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Protein-protein interactions in model systems: design, control of catalytic activity and biosensor applications
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the design of polypeptides, unordered in the monomeric state but capable of folding into helix-loop-helix motifs and dimerise to form four-helix bundles. The goal of the design was to encode them with the capacity to form dimers highly selectively and the ability to carry out molecular functions in the folded state but not in the unordered state, and thus to establish a molecular link between recognition and function. The 42-residue sequences JR2E and JR2K were both shown by CD spectroscopy to adopt unordered conformations under single solute conditions at pH 7 but to form helical conformations in a 1:1 mixture. Analytical ultracentrifugation showed that JR2E and JR2K formed a clean heterodimer and the dissociation constant Kd, measured by CD spectroscopy, was found to be 5 ± 1 μM. Discrimination was enabled by the incorporation of charged residues at the dimer interface in the helical segments of the helix-loop-helix motif. Glutamic acids were incorporated in JR2E and lysines in JR2K, and charge repulsion prevented the monomeric subunits from forming homodimers. In mixtures, however, highly helical heterodimers were formed. The cooperative transition from unordered conformation to heterodimeric four-helix bundle was exploited in the design of a signal response system by incorporating a reactive site, capable of catalysing the hydrolysis of a m-nitrophenyl ester, into the negatively charged polypeptide. In the unfolded state the functionalised polypeptide was virtually inactive but in the folded state, induced by the interaction with JR2K, the substrate was hydrolysed approximately an order of magnitude more efficiently.

Interactions between the designed polypeptides and a functionalised polythiophene polymer were studied and it was found that the conformation of the polymer was controlled by the polypeptides, largely by electrostatic interactions. The negatively charged JR2E forced the polymer to adopt a planar conformation while the positively charged JR2K induced a more twisted conformation of the polymer. The spectral changes coupled to the conformational transitions of the polymer were used to measure the binding of human Carbonic anhydrase II by JR2E functionalised with a benzenesulphonamide ligand, in demonstration of its use as a tool for high-throughput screening.

JR2E immobilised on gold nanoparticles was shown to form homodimers reversibly under pH control, with affinities large enough to determine the state of aggregation of the gold nanoparticles.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2006.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1037
Keyword [en]
Chemistry, Protein interactions, design, catalysis, biosensors, hybride materials, nanoparticles
Keyword [sv]
Kemi
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:liu:diva-7485ISBN: 91-85523-19-4 (print)OAI: oai:DiVA.org:liu-7485DiVA: diva2:22505
Public defence
2006-09-22, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 09:15 (English)
Opponent
Supervisors
Available from: 2006-09-28 Created: 2006-09-28 Last updated: 2009-04-01Bibliographically approved
List of papers
1. Intrinsically unstructured proteins by design: electrostatic interactions can control binding, folding and function of a helix-loop-helix heterodimer
Open this publication in new window or tab >>Intrinsically unstructured proteins by design: electrostatic interactions can control binding, folding and function of a helix-loop-helix heterodimer
2006 (English)Article in journal (Refereed) Submitted
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-14038 (URN)
Available from: 2006-09-28 Created: 2006-09-28
2. Self-assembly of synthetic peptides control conformation and optical properties of a zwitterionic polythiophene derivative
Open this publication in new window or tab >>Self-assembly of synthetic peptides control conformation and optical properties of a zwitterionic polythiophene derivative
2003 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 18, 10170-10174 p.Article in journal (Refereed) Published
Abstract [en]

The optical transitions of a chiral, three-substituted polythiophene with an amino acid function can be tuned by interactions with synthetic peptides. The addition of a positively charged peptide with a random-coil formation will force the polymer to adopt a nonplanar conformation, and the intensity of the emitted light is increased and blue-shifted. After the addition of a negatively charged peptide with a random-coil conformation, the backbone of the polymer adopts a planar conformation and an aggregation of the polymer chains occurs, seen as a red shift and a decrease of the intensity of the emitted light. By adding the positively charged peptide designed to form a four-helix bundle with the negatively charged peptide, the polymer aggregates are disrupted and the intensity of the emitted light is increased because of separation of the polymer chains. This technique could be used as a platform for making novel sensors and biomolecular switches.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-14039 (URN)10.1073/pnas.1834422100 (DOI)
Available from: 2006-09-28 Created: 2006-09-28 Last updated: 2017-12-13
3. Self-assembled quaternary complexes from designed polypeptides, organic polymers and human Carbonic Anhydrase II: implications for high-affinity screening of small molecule libraries in drug discovery
Open this publication in new window or tab >>Self-assembled quaternary complexes from designed polypeptides, organic polymers and human Carbonic Anhydrase II: implications for high-affinity screening of small molecule libraries in drug discovery
Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-14040 (URN)
Available from: 2006-09-28 Created: 2006-09-28 Last updated: 2010-01-13
4. Aggregation-Induced Folding of a de novo Designed Polypeptide Immobilized on Gold Nanoparticles
Open this publication in new window or tab >>Aggregation-Induced Folding of a de novo Designed Polypeptide Immobilized on Gold Nanoparticles
Show others...
2006 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, no 7, 2194 -2195 p.Article in journal (Refereed) Published
Abstract [en]

This communication reports the first steps in the construction of a novel, nanoparticle-based hybrid material for biomimetic and biosensor applications. Gold nanoparticles were modified with synthetic polypeptides to enable control of the particle aggregation state in a switchable manner, and particle aggregation was, in turn, found to induce folding of the immobilized peptides.

Place, publisher, year, edition, pages
ACS Publications, 2006
Keyword
Not aviable
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:liu:diva-14041 (URN)10.1021/ja057056j (DOI)
Available from: 2006-09-28 Created: 2006-09-28 Last updated: 2017-12-13Bibliographically approved

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Rydberg, Johan

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