| 1. |
- Håversen, Liliana, 1963, et al.
(författare)
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Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial alpha-helix of human lactoferrin.
- 2010
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Ingår i: Antimicrobial agents and chemotherapy. - 1098-6596 .- 0066-4804. ; 54:1, s. 418-25
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Tidskriftsartikel (refereegranskat)abstract
- There is a need for new microbicidal agents with therapeutic potential due to antibiotic resistance in bacteria and fungi. In this study, the structure-microbicidal activity relationship of amino acid residues 14 to 31 (sequence 14-31) from the N-terminal end, corresponding to the antibacterial alpha-helix of human lactoferrin (LF), was investigated by downsizing, alanine scanning, and substitution of amino acids. Microbicidal analysis (99% killing) was performed by a microplate assay using Escherichia coli, Staphylococcus aureus, and Candida albicans as test organisms. Starting from the N-terminal end, downsizing of peptide sequence 14-31 showed that the peptide sequence 19-31 (KCFQWQRNMRKVR, HL9) was the optimal length for antimicrobial activity. Furthermore, HL9 bound to lipid A/lipopolysaccharide, as shown by neutralizing endotoxic activity in a Limulus assay. Alanine scanning of peptide sequence 20-31 showed that Cys20, Trp23, Arg28, Lys29, or Arg31 was important for expressing full killing activity, particularly against C. albicans. Substituting the neutral hydrophilic amino acids Gln24 and Asn26 for Lys and Ala (HLopt2), respectively, enhanced microbicidal activity significantly against all test organisms compared to the amino acids natural counterpart, also, in comparison with HL9, HLopt2 had more than 10-fold-stronger fungicidal activity. Furthermore, HLopt2 was less affected by metallic salts than HL9. The microbicidal activity of HLopt2 was slightly reduced only at pH 7.0, as tested in the pH range of 4.5 to 7.5. The results showed that the microbicidal activity of synthetic peptide sequences, based on the antimicrobial alpha-helix region of LF, can be significantly enhanced by optimizing the length and substitution of neutral amino acids at specific positions, thus suggesting a sequence lead with therapeutic potential.
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| 2. |
- T. Tegler, Lotta, et al.
(författare)
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A highly selective polypeptide binder for human Acetylcholine esterase
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A highly selective high-affinity polypeptide conjugate binder for human Acetylcholine Esterase (hAChE) has been obtained by coupling a derivative of acridine, a known medium-affinity inhibitor of hAChE, to each member of a 16-membered set of 42-residue polypeptide scaffolds. The best candidate, 4-C10L17-Ac, was identified to have a KD of 10 nM or less in an assay where each polypeptide conjugate was titrated with hAChE in 50 mM phosphate buffer at pH 7.0 and 298K. It was found in a sandwich ELISA to have high selectivity for hAChE in cerebrospinal fluid. Targeting the active site of hAChE by a polypeptide conjugate binder presents a special problem as it is buried deep inside the protein in a cavity that is approximately 20 Å deep. In order to permit simultaneous and cooperative binding of the acridine and the polypeptide to the active site and the AChE surface a fourteen atom spacer was needed. The 9-aminoacridine group was linked to the side chain of a lysine residue in each polypeptide via a spacer prepared from two aminohexanoic acid fragments. The results reinforce the impression that polypeptide conjugates are excellent alternatives to currently used protein binder technologies in diagnostic and therapeutic applications and that the conjugation of enzyme inhibitors to polypeptide scaffolds is a strategy of general applicability in the design of high-affinity binders for enzymes.
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| 3. |
- Andersson, Per Ola, et al.
(författare)
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A Novel ATR-FTIR Approach for Characterisation and Identification of Ex Situ Immobilised Species
- 2007
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Ingår i: ChemPhysChem. - : Wiley. - 1439-4235 .- 1439-7641. ; 8:5, s. 712-722
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a novel method to analyse ex situ prepared protein chips by attenuated total reflection Fourier IR spectroscopy (ATR-FTIR), which circumvents tedious functionalisation steps of internal reflection elements (IREs), and simultaneously allows for complementary measurements by other analytical techniques. This concept is proven by utilising immobilised metal affinity capture (IMACTM) chips containing about 10 m thick films of copolymers coated with nitrilotriacetic acid (NTA) groups, which originally was manufactured for surface enhanced laser desorption ionisation (SELDI) spectrometry. Three immobilisation steps were analysed by ATR-FTIR spectroscopy: 1) NTA complexation with nickel(II) ions 2) binding of two histidine (His)-tagged synthetic peptides of 25 (25-His6) and 48 (48-His6) amino acids to the NTA-groups and 3) attachment of a ligand, mesyl amide, to the surface-bound 48-His6. Despite interference from H2O, both amide I and II were well resolved. Utilising peptide adsorption in the thick copolymer matrix yields a high saturation peptide concentration of 100 mg mL-1 and a dissociation constant of 116±11 M, as determined by a detailed analysis of the Langmuir adsorption isotherm. The mesyl amide ligand was directly seen in the raw ATR-FTIR spectrum with specific peaks in the fingerprint region at 1172 and 1350 cm-1. Several aspects of the fine structure of the amide I band of the peptide were analysed: influences from secondary structure, amino side chains and competing contamination product. We believe that this approach has great potential as a stand-alone or complementary analytical tool for determination of the chemical composition of functionalised surfaces. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and applied in other experimental set-ups.
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| 4. |
- Kattge, Jens, et al.
(författare)
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TRY plant trait database - enhanced coverage and open access
- 2020
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Ingår i: Global Change Biology. - : Wiley-Blackwell. - 1354-1013 .- 1365-2486. ; 26:1, s. 119-188
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Tidskriftsartikel (refereegranskat)abstract
- Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives.
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| 5. |
- Kondori, Nahid, 1967, et al.
(författare)
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Fungicidal activity of human lactoferrin-derived peptides based on the antimicrobial alpha beta region
- 2011
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Ingår i: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 37:1, s. 51-57
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Tidskriftsartikel (refereegranskat)abstract
- Owing to the increasing number of infections in hospitalised patients caused by resistant strains of fungi, there is a need to develop new therapeutic agents for these infections. Naturally occurring antimicrobial peptides may constitute models for developing such agents. A modified peptide sequence (CFQWKRAM-RKVR; HLopt2) based on amino acid residues 20-31 of the N-terminal end of human lactoferrin (hLF) as well as a double-sized human lactoferricin-like peptide (amino acid residues 16-40; HLBD1) were investigated for their antifungal activities in vitro and in vivo. By in vitro assay, HLopt2 was fungicidal at concentrations of 12.5-25 mu g/mL against Cryptococcus neoformans, Candida albicans, Candida krusei, Candida kefyr and Candida parapsilosis, but not against Candida glabrata. HLopt2 was demonstrated to have >= 16-fold greater killing activity than HLBD1. By inducing some helical formation caused by lactam bridges or by extending the assay time (from 2 h to 20 h), HLBD1 became almost comparable with HLopt2 in its fungicidal activity. Killing of C. albicans yeast cells by HLopt2 was rapid and was accompanied by cytoplasmic and mitochondrial membrane permeabilisation as well as formation of deep pits on the yeast cell surface. In a murine C. albicans skin infection model, atopic treatment with the peptides resulted in significantly reduced yields of Candida from the infected skin areas. The antifungal activities of HLopt2 in vitro and in vivo suggest possible potential as a therapeutic agent against most Candida spp. and C. neoformans. The greatly improved antifungal effect of the lactam-modified HLBD1 indicates the importance of amphipathic helix formation for lethal activity.
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| 6. |
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| 7. |
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| 8. |
- Aili, Daniel, 1977-, et al.
(författare)
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Aggregation-Induced Folding of a de novo Designed Polypeptide Immobilized on Gold Nanoparticles
- 2006
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Ingår i: Journal of the American Chemical Society. - : ACS Publications. - 0002-7863 .- 1520-5126. ; 128:7, s. 2194 -2195
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
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| 10. |
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| 11. |
- Aili, Daniel, 1977-, et al.
(författare)
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Assembly of Polypeptide-Functionalized Gold Nanoparticles through a Heteroassociation- and Folding-Dependent Bridging
- 2008
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Ingår i: Nano letters (Print). - : ACS Publications. - 1530-6984 .- 1530-6992. ; 8:8, s. 2473-2478
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 12. |
- Aili, Daniel, 1977-, et al.
(författare)
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Colorimetric Protein Sensing by Controlled Assembly of Gold Nanoparticles Functionalized with Synthetic Receptors
- 2009
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 5:21, s. 2445-2452
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Tidskriftsartikel (refereegranskat)abstract
- 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 Zn2+-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 approximate to 10 nM and approximate to 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.
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| 13. |
- Aili, Daniel, et al.
(författare)
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Colorimetric sensing: Small 21/2009
- 2009
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Ingår i: Small. - : John Wiley & Sons. - 1613-6810 .- 1613-6829. ; 5:21
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The cover picture illustrates a novel concept for colorimetric protein sensing based on the controllable assembly of polypeptide-functionalized gold nanoparticles. Recognition of the analyte is accomplished by polypeptide-based synthetic receptors immobilized on gold nanoparticles. Also present on the particle surface is a de novo-designed helix-loop-helix polypeptide that homodimerizes and folds into four-helix bundles in the presence of Zn2+, resulting in particle aggregation. Analyte binding interferes with the folding-induced aggregation, giving rise to a clearly detectable colorimetric response.
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| 14. |
- Aili, Daniel, 1977-, et al.
(författare)
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Controlled Assembly of Gold Nanoparticles using De Novo Designed Polypeptide Scaffolds
- 2008
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Ingår i: Proceedings SPIE, Vol. 6885, Photonic Biosensing and Microoptics. - : SPIE. ; , s. 688506-1-688506-8
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Konferensbidrag (refereegranskat)abstract
- 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.
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| 15. |
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| 16. |
- Aili, Daniel, 1977-, et al.
(författare)
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Folding Induced Assembly of Polypeptide Decorated Gold Nanoparticles
- 2008
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 130:17, s. 5780-5788
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 17. |
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| 18. |
- Aili, Daniel, et al.
(författare)
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Polypeptide Folding-Mediated Tuning of the Optical and Structural Properties of Gold Nanoparticle Assemblies
- 2011
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Ingår i: Nano letters (Print). - : American Chemical Society (ACS). - 1530-6984 .- 1530-6992. ; 11:12, s. 5564-5573
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Tidskriftsartikel (refereegranskat)abstract
- Responsive hybrid nanomaterials with well-defined properties are of significant interest for the development of biosensors with additional applications in tissue engineering and drug delivery. Here, we present a detailed characterization using UV-vis spectroscopy and small angle X-ray scattering of a hybrid material comprised of polypeptide-decorated gold nanoparticles with highly controllable assembly properties. The assembly is triggered by a folding-dependent bridging of the particles mediated by the heteroassociation of immobilized helix-loop-helix polypeptides and a complementary nonlinear polypeptide present in solution. The polypeptides are de novo designed to associate and fold into a heterotrimeric complex comprised of two disulfide-linked four-helix bundles. The particles form structured assemblies with a highly defined interparticle gap (4.8 +/- 0.4 nm) that correlates to the size of the folded polypeptides. Transitions in particle aggregation dynamics, mass-fractal dimensions and ordering, as a function of particle size and the concentration of the bridging polypeptide, are observed; these have significant effects on the optical properties of the assemblies. The assembly and ordering of the particles are highly complex processes that are affected by a large number of variables including the number of polypeptides bridging the particles and the particle mobility within the aggregates. A fundamental understanding of these processes is of paramount interest for the development of novel hybrid nanomaterials with tunable structural and optical properties and for the optimization of nanoparticle-based colorimetric biodetection strategies.
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| 19. |
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| 20. |
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| 21. |
- Aili, Daniel, et al.
(författare)
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Synthetic de novo designed polypeptides for control of nanoparticle assembly and biosensing
- 2007
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Ingår i: Biochemical Society Transactions. - 0300-5127 .- 1470-8752. ; 35:3, s. 532-534
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Tidskriftsartikel (refereegranskat)abstract
- This contribution describes how de novo designed synthetic helix–loop–helix polypeptides are utilized tocontrol the assembly of gold nanoparticles and as scaffolds for biosensing. The synthetic polypeptides aredesigned to fold into a four-helix bundle upon dimerization. When immobilized on gold nanoparticles,dimerization and folding occur between peptides on neighbouring particles as an effect of particleaggregation and the folded polypeptides are rigid enough to keep the particles separated at a distancecorresponding to the size of the four-helix bundle. Moreover, peptide dimerization offers a convenientroute to assemble nanoparticles into hybrid multilayers on planar substrates. The drastic change in theresonance conditions of the localized nanoparticle surface plasmon upon particle aggregation is shown tobe useful for optical detection of biomolecular interactions.
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| 22. |
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| 23. |
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| 24. |
- Allert, M., et al.
(författare)
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Noncovalent binding of a reaction intermediate by a designed helix-loop-helix motif - Implications for catalyst design
- 2003
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 4:4, s. 306-318
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Tidskriftsartikel (refereegranskat)abstract
- In our search for a catalyst for the transamination reaction of asparatic acid to form oxaloacetate, twenty-five forty-two-residue sequences were designed to fold into helix-loop-helix dimers and form binding sites for the key intermediate along the reaction pathway, the aldimine. This intermediate is formed from aspartic acid and the cofactor pyridoxal phosphate. The design of the binding sites followed a strategy in which exclusively noncovalent forces were used for binding the aldimine. Histidine residues were incorporated to catalyse the rate-limiting 1,3 proton transfer reactions that converts the aldimine into the ketimine, an intermediate that is subsequently hydrolysed to form oxaloacetate and pyridoxamine phosphate. The two most efficient catalysts, T-4 and T-16, selected from the pool of sequences by a simple screening procedure, were shown by CD and NMR spectroscopies to bind the aldimine intermediate with dissociation constants in the millimolar range. The mean residue ellipticity of T-4 in aqueous solution at pH 7.4 and a concentration of 0.75 mM was -18 500 deg cm-2 dmol-1. Upon addition of 6 mM L-aspartic acid and 1.5 mM pyridoxal phosphate to form the aldimine, the mean residue ellipticity changed to -19 900 deg cm2 dmol-1. The corresponding mean residue ellipticities of T-16 were -21 200 deg cm2 dmol-1 and -24 000 deg cm2 dmol-1. These result show that the helical content increased in the presence of the aldimine, and that the folded polypeptides bound the aldimine. The 1H NMR relaxation time of the imine CH proton of the aldimine was affected by the presence of T-4 as was the 31P NMR resonance linewidth. The catalytic efficienceis of T-4 and T-16 were compared to that of imidazole and found to be more than three orders of magnitude larger. The designed binding sites were thus shown to be capable of binding the aldimine in close proximity to His residues, by noncovalent forces, into conformations that proved to be catalytically active. The results show the first time the design of well-defined catalytic sites that bind a reaction intermediate with enzyme-like affinities under equilibrium conditions and represent an important advance in de novo catalyst design.
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| 25. |
- Allert, M, et al.
(författare)
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Setting the stage for new catalytic functions in designed proteins - Exploring the imine pathway in the efficient decarboxylation of oxaloacetate by an Arg-Lys site in a four-helix bundle protein scaffold
- 2002
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Ingår i: Chemistry - A European Journal. - 0947-6539 .- 1521-3765. ; 8:11, s. 2549-2560
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Tidskriftsartikel (refereegranskat)abstract
- Fourteen 42-residue polypeptides have been designed to identify reactive sites for the catalysis of the decarboxylation of oxaloacetate, a chemical transformation that proceeds through the formation of an imine intermediate. The sequences fold into helix-loop-helix motifs and dimerise to four-helix bundles. The catalytically active lysine residues were incorporated in several surface exposed positions, but also in positions characterised by hydrophobic properties to reduce their pK(a) values. The molecular environments of the Lys residues were systematically varied, to find which residues were able to stabilise and bind the imine intermediate in the decarboxylation reaction. A two-residue Arg-Lys site formed the main component of the reactive site of the helix-loop-helix dimer Decarb-K34_R33, which obeyed saturation kinetics in catalysing the reaction with a k(cat)/K-M of 0.59m(-1) s(-1). The rate constant measured was nearly three orders of magnitude larger than the second-order rate constant of the butylamine-catalysed reaction (0.0011 M-1 s(-1)), and four orders of magnitude larger than the pseudo first-order rate constant of the uncatalysed reaction (1.3 x 10(-5) s(-1)). The sequence of Decarb-K34_R33 contained only a single lysine residue. It was flanked by an arginine in the preceding position in the sequence. A flanking Arg residue provided more efficient catalysis than a flanking Lys or Gln residue. Arginines in flanking positions in the helix, in positions four residues before or after the Lys in the sequence, are not as important in catalysis as the Arg of the Arg-Lys pair. The effect of pK(a) on the catalytic efficiency of the Lys residue in the decarboxylation reaction is well known. The identification of the role of the flanking Arg residue in catalysing decarboxylation, its optimal position, and the importance of conformational stability reported here sets the stage for developing a number of catalytic systems that depend on the formation of imine intermediates, but that lead to different reaction products.
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| 26. |
- Anderson, LK, et al.
(författare)
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Control of lysine reactivity in four-helix bundle proteins by site-selective pK(a) depression : Expanding the versatility of proteins by postsynthetic functionalisation
- 2002
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Ingår i: Chemistry - A European Journal. - 0947-6539 .- 1521-3765. ; 8:16, s. 3687-3697
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Tidskriftsartikel (refereegranskat)abstract
- Five 42-residue polypeptides have been designed to fold into hairpin helix-loop-helix motifs that dimerise to form four-helix bundles, and to serve as protein scaffolds for the elucidation at the molecular level of the principles that control and fine-tune lysine and ornithine reactivities in a protein context. Site-selective control of Lys and Orn reactivity provides a mechanism for addressing directly individual residues and is a prerequisite for the site-selective functionalisation of folded proteins. Several lysine and one ornithine residues were introduced on the surface and in the hydrophobic core of the folded motif. The reactivity of each residue was determined by measuring the degree of acylation of the trypsin cleaved fragments by HPLC and mass spectrometry. The most reactive residues were Orn34 and Lys19, both of which were located in d positions in the heptad repeat, and therefore in hydrophobic environments. Upon reaction of the helix-loop-helix dimer KA-I with one equivalent of mono-p-nitrophenyl fumarate, Orn34 was acylated approximately three times more efficiently than Lys19, whereas Lys10 (b position), Lys15 (g position), and Lys33 (c position) remained unmodified. In the sequence KA-I-A(15) Lys15 was replaced by an alanine residue and the selectivity of Orn34 over Lys19 increased to approximately a factor of six, probably because Lys15 had the capacity to reduce the pK(a) value of Lys19 and 85 % of site-selectively monoacylated product was obtained. The pH dependence of the acylation reaction was determined and showed that the pK(a) of the reactive residues were 9.3, more than a pK(a) unit below the magnitude of the corresponding residue in a solvent exposed position. Introducing Lys and Orn residues into a or d positions of the heptad repeat therefore serves as a mechanism of depressing their pKa to increase their reactivity site selectively. Extensive NMR and CD spectroscopic analyses showed that the sequences fold according to prediction.
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| 27. |
- Andersson, LK, et al.
(författare)
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Multifunctional folded polypeptides from peptide synthesis and site-selective self-functionalization - Practical scaffolds in aqueous solution
- 2002
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Ingår i: ChemBioChem. - 1439-4227 .- 1439-7633. ; 3:8, s. 741-751
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Tidskriftsartikel (refereegranskat)abstract
- The site selectivity of His-mediated lysine and ornithine side-chain acylation in a designed four-helix bundle protein scaffold was mapped by reaction of several polypeptides with one equivalent of mono-p-nitrophenyl fumarate in aqueous solution at pH 5.9 and room temperature followed by an analysis of the degrees and sites of acylation. Integration of the HPLC chromatograms of the acylated polypeptides and trypsin cleavage followed by mass spectrometry analysis of the tryptic fragments provided the experimental evidence. Based on these and previously published results a strategy was developed for the site-selective and stepwise incorporation of three residues into a folded polypeptide in aqueous solution at room temperature. The first substituent was incorporated by reaction of a 1.7-fold excess of the corresponding active ester with the polypeptide at pH 5.9, the second substituent was introduced in a 3-fold excess after the pH value was raised to 8, and the third substituent was incorporated by reaction of a 10-fold excess with the polypeptide at pH 5.9. No intermediate steps of purification were taken and the overall yield was 30% or more. Examples of the substituents included are carbohydrates, an enzyme inhibitor, a fumarate, and an acetate group. The introduction of different substituents into three individually addressable positions in a stepwise, efficient, and controllable reaction demonstrates that designed folded polypeptides are practically useful scaffolds that can be functionalized by using very simple chemistry in aqueous solution. Predicted applications include designed receptors, biosensors, and molecular devices.
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| 28. |
- Andersson, LK, et al.
(författare)
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The effect of glycosylation on the structure of designed four-helix bundle motifs
- 2000
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Ingår i: JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2. - 0300-9580. ; :3, s. 459-464
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Tidskriftsartikel (refereegranskat)abstract
- A galactose-, 1, and a cellobiose derivative, 2, have been site selectively, post-translationally, incorporated into a folded helix-loop-helix dimer LA-42b in a one step reaction at room temperature. The structural effects on the folded peptide upon glycosylation have been studied by CD and NMR spectroscopy. The negative value of the mean residue ellipticity of the folded peptide, LA-42b, was raised from -19000 +/- 1000 to -21200 +/- 1000 deg cm(2) dmol(-1) upon introduction of the galactose derivative and to -19500 +/- 1000 deg cm(2) dmol(-1) upon introduction of the cellobiose derivative, showing that the helical content was increased. The dissociation constant of the dimer decreased from 120 to 30 mu M upon glycosylation. The introduction of 1 into GTD-C, a folded helix-loop-helix dimer with a well defined tertiary structure, had little structural impact. Glycosylation stabilises the folded structure of proteins with partially exposed hydrophobic cores but has little effect on well-packed proteins.
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| 29. |
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| 30. |
- Andersson, Theresa, et al.
(författare)
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The binding of human Carbonic Anhydrase II by functionalized folded polypeptide receptors
- 2005
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 12:11, s. 1245-1252
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Tidskriftsartikel (refereegranskat)abstract
- Several receptors for human carbonic anhydrase II (HCAII) have been prepared by covalently attaching benzenesulfonamide carboxylates via aliphatic aminocarboxylic acid spacers of variable length to the side chain of a lysine residue in a designed 42 residue helix-loop-helix motif. The sulfonamide group binds to the active site zinc ion of human carbonic anhydrase II located in a 15 Å deep cleft. The dissociation constants of the receptor-HCAII complexes were found to be in the range from low micromolar to better than 20 nM, with the lowest affinities found for spacers with less than five methylene groups and the highest affinity found for the spacer with seven methylene groups. The results suggest that the binding is a cooperative event in which both the sulfonamide residue and the helix-loop-helix motif contribute to the overall affinity.
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| 31. |
- Balliu, Aleksandra, et al.
(författare)
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Conjugation of a Dipicolyl Chelate to Polypeptide Conjugates Increases Binding Affinities for Human Serum Albumin and Survival Times in Human Serum
- 2017
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 18:14, s. 1408-1414
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Tidskriftsartikel (refereegranskat)abstract
- The affinity for human serum albumin (HSA) of a series of 2–5 kDa peptides covalently linked to 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, a dipicolyl chelator with micromolar affinity for Zn2+, was found by surface plasmon resonance to increase in the presence of 1 μm ZnCl2 at physiological pH. The dependence on polypeptide hydrophobicity was found to be minor, thus suggesting that the conjugates bound to the metal-binding site and not to the fatty-acid-binding site. The affinity of the conjugates increased strongly with the positive charge of the polypeptides, thus implicating the negatively charged protein surface surrounding the metal-binding site. The survival times of the peptides in human serum were extended as a consequence of stronger binding to HSA, thus suggesting that Zn2+-chelating agents might provide a general route to increased survival time of peptides in serum in therapeutic and diagnostic applications without significantly increasing their molecular weights.
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| 32. |
- Balliu, Aleksandra
(författare)
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Exploring molecular interactions between polypeptide conjugates and protein targets : Manipulating affinity by chemical modifications
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis molecular interactions between polypeptide conjugates and protein targets were investigated. Polypeptides were derivatized with small organic molecules, peptides and oligonucleotides. New strategies were developed with the aim to increase affinities for proteins of biological interest.A 42-residue polypeptide (4-C15L8) conjugated to a small organic molecule 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid (PP1), was shown to bind glycogen phosphorylase a (GPa) in the presence of zinc ions. Under the assumption that hydrophobic interactions dominated the binding energy, the hydrophobic residues of 4-C15L8-PP1 were systematically replaced in order to study their contribution to the affinity enhancement. The replacement of the Nle, Ile and Leu residues by Ala amino acids reduced affinities. The introduction of non-natural L-2-aminooctanoic acid (Aoc) residues into the peptide sequence enhanced the binding affinity for GPa. A decreased KD of 27nM was obtained when Nle5, Ile9 and Leu12 were replaced by Aoc residues, in comparison to the KD value of 280nM obtained for the unmodified 4-C15L8-PP1. It is evident that there are non-obvious hydrophobic binding sites on the surfaces of proteins that could be identified by introducing the more hydrophobic and conformationally flexible Aoc residues. The downsizing of the 42-mer peptide to an 11-mer and the incorporation of three Aoc residues gave rise to a KD of 550 nM, comparable to that of 4-C15L8-PP1 suggesting that bioactive peptides can be downsized by the introduction of Aoc.Aiming to improve in vivo stability, the affinity for human serum albumin (HSA) of hydrophobic, positively and negatively charged polypeptide-PP1 conjugates was evaluated. Increased hydrophobicity due to the introduction of Aoc residues did not significantly increase the affinity for HSA. No binding was observed in the case of the most negatively charged polypeptides whereas the slightly negatively and positively charged polypeptides conjugated to PP1 bound HSA with affinities that increased with the positive charge. It was found that polypeptide-PP1 conjugates target the zinc binding site of the HSA. Affinity enhancement was obtained due to the incorporation of PP1 and increased by charge to charge interactions between the positively charged amino acids of the polypeptide and the negatively charged residues of HSA, in close proximity to the HSA zinc binding site. The survival times of the peptide-PP1 conjugates in human serum were extended as a result of binding to HSA. Zn2+ ion chelating agents can be incorporated in potential peptide therapeutics with a short plasma half-life, without increasing their molecular weights.
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| 33. |
- Balliu, Aleksandra, et al.
(författare)
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Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces : Increasing Affinities in Peptide–Protein Interactions
- 2017
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 18:14, s. 1396-1407
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Tidskriftsartikel (refereegranskat)abstract
- A 42-residue polypeptide conjugated to a small-molecule organic ligand capable of targeting the phosphorylated side chain of Ser15 was shown to bind glycogen phosphorylase a (GPa) with a KD value of 280 nm. The replacement of hydrophobic amino acids by Ala reduced affinities, whereas the incorporation of l-2-aminooctanoic acid (Aoc) increased them. Replacing Nle5, Ile9 and Leu12 by Aoc reduced the KD value from 280 to 27 nm. “Downsizing” the 42-mer to an undecamer gave rise to an affinity for GPa an order of magnitude lower, but the undecamer in which Nle5, Ile9 and Leu12 were replaced by Aoc showed a KD value of 550 nm, comparable with that of the parent 42-mer. The use of Aoc residues offers a convenient route to increased affinity in protein recognition as well as a strategy for the “downsizing” of peptides essentially without loss of affinity. The results show that hydrophobic binding sites can be found on protein surfaces by comparing the affinities of polypeptide conjugates in which Aoc residues replace Nle, Ile, Leu or Phe with those of their unmodified counterparts. Polypeptide conjugates thus provide valuable opportunities for the optimization of peptides and small organic compounds in biotechnology and biomedicine.
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| 34. |
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| 35. |
- Baltzer, Lars
(författare)
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Crossing borders to bind proteins-a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set
- 2011
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 400:6, s. 1653-1664
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Forskningsöversikt (refereegranskat)abstract
- A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.
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| 36. |
- Baltzer, Lars, et al.
(författare)
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De novo design of proteins - What are the rules?
- 2001
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Ingår i: Chemical Reviews. - : American Chemical Society (ACS). - 0009-2665 .- 1520-6890. ; 101:10, s. 3153-3163
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Forskningsöversikt (refereegranskat)abstract
- The different techniques used for analyzing protein folding were discussed. The design of polypeptides that fold into structures that are preorganized to form specific protein-protein interactions was also discussed. The construction of cavities was found to be a necessary step in the design of efficient catalysts and selective receptors.
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| 37. |
- Baltzer, Lars, et al.
(författare)
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Emerging principles of de novo catalyst design
- 2001
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Ingår i: Current Opinion in Biotechnology. - 0958-1669 .- 1879-0429. ; 12:4, s. 355-360
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Forskningsöversikt (refereegranskat)abstract
- Considerable progress has been made in the understanding of how to exploit hydrophobic and charge - charge interactions in forming binding sites for peptides and small molecules in folded polypeptide catalysts. This knowledge has enabled the introduction of feedback and control functions into catalytic cycles and the construction of folded polypeptide catalysts that follow saturation kinetics. Major advances have also been made in the design of metalloproteins and metallopeptides, especially with regards to understanding redox potential control.
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| 38. |
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| 39. |
- Baltzer, Lars
(författare)
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Polypeptide Conjugate Binders for Protein Recognition
- 2007
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Ingår i: Topics in current chemistry. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0340-1022 .- 1436-5049. ; 277, s. 89-106
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Forskningsöversikt (refereegranskat)abstract
- A new class of hybrid molecules for protein recognition is presented, where polypeptides are covalently linked to small organic molecules to form polypeptide conjugates that bind proteins with high affinity and selectivity. To illustrate the concept, a binder for human carbonic anhydrase 11 with a dissociation constant of 4 nM is described. The affinity of the polypeptide conjugate arises from cooperativity in binding between a benzenesulfonamide residue, with a dissociation constant of 1.5 mu M, and the polypeptide scaffold with a dissociation constant of < 1 mM. The combination of a ligand with moderate affinity for a target protein with a polypeptide relaxes considerably the need for high affinity on the part of the polypeptide, and thus the need for structural complexity and preorganization. At the same time, the requirement for high affinity on the part of ligand is relaxed. As a consequence, the time for development of robust, high affinity, selective binder is shortened. The chemical approach to protein recognition provides well-defined molecular entities that are conveniently handled, stored and site-specifically functionalized.
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| 40. |
- Barman, Jharna, 1979-
(författare)
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Targeting RNA by the Antisense Approach and a Close Look at RNA Cleavage Reaction
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis summarizes the results of studies on two aspects of nucleic acids. Chemically modified antisense oligonucleotides (AONs) have been evaluated with regards to their suitability for mRNA targeting in an antisense approach (Paper I – III). The chemically modified nucleotidic units 2'-O-Me-T, 2'-O-MOE-T, oxetane-T, LNA-T, azetidine-T, aza-ENA-T, carbocyclic-ENA-T and carbocyclic-LNA-T were incorporated into 15-mer AONs and targeted against a 15-mer RNA chosen from the coding region of SV-40 large T antigen. The comparative study showed that a single modified nucleotide in the AON with North-East locked sugar (oxetane-T and azetidine-T) lowered the affinity for the complementary RNA whereas North locked sugars (LNA-T, aza-ENA-T, carbocyclic-ENA-T, and carbocyclic-LNA-T) significantly improved the affinity. A comparative RNase H digestion study showed that modifications of the same type (North-East type or North type) in different sequences gave rise to similar cleavage patterns. Determination of the Michaelis-Menten parameters by kinetic experiments showed that the modified AONs recruit RNase H resulting in enhanced turnover numbers (kcat) although with weaker enzyme-substrate binding (1/Km) compared to the unmodified AON. The modified AONs were also evaluated with regards to resistance towards snake venom phosphodiesterase and human serum to estimate their stability toward exonucleases. The aza-ENA-T and carbocyclic-ENA-T modified AONs showed improved stability compared to all other modified AONs. In general, the modified AONs with North type nucleotides (except LNA-T) were found to be superior to the North-East type as they showed improved target affinity, comparable RNase H recruitment capability and improved exonuclease stability.The second aspect studied in this thesis is based on physicochemical studies of short RNA molecules utilizing NMR based pH titration and alkaline hydrolysis reactions (Paper IV – V). The NMR based (1H and 31P) pH titration studies revealed the effect of guaninyl ion formation, propagated electrostatically through a single stranded chain in a sequence dependent manner. The non-identical electronic character of the internucleotidic phosphodiesters was further verified by alkaline hydrolysis experiments. The internucleotidic phosphodiesters, which were influenced by guaninyl ion formation, were hydrolyzed at a faster rate than those sequences where such guaninyl ion formation was prevented by replacing G with N1-Me-G.
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| 41. |
- Enander, Karin, 1972-, et al.
(författare)
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A versatile polypeptide platform for integrated recognition and reporting : affinity arrays for protein-ligand interaction analysis
- 2004
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Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 10:10, s. 2375-2385
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 42. |
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| 43. |
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| 44. |
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| 45. |
- Enander, Karin, et al.
(författare)
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Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold
- 2005
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Ingår i: Langmuir. - : ACS Publications. - 0743-7463 .- 1520-5827. ; 21:6, s. 2480-2487
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 46. |
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| 47. |
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