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Polypeptide-Based Nanoscale Materials
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
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 [en]
Gold nanoparticle, polypeptide, helix-loop-helix, four-helix bundle, self-assembly, folding
National Category
Other Basic Medicine
Identifiers
URN: urn:nbn:se:liu:diva-15124ISBN: 978-91-7393-818-1 (print)OAI: oai:DiVA.org:liu-15124DiVA: diva2:54450
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
List of papers
1. 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
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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
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
2. 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
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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
3. Folding Induced Assembly of Polypeptide Decorated Gold Nanoparticles
Open this publication in new window or tab >>Folding Induced Assembly of Polypeptide Decorated Gold Nanoparticles
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2008 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 130, no 17, 5780-5788 p.Article in journal (Refereed) Published
Abstract [en]

Reversible assembly of gold nanoparticles controlled by the homodimerization and folding of an immobilized de novo designed synthetic polypeptide is described. In solution at neutral pH, the polypeptide folds into a helix–loop–helix four-helix bundle in the presence of zinc ions. When immobilized on gold nanoparticles, the addition of zinc ions induces dimerization and folding between peptide monomers located on separate particles, resulting in rapid particle aggregation. The particles can be completely redispersed by removal of the zinc ions from the peptide upon addition of EDTA. Calcium ions, which do not induce folding in solution, have no effect on the stability of the peptide decorated particles. The contribution from folding on particle assembly was further determined utilizing a reference peptide with the same primary sequence but containing both D and L amino acids. Particles functionalized with the reference peptide do not aggregate, as the peptides are unable to fold. The two peptides, linked to the nanoparticle surface via a cysteine residue located in the loop region, form submonolayers on planar gold with comparable properties regarding surface density, orientation, and ability to interact with zinc ions. These results demonstrate that nanoparticle assembly can be induced, controlled, and to some extent tuned, by exploiting specific molecular interactions involved in polypeptide folding.

Place, publisher, year, edition, pages
ACS Publications, 2008
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:liu:diva-15116 (URN)10.1021/ja711330f (DOI)
Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2017-12-07Bibliographically approved
4. Controlled Assembly of Gold Nanoparticles using De Novo Designed Polypeptide Scaffolds
Open this publication in new window or tab >>Controlled Assembly of Gold Nanoparticles using De Novo Designed Polypeptide Scaffolds
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2008 (English)In: Proceedings SPIE, Vol. 6885, Photonic Biosensing and Microoptics, 2008, 688506-1-688506-8 p.Conference paper, Published paper (Refereed)
Abstract [en]

Heterodimerization between designed helix-loop-helix polypeptides was utilized in order to assemble gold nanoparticles on planar substrates. The peptides were designed to fold into four-helix bundles upon dimerization. A Cys-residue in the loop region was used to immobilize one of the complementary peptides on a maleimide containing SAM on planar gold substrates whereas the second peptide was immobilized directly on gold nanoparticles. Introducing the peptide decorated particles over a peptide functionalized surface resulted in particle assembly. Further, citrate stabilized particles were assembled on amino-silane modified glass and silicon substrates. By subsequently introducing peptides and gold nanoparticles, particle-peptide hybrid multi layers could be formed.

Keyword
Heterodimerization, polypeptides, gold nanoparticles, four-helix bundle, helix-loop-helix, self-assembly
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:liu:diva-15118 (URN)10.1117/12.775806 (DOI)
Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2014-10-08Bibliographically approved
5. Self-Assembly of Fibers and Nanorings from Disulfide-Linked Helix–Loop–Helix Polypeptides
Open this publication in new window or tab >>Self-Assembly of Fibers and Nanorings from Disulfide-Linked Helix–Loop–Helix Polypeptides
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2008 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 47, no 30, 5554-5556 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
Wiley InterScience, 2008
Keyword
fibers, helical structures, nanostructures, polypeptides, self-assembly
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:liu:diva-15120 (URN)10.1002/anie.200801155 (DOI)
Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2017-12-07Bibliographically approved
6. Assembly of Polypeptide-Functionalized Gold Nanoparticles through a Heteroassociation- and Folding-Dependent Bridging
Open this publication in new window or tab >>Assembly of Polypeptide-Functionalized Gold Nanoparticles through a Heteroassociation- and Folding-Dependent Bridging
2008 (English)In: Nano letters (Print), ISSN 1530-6984, E-ISSN 1530-6992, Vol. 8, no 8, 2473-2478 p.Article in journal (Refereed) Published
Abstract [en]

Gold nanoparticles were functionalized with a synthetic polypeptide, de novo-designed to associate with a charge complementary linker polypeptide in a folding-dependent manner. A heterotrimeric complex that folds into two disulphide-linked four-helix bundles is formed when the linker polypeptide associates with two of the immobilized peptides. The heterotrimer forms in between separate particles and induces a rapid and extensive aggregation with a well-defined interparticle spacing. The aggregated particles are redispersed when the disulphide bridge in the linker polypeptide is reduced.

Place, publisher, year, edition, pages
ACS Publications, 2008
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-15121 (URN)10.1021/nl8014796 (DOI)
Note
The original title of this article was "Assembly of Decorated Gold Nanoparticles through a Hetero-Association and Folding-Dependent Bridging".Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2017-12-07
7. Colorimetric Protein Sensing by Controlled Assembly of Gold Nanoparticles Functionalized with Synthetic Receptors
Open this publication in new window or tab >>Colorimetric Protein Sensing by Controlled Assembly of Gold Nanoparticles Functionalized with Synthetic Receptors
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2009 (English)In: Small, ISSN 1613-6810, Vol. 5, no 21, 2445-2452 p.Article in journal (Refereed) Published
Abstract [en]

A strategy for colorimetric sensing of proteins, based on the induced assembly of polypeptide-functionalized gold nanoparticles, is described. Recognition was accomplished using a polypeptide sensor scaffold designed to specifically bind the model analyte, human carbonic anhydrase II (HCAII). The extent of particle aggregation, induced by the Zn2+-triggered dimerization and folding of a second polypeptide also present on the surface of the gold nanoparticle, gave a readily detectable colorimetric shift that was dependent on the concentration of the target protein. In the absence of HCAII, particle aggregation resulted in a major redshift of the plasmon peak whereas analyte binding prevented formation of dense aggregates, significantly reducing the magnitude of the redshift. The limit of detection of HCAII was estimated to be around 15 nM. The versatility of the technique was demonstrated using a second model system based on the recognition of a peptide sequence from the tobacco mosaic virus coat protein (TMVP by a recombinant antibody fragment. This strategy is proposed as a generic platform for robust and specific protein analysis that can be further developed for monitoring a wide range of target proteins.

Keyword
Not available.
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-15122 (URN)10.1002/smll.200900530 (DOI)
Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2015-05-29Bibliographically approved

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