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Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold
Division of Organic Chemistry, Uppsala University.
Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.ORCID iD: 0000-0002-7001-9415
Division of Organic Chemistry, Department of Chemistry, BMC, Uppsala University, Uppsala, Sweden.
Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
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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. Vol. 21, no 6, 2480-2487 p.
National Category
Other Basic Medicine
Identifiers
URN: urn:nbn:se:liu:diva-15115DOI: 10.1021/la048029uOAI: oai:DiVA.org:liu-15115DiVA: diva2:54434
Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Polypeptide-Based Nanoscale Materials
Open this publication in new window or tab >>Polypeptide-Based Nanoscale Materials
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Self-assembly has emerged as a promising technique for fabrication of novel hybrid materials and nanostructures. The work presented in this thesis has been focused on developing nanoscale materials based on synthetic de novo designed polypeptides. The polypeptides have been utilized for the assembly of gold nanoparticles, fibrous nanostructures, and for sensing applications.

The 42-residue polypeptides are designed to fold into helix-loop-helix motifs and dimerize to form four-helix bundles. Folding is primarily driven by the formation of a hydrophobic core made up by the hydrophobic faces of the amphiphilic helices. The peptides have either a negative or positive net charge at neutral pH, depending on the relative abundance of Glu and Lys. Charge repulsion thus prevents homodimerization at pH 7 while promoting hetero-dimerization through the formation of stabilising salt bridges. A Cys incorporated in position 22, located in the loop region, allowed for directed, thiol-dependent, immobilization on planar gold surfaces and gold nanoparticles. The negatively charged (Glu-rich) peptide formed homodimers and folded in solution at pH < 6 or in the presence of certain metal ions, such as Zn2+. The folding properties of this peptide were retained when immobilized directly on gold, which enabled reversible assembly of gold nanoparticles resulting in aggregates with well-defined interparticle separations. Particle aggregation was found to induce folding of the immobilized peptides but folding could also be utilized to induce aggregation of the particles by exploiting the highly specific interactions involved in both homodimerization and hetero-association. The possibility to control the assembly of polypeptide-functionalized gold nanoparticles was utilized in a colorimetric protein assay. Analyte binding to immobilized ligands prevented the formation of dense particle aggregates when subjecting the particles to conditions normally causing extensive aggregation. Analyte binding could hence easily be distinguished by the naked eye. Moreover, the peptides were utilized to assemble gold nanoparticles on planar gold and silica substrates.

Fibrous nanostructures were realized by linking monomers through a disulphide-bridge. The disulphide-linked peptides were found to spontaneously assemble into long and extremely thin peptide fibres as a result of a propagating association mediated by folding into four-helix bundles.

Abstract [en]

Ingenjörer och vetenskapsmän har ofta inspirerats av naturen i sökandet efter lösningar på tekniska problem. Allt ifrån byggnadskonstruktioner, flygplansvingar, kompositmaterial till kardborrebandet har skapats med utgångspunkt från förebilder i naturen. Många av de material och konstruktioner som återfinns i naturen har åtråvärda egenskaper som är svåra att erhålla i syntetiska matrial med traditionell teknik. Även om vi i flera fall kan härma sammansättningen och formen blir resultatet inte nödvändigtvis det samma. Den största skillnaden mellan syntetiska material och material producerade av levande organismer är hur deras komponenter sinsemellan är organiserade och sammansatta. I syntetiska material är komponenterna ofta inbördes mer eller mindre slumpvis ordnade medan de i biologiska material är organiserade med en oerhörd precision som sträcker sig ända ned på molekyl- och atomnivå. Naturens byggstenar har genom evolutionens gång förfinats för att spontant kunna organisera sig och bilda komplexa material  och strukturer. Denna process, som styrs genom att många svaga krafter inom och mellan byggstenarna samverkar, kallas ofta för självorganisering och är en förutsättning för allt liv. Självorganisering har också blivit en allt viktigare metod inom nanotekniken för att konstruera material och strukturer med nanometerprecision.

I den här avhandlingen beskrivs en typ av självorganiserande material där byggstenarna utgörs av nanometerstora guldpartiklar och syntetiska proteiner. De syntetiska proteinerna är designade för att efterlikna naturliga biomolekyler och antar en välbestämd tredimensionell struktur när två av dem interagerar med varandra. Denna interaktion är mycket specifik men kan styras genom att variera kemiska parametrar som surhet och jonstyrka vilket ger en möjlighet att påverka och kontrollera proteinernas struktur. Proteinerna har vidare modifierats för att spontant organisera sig till fibrer som är flera mikrometer långa men endast några nanometer tjocka. Proteinfibrer utgör en mycket viktig typ av strukturer i biologiska system och finns i alltifrån spindelväv till muskler. Syntetiska proteinfibrer är därför både ett intressant modellsystem och ett material med många potentiellt intressanta användningsområden.

Genom att fästa de syntetiska proteinerna på ytan av guldnanopartiklar går interaktionerna mellan partiklarna att kontrollera på samma sätt som interaktionerna mellan proteinerna. Krafterna mellan proteinerna och interaktionerna involverade i proteinernas veckning har använts för att reversibelt aggregera och organisera nanopartiklarna. Ett antal olika byggstenar har studerats och utvecklats till något som liknar ett mycket enkelt nano-Lego, som på en given signal spontant bygger ihop sig eller trillar isär.

Guldnanopartiklar är intressanta eftersom de är stabila och lätta att modifiera kemiskt men också på grund av deras optiska egenskaper som ger dem en ovanligt vacker vinröd färg. Färgen uppstår på grund av partiklarnas ringa storlek och varierar naturligt med egenskaperna hos den omgivande miljön. Detta gör det enkelt att studera hur partiklarna interagerar eftersom de byter färg när de närmar sig varandra, men gör dem också intressanta för sensortillämpningar. En enkel och robust sensor beskrivs i avhandlingen där syntetiska proteiner, speciellt utformade för att upptäcka och binda andra molekyler, har fästs på nanopartiklarna. Med partiklarnas hjälp går det att med blotta ögat detektera ett mänskligt protein i koncentrationer under ett tusendels gram per liter. En tidig diagnos av sjukdomstillstånd kan i de flesta fall avsevärt underlätta behandlingen och behovet av enkla sensorer för att bestämma närvaro och koncentration av medicinskt intressanta molekyler är därför mycket stort.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2008. 74 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1207
Keyword
Gold nanoparticle, polypeptide, helix-loop-helix, four-helix bundle, self-assembly, folding
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:liu:diva-15124 (URN)978-91-7393-818-1 (ISBN)
Public defence
2008-10-03, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2014-10-08Bibliographically approved
2. Folded polypeptide scaffolds for biosensor and biochip applications: design, synthesis, functionalisation and characterisation
Open this publication in new window or tab >>Folded polypeptide scaffolds for biosensor and biochip applications: design, synthesis, functionalisation and characterisation
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:nbn:se:liu:diva-43337 (URN)73560 (Local ID)91-7373-762-3 (ISBN)73560 (Archive number)73560 (OAI)
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

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Enander, KarinAili, DanielBaltzer, LarsLundström, IngemarLiedberg, Bo

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