integrated process. The well-characterised binding of carbonic anhydrase by its benzenesulphonamide inhibitor was employed as a model interaction, and the emission intensity of the probe(s) was found to correlate with carbonic anhydrase concentration. A molecular array, spanning two orders of magnitude in affmity and useful for one-step target quantification, was designed by varying the spacer of the benzenesulphonamide derivative. The scaffold itself was found to contribute to binding, expanding the parameters available for affmity modulation. In a separate study focused on the interaction model system, it was revealed that a destabilising point mutation distant from the carbonic anhydrase active site resulted in faster dissociation rates of the benzenesulphonamide ligand. and that this effect was mediated by increased molecular dynamics caused by destabilisation.

The fluorescence intensity difference displayed by free and target-bound peptides was found to be critically dependent on the position of the probe(s) in the scaffold, showing that the polypeptide fold, providing directionality of incorporated moieties, contributed considerably to peptide function. Dual labelling of the scaffold with different probes in positions where they displayed increased intensity in the corresponding single-probe peptides resulted in a synergistic emission increase upon target protein binding, significantly enhancing sensitivity. The peptides were shown to bind the target protein as monomers, and the molecular basis for sensing was a combination of specific peptide-protein interactions and dimer dissociation. The photochemical crosstalk between the probes was interrupted upon expulsion of one of the monomers upon binding.

Strategies for thiol-dependent attachment of the peptides to modified gold surfaces were explored, and folding of immobilised scaffolds was demonstrated in the case of a model system with controllable dirnerisation properties. Results indicating that the sensing ability was retained upon peptide immobilisation were encouraging and prompted future studies on the relation between peptide structure and function, aiming at successful sensor surface and rnicroarray designs for the identification, quantification and characterisation of a wide variety of target biomolecules.

Ort, förlag, år, upplaga, sidor
Linköping: Linköpings universitet, 2003. s. 96
Serie
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 848
Nationell ämneskategori
Naturvetenskap
Identifikatorer
urn:nbn:se:liu:diva-43337 (URN)73560 (Lokalt ID)91-7373-762-3 (ISBN)73560 (Arkivnummer)73560 (OAI)
Disputation
2003-12-12, Hörsal Planck, Fysikhuset, Linköpings Universitet, Linköping, 09:15 (Svenska)
Opponent
Tillgänglig från: 2009-10-10 Skapad: 2009-10-10 Senast uppdaterad: 2013-01-30

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