Karin Enander

Colorimetric Protein Sensing by Controlled Assembly of Gold Nanoparticles Functionalized with Synthetic Receptors

A novel strategy is described for the colorimetric sensing of proteins, based on polypeptide-functionalized gold nanoparticles. Recognition is accomplished using a polypeptide sensor scaffold designed to specifically bind to the model analyte, human carbonic anhydrase II (HCAII). The extent of particle aggregation, induced by the Zn(2+)-triggered dimerization and folding of a second polypeptide also present on the surface of the gold nanoparticle, gives a readily detectable colorimetric shift that is dependent on the concentration of the target protein. In the absence of HCAII, particle aggregation results in a major redshift of the plasmon peak, whereas analyte binding prevented the formation of dense aggregates, significantly reducing the magnitude of the redshift. The versatility of the technique is 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 (Fab57P). Concentrations down to approximately 10 nM and approximately 25 nM are detected for HCAII and Fab57P, respectively. This strategy is proposed as a generic platform for robust and specific protein analysis that can be further developed to monitor a wide range of target proteins.

Folding Induced Assembly of Polypeptide Decorated Gold Nanoparticles

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.

A Peptide-Based, Ratiometric Biosensor Construct for Direct Fluorescence Detection of a Protein Analyte

We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore ( I(N*)/I(T*)) upon binding of Fab57P. When the peptide was labeled in the C terminus, the I(N*)/I(T*) ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats.

Assembly of Polypeptide-Functionalized Gold Nanoparticles through a Heteroassociation-and Folding-Dependent Bridging

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.

Self-Assembly of Fibers and Nanorings from Disulfide-Linked Helix–Loop–Helix Polypeptides

Ingenjorer och vetenskapsman har ofta inspirerats av naturen i sokandet efter losningar pa tekniska problem. Allt ifran byggnadskonstruktioner, flygplansvingar, kompositmaterial till kardborrebandet har skapats med utgangspunkt fran forebilder i naturen. Manga av de material och konstruktioner som aterfinns i naturen har atravarda egenskaper som ar svara att erhalla i syntetiska matrial med traditionell teknik. Aven om vi i flera fall kan harma sammansattningen och formen blir resultatet inte nodvandigtvis det samma. Den storsta skillnaden mellan syntetiska material och material producerade av levande organismer ar hur deras komponenter sinsemellan ar organiserade och sammansatta. I syntetiska material ar komponenterna ofta inbordes mer eller mindre slumpvis ordnade medan de i biologiska material ar organiserade med en oerhord precision som stracker sig anda ned pa molekyl- och atomniva. Naturens byggstenar har genom evolutionens gang forfinats for att spontant kunna organisera sig och bilda komplexa material och strukturer. Denna process, som styrs genom att manga svaga krafter inom och mellan byggstenarna samverkar, kallas ofta for sjalvorganisering och ar en forutsattning for allt liv. Sjalvorganisering har ocksa blivit en allt viktigare metod inom nanotekniken for att konstruera material och strukturer med nanometerprecision. I den har avhandlingen beskrivs en typ av sjalvorganiserande material dar byggstenarna utgors av nanometerstora guldpartiklar och syntetiska proteiner. De syntetiska proteinerna ar designade for att efterlikna naturliga biomolekyler och antar en valbestamd tredimensionell struktur nar tva av dem interagerar med varandra. Denna interaktion ar mycket specifik men kan styras genom att variera kemiska parametrar som surhet och jonstyrka vilket ger en mojlighet att paverka och kontrollera proteinernas struktur. Proteinerna har vidare modifierats for att spontant organisera sig till fibrer som ar flera mikrometer langa men endast nagra nanometer tjocka. Proteinfibrer utgor en mycket viktig typ av strukturer i biologiska system och finns i alltifran spindelvav till muskler. Syntetiska proteinfibrer ar darfor bade ett intressant modellsystem och ett material med manga potentiellt intressanta anvandningsomraden. Genom att fasta de syntetiska proteinerna pa ytan av guldnanopartiklar gar interaktionerna mellan partiklarna att kontrollera pa samma satt som interaktionerna mellan proteinerna. Krafterna mellan proteinerna och interaktionerna involverade i proteinernas veckning har anvants for att reversibelt aggregera och organisera nanopartiklarna. Ett antal olika byggstenar har studerats och utvecklats till nagot som liknar ett mycket enkelt nano-Lego, som pa en given signal spontant bygger ihop sig eller trillar isar. Guldnanopartiklar ar intressanta eftersom de ar stabila och latta att modifiera kemiskt men ocksa pa grund av deras optiska egenskaper som ger dem en ovanligt vacker vinrod farg. Fargen uppstar pa grund av partiklarnas ringa storlek och varierar naturligt med egenskaperna hos den omgivande miljon. Detta gor det enkelt att studera hur partiklarna interagerar eftersom de byter farg nar de narmar sig varandra, men gor dem ocksa intressanta for sensortillampningar. En enkel och robust sensor beskrivs i avhandlingen dar syntetiska proteiner, speciellt utformade for att upptacka och binda andra molekyler, har fasts pa nanopartiklarna. Med partiklarnas hjalp gar det att med blotta ogat detektera ett manskligt protein i koncentrationer under ett tusendels gram per liter. En tidig diagnos av sjukdomstillstand kan i de flesta fall avsevart underlatta behandlingen och behovet av enkla sensorer for att bestamma narvaro och koncentration av medicinskt intressanta molekyler ar darfor mycket stort.

Self-sorting heterodimeric coiled coil peptides with defined and tuneable self-assembly properties

Coiled coils with defined assembly properties and dissociation constants are highly attractive components in synthetic biology and for fabrication of peptide-based hybrid nanomaterials and nanostructures. Complex assemblies based on multiple different peptides typically require orthogonal peptides obtained by negative design. Negative design does not necessarily exclude formation of undesired species and may eventually compromise the stability of the desired coiled coils. This work describe a set of four promiscuous 28-residue de novo designed peptides that heterodimerize and fold into parallel coiled coils. The peptides are non-orthogonal and can form four different heterodimers albeit with large differences in affinities. The peptides display dissociation constants for dimerization spanning from the micromolar to the picomolar range. The significant differences in affinities for dimerization make the peptides prone to thermodynamic social self-sorting as shown by thermal unfolding and fluorescence experiments, and confirmed by simulations. The peptides self-sort with high fidelity to form the two coiled coils with the highest and lowest affinities for heterodimerization. The possibility to exploit self-sorting of mutually complementary peptides could hence be a viable approach to guide the assembly of higher order architectures and a powerful strategy for fabrication of dynamic and tuneable nanostructured materials.

An indirect competitive immunoassay for insulin autoantibodies based on surface plasmon resonance

We have developed a sensitive and specific method based on surface plasmon resonance (SPR) for detection of insulin autoantibodies (IAA) in serum samples from individuals at high risk of developing type 1 diabetes (T1D). When measuring trace molecules in undiluted sera with label-free techniques like SPR, non-specific adsorption of matrix proteins to the sensor surface is often a problem, since it causes a signal that masks the analyte response. The developed method is an indirect competitive immunoassay designed to overcome these problems. Today, IAA is mainly measured in radio immunoassays (RIAs), which are time consuming and require radioactively labeled antigen. With our SPR-based immunoassay the overall assay time is reduced by a factor of >100 (4 days to 50min), while sensitivity is maintained at a level comparable to that offered by RIA.

A multiple-ligand approach to extending the dynamic range of analyte quantification in protein microarrays.

This work describes a concept for extending the dynamic range of quantification in an affinity biosensor assay by using a set of ligands with different affinities toward a common analyte. Three synthetic, biotinylated polypeptides capable of binding a model protein analyte with different affinities (10(-9) M < or = K(d) < or = 10(-7) M) were immobilized in a microarray format on a gold slide covered with an oligo(ethylene glycol)-containing alkane thiolate self-assembled monolayer. A five-element affinity array, comprising single-peptide spots as well as spots where peptides were immobilized in mixtures, was realized by means of piezodispensation. Imaging surface plasmon resonance was used to study binding of the analyte to the different spots. The lower limit of analyte quantification was approximately 3 nM and the corresponding upper limit was increased by more than an order of magnitude compared to if only the highest affinity ligand would have been used. Affinity array sensors with multiple ligands for each analyte are particularly interesting for omitting dilution steps and providing highly accurate data in assays where several analytes such as disease biomarkers with extremely variable concentrations are quantified in parallel.

Environmentally Sensitive Fluorescent Sensors Based on Synthetic Peptides

Biosensors allow the direct detection of molecular analytes, by associating a biological receptor with a transducer able to convert the analyte-receptor recognition event into a measurable signal. We review recent work aimed at developing synthetic fluorescent molecular sensors for a variety of analytes, based on peptidic receptors labeled with environmentally sensitive fluorophores. Fluorescent indicators based on synthetic peptides are highly interesting alternatives to protein-based sensors, since they can be synthesized chemically, are stable, and can be easily modified in a site-specific manner for fluorophore coupling and for immobilization on solid supports.

Chemistry-specific surface adsorption of the barnacle settlement-inducing protein complex

Gregarious settlement in barnacle larvae (cyprids) is induced by a contact pheromone, the settlement-inducing protein complex (SIPC). The SIPC has been identified both in the cuticle of adult barnacles and in the temporary adhesive secretion (footprint) of cyprids. Besides acting as a settlement inducer, the presence of the SIPC in footprints points to its additional involvement in the adhesion process. SIPC adsorption behaviour was therefore investigated on a series of self-assembled monolayers (SAMs) by surface plasmon resonance at the pH of seawater (8.3). Fibrinogen and α2-macroglobulin (A2M) (blood complement protease inhibitors with which the SIPC shares 29% sequence homology) were used in the adsorption experiments as positive and negative standards, respectively. The mass uptake of the SIPC was comparable to that of fibrinogen, with adsorption observed even on the protein-resistant oligo(ethylene glycol) surface. Notably, on the positively charged SAM the SIPC showed a kinetic overshoot, indicating a metastable configuration causing the amount of adsorbed protein to temporarily exceed its equilibrium value. A2M adsorption was low or negligible on all SAMs tested, except for the positively charged surface, indicating that A2M adsorption is mainly driven by electrostatics. Evaluation of SIPC non-specific adsorption kinetics revealed that it adsorbed irreversibly and non-cooperatively on all surfaces tested.