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Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor
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.
Biacore AB, Uppsala, Sweden.
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2001 (Engelska)Ingår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 296, nr 2, s. 188-196Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s−1. For the mutants, dissociation rates were faster (0.022–0.025 s−1), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.

Ort, förlag, år, upplaga, sidor
2001. Vol. 296, nr 2, s. 188-196
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:liu:diva-25364DOI: 10.1006/abio.2001.5301Lokalt ID: 9807OAI: oai:DiVA.org:liu-25364DiVA, id: diva2:245693
Tillgänglig från: 2009-10-07 Skapad: 2009-10-07 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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Svedhem, SofiaEnander, KarinKarlsson, MartinLiedberg, BoMårtensson, Lars-GöranSjöstrand, Sven-ErikSvensson, StefanCarlsson, UnoLundström, Ingemar

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Svedhem, SofiaEnander, KarinKarlsson, MartinLiedberg, BoMårtensson, Lars-GöranSjöstrand, Sven-ErikSvensson, StefanCarlsson, UnoLundström, Ingemar
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