liu.seSearch for publications in DiVA
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Folded polypeptide scaffolds for biosensor and biochip applications: design, synthesis, functionalisation and characterisation
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 96 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 848
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-43337Local ID: 73560ISBN: 91-7373-762-3 (print)OAI: oai:DiVA.org:liu-43337DiVA: diva2:264196
Public defence
2003-12-12, Hörsal Planck, Fysikhuset, Linköpings Universitet, Linköping, 09:15 (Swedish)
Opponent
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2013-01-30
List of papers
1. 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
Open this publication in new window or tab >>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
Show others...
2001 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 296, no 2, 188-196 p.Article in journal (Refereed) 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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25364 (URN)10.1006/abio.2001.5301 (DOI)9807 (Local ID)9807 (Archive number)9807 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13
2. Designed, folded polypeptide scaffolds that combine key biosensing events of recognition and reporting
Open this publication in new window or tab >>Designed, folded polypeptide scaffolds that combine key biosensing events of recognition and reporting
Show others...
2002 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 67, no 9, 3120-3123 p.Article in journal (Refereed) Published
Abstract [en]

No abstract available.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-42053 (URN)10.1021/jo010954n (DOI)59995 (Local ID)59995 (Archive number)59995 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
3. A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis
Open this publication in new window or tab >>A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis
Show others...
2004 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 10, no 10, 2375-2385 p.Article in journal (Refereed) Published
Abstract [en]

A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix–loop–helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02–3 μM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-42052 (URN)10.1002/chem.200305391 (DOI)59994 (Local ID)59994 (Archive number)59994 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
4. Designed, functionalized helix-loop-helix motifs that bind human carbonic anhydrase II: a new class of synthetic receptor molecules
Open this publication in new window or tab >>Designed, functionalized helix-loop-helix motifs that bind human carbonic anhydrase II: a new class of synthetic receptor molecules
2004 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 14, 4464-4465 p.Article in journal (Refereed) 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.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-45770 (URN)10.1021/ja038799c (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
5. Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold
Open this publication in new window or tab >>Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold
Show others...
2005 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, no 6, 2480-2487 p.Article in journal (Refereed) Published
Abstract [en]

Designed, synthetic polypeptides that assemble into four-helix bundles upon dimerization in solution were studied with respect to folding on planar gold surfaces. A model system with controllable dimerization properties was employed, consisting of negatively and positively charged peptides. Circular dichroism spectroscopy and surface plasmon resonance based measurements showed that at neutral pH, the peptides were able to form heterodimers in solution, but unfavorable electrostatic interactions prevented the formation of homodimers. The dimerization propensity was found to be both pH- and buffer-dependent. A series of infrared absorption−reflection spectroscopy experiments of the polypeptides attached to planar gold surfaces revealed that if the negatively charged peptide was immobilized from a loading solution where it was folded, its structure was retained on the surface provided it had a cysteine residue available for anchoring to gold. If it was immobilized as random coil, it remained unstructured on the surface but was able to fold through heterodimerization if subsequently exposed to a positively charged polypeptide. When the positively charged peptide was immobilized as random coil, heterodimerization could not be induced, probably because of high-affinity interactions between the charged primary amine groups and the gold surface. These observations are intended to pave the way for future engineering of functional surfaces based on polypeptide scaffolds where folding is known to be crucial for function.

Place, publisher, year, edition, pages
ACS Publications, 2005
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:liu:diva-15115 (URN)10.1021/la048029u (DOI)
Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2017-12-07Bibliographically approved

Open Access in DiVA

No full text

Authority records BETA

Enander, Karin

Search in DiVA

By author/editor
Enander, Karin
By organisation
ChemistryThe Institute of Technology
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 158 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf