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  • 1.
    Aili, Daniel
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Enander, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Synthetic de novo designed polypeptides for control of nanoparticle assembly and biosensing2007In: Bionanotechnology; from self-assembly to cellbiology,2007, London: Biochemical Society Transactions , 2007, p. 532-Conference paper (Refereed)
    Abstract [en]

         

  • 2.
    Aili, Daniel
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Enander, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Towards novel functional materials and sensors using de novo designed polypeptides on gold nanoparticles2006In: Europtrode VIII,2006, 2006Conference paper (Other academic)
    Abstract [en]

        

  • 3.
    Aili, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Rydberg, Johan
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics . Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Aggregation-Induced Folding of a de novo Designed Polypeptide Immobilized on Gold Nanoparticles2006In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, no 7, p. 2194 -2195Article in journal (Refereed)
    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.

  • 4.
    Allert, M.
    et al.
    Department of Chemistry, Göteborg University, 41296 Göteborg, Sweden.
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Noncovalent binding of a reaction intermediate by a designed helix-loop-helix motif - Implications for catalyst design2003In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 4, no 4, p. 306-318Article in journal (Refereed)
    Abstract [en]

    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.

  • 5.
    Allert, M
    et al.
    Univ Gothenburg, Dept Chem Organ Chem, S-41296 Gothenburg, Sweden Linkoping Univ, Dept Chem, S-58183 Linkoping, Sweden.
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    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 scaffold2002In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 8, no 11, p. 2549-2560Article in journal (Refereed)
    Abstract [en]

    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.

  • 6.
    Anderson, LK
    et al.
    Univ Gothenburg, Dept Chem, S-41296 Gothenburg, Sweden Linkoping Univ, Dept Chem, S-58183 Linkoping, Sweden.
    Caspersson, M
    Univ Gothenburg, Dept Chem, S-41296 Gothenburg, Sweden Linkoping Univ, Dept Chem, S-58183 Linkoping, Sweden.
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Control of lysine reactivity in four-helix bundle proteins by site-selective pK(a) depression: Expanding the versatility of proteins by postsynthetic functionalisation2002In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 8, no 16, p. 3687-3697Article in journal (Refereed)
    Abstract [en]

    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.

  • 7. Andersson, LK
    et al.
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Multifunctional folded polypeptides from peptide synthesis and site-selective self-functionalization - Practical scaffolds in aqueous solution2002In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 3, no 8, p. 741-751Article in journal (Refereed)
    Abstract [en]

    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.

  • 8. Andersson, LK
    et al.
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Kihlberg, J
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    The effect of glycosylation on the structure of designed four-helix bundle motifs2000In: JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2, ISSN 0300-9580, no 3, p. 459-464Article in journal (Refereed)
    Abstract [en]

    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.

  • 9.
    Andersson, Theresa
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Dolphin, Gunnar T.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Nilsson, Jonas W.
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    The binding of human Carbonic Anhydrase II by functionalized folded polypeptide receptors2005In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 12, no 11, p. 1245-1252Article in journal (Refereed)
    Abstract [en]

    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.

  • 10.
    Baltzer, Lars
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Nilsson, Helena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry .
    Nilsson, Jonas
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry .
    De novo design of proteins - What are the rules?2001In: Chemical Reviews, ISSN 0009-2665, E-ISSN 1520-6890, Vol. 101, no 10, p. 3153-3163Article, review/survey (Refereed)
    Abstract [en]

    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.

  • 11.
    Baltzer, Lars
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Nilsson, Jonas
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry .
    Emerging principles of de novo catalyst design2001In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 12, no 4, p. 355-360Article, review/survey (Refereed)
    Abstract [en]

    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.

  • 12.
    Enander, Karin
    et al.
    Division of Organic Chemistry, Uppsala University.
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Division of Organic Chemistry, Department of Chemistry, BMC, Uppsala University, Uppsala, Sweden.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold2005In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, no 6, p. 2480-2487Article in journal (Refereed)
    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.

  • 13.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Andersson, Linda
    Göteborg universitet.
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Designed, folded polypeptides for bioanalytical purposes - molecular scaffolds that combine recognition and reporting2001In: 4th International Conference on Structural Molecular Biology,2001, 2001Conference paper (Other academic)
    Abstract [en]

      

  • 14.
    Enander, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Dolphin, Gunnar
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Andersson, Linda
    Department of Organic Chemistry Göteborg University.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Designed, folded polypeptide scaffolds that combine key biosensing events of recognition and reporting2002In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 67, no 9, p. 3120-3123Article in journal (Refereed)
    Abstract [en]

    No abstract available.

  • 15.
    Enander, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Dolphin, Gunnar
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Designed, functionalized helix-loop-helix motifs that bind human carbonic anhydrase II: a new class of synthetic receptor molecules2004In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 14, p. 4464-4465Article in journal (Refereed)
    Abstract [en]

    Polypeptides designed to fold into helix−loop−helix motifs and to dimerize to form four-helix bundles were functionalized by the introduction of a sulfonamide derivative known to bind human carbonic anhydrase II (HCAII) and one or both of the dansyl- and methoxycoumarin fluorescent probes. The 42-residue sequence DC that carries all three substituents in solvent-exposed positions was found to bind HCAII with a dissociation constant of 5 nM in aqueous solution at pH 7. At 2 μM concentration, DC was mainly dimeric in aqueous solution but bound HCAII as a monomer. Upon addition of a large excess of a helix−loop−helix motif without a high-affinity ligand, KE2-Q, a ternary complex was formed between HCAII, DC, and KE2-Q. Hydrophobic interactions between DC and HCAII and coordination of the sulfonamide group to the zinc ion of HCAII contributed cooperatively to binding in a demonstration of the usefulness of folded polypeptide−small organic molecule chimera as novel protein receptors. The DC homodimer was found to be a very sensitive biosensor component due to intermolecular quenching of its fluorescence that was inhibited upon binding to HCAII.

  • 16.
    Enander, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Dolphin, Gunnar
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis2004In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 10, no 10, p. 2375-2385Article in journal (Refereed)
    Abstract [en]

    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.

  • 17.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    De novo designed helix-loop-helix polypeptides - a structure-function-based design strategy for microarray and biosensor applications2003In: 1st World Congress on Synthetic Receptors,2003, 2003Conference paper (Refereed)
  • 18.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Designed, folded polypeptides as functional units in biosensor applications2002In: 18:e Organikerdagarna,2002, 2002Conference paper (Other academic)
  • 19.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Löfdahl, Mikael
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Designed, folded polypeptides as functional units in surface-based biosensors - versatile scaffolds connecting recognition and reporting2002In: Europtrode VI,2002, 2002Conference paper (Refereed)
  • 20.
    Enander, Karin
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Dolphin, Gunnar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Löfdahl, Mikael
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Lundström, Ingemar
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics .
    Liedberg, Bo
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics .
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Helix-loop-helix polypeptides as scaffolds for designed biosensing surfaces2002In: 6th World Congress on Biosensors,2002, 2002Conference paper (Other academic)
  • 21.
    Haversen, L.
    et al.
    Univ Gothenburg, Dept Infect Med Clin Bacteriol, S-41346 Gothenburg, Sweden.
    Kondori, N.
    Univ Gothenburg, Dept Infect Med Clin Bacteriol, S-41346 Gothenburg, Sweden.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    A Hanson, L A
    Univ Gothenburg, Dept Clin Immunol, S-41346 Gothenburg, Sweden.
    Dolphin, Gunnar
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Duner, K.
    Univ Gothenburg, Dept Infect Med Clin Bacteriol, S-41346 Gothenburg, Sweden.
    Mattsby-Baltzer, I.
    Univ Gothenburg, Dept Infect Med Clin Bacteriol, S-41346 Gothenburg, Sweden.
    Structure-Microbicidal Activity Relationship of Synthetic Fragments Derived from the Antibacterial alpha-Helix of Human Lactoferrin2010In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 54, no 1, p. 418-425Article in journal (Refereed)
    Abstract [en]

    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.

  • 22.
    Hederos (Håkansson), Sofia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Department of Chemistry, Biomedical Centre, Uppsala University, Uppsala, Sweden.
    Nucleophile Selectivity in the Acyl Transfer Reaction of a Designed Enzyme2005In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 79, no 6, p. 292-299Article in journal (Refereed)
    Abstract [en]

    The acyl transfer reaction of S-glutathionyl benzoate (GSB) is catalyzed by a rationally designed mutant of human glutathione transferase A1-1, A216H. The catalyzed reaction proceeds via the formation of an acyl intermediate and has been studied in the presence of nitrogen, oxygen, and sulfur nucleophiles to determine the selectivity with regards to nucleophile structure. Methanol was previously shown to react with the acyl intermediate and form the corresponding ester, methylbenzoate, under a significant rate enhancement. In the present investigation, the dependence on nucleophile structure and reactivity has been investigated. Ethane thiol gave rise to a larger rate enhancement in the enzyme-catalyzed reaction than ethanol, whereas ethylamine did not increase the reaction rate. The reactivities toward the acyl intermediate of primary and secondary alcohols with similar pKa values depended on the structure of the aliphatic chain, and 1-propanol was the most efficient alcohol. The reactivity of the oxygen nucleophiles was also found to depend strongly on pKa as 2,2,2-trifluoroethanol, with a pKa of 12.4, was the most efficient nucleophile of all that were tested. Saturation kinetics was observed in the case of 1-propanol, indicating a second binding site in the active site of A216H. The nucleophile selectivity of A216H provides the knowledge base needed for the further reengineering of A216H towards alternative substrate specificities.

  • 23.
    Höst, Gunnar
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Razkin, Jesus
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Uppsala University, Department of Chemistry, Uppsala, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Combined Enzyme and Substrate Design: Grafting of a Cooperative Two-Histidine Catalytic Motif into a Protein Targeted at the Scissile Bond in a Designed Ester Substrate2007In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 8, no 13, p. 1570-1576Article in journal (Refereed)
    Abstract [en]

    A histidine-based, two-residue reactive site for the catalysis of hydrolysis of designed sulfonamide-containing para-nitrophenyl esters has been engineered into a scaffold protein. A matching substrate was designed to exploit the natural active site of human carbonic anhydrase II (HCAII) for well-defined binding. In this we took advantage of the high affinity between the active site zinc atom and sulfonamides. The ester substrate was designed to position the scissile bond in close proximity to the His64 residue in the scaffold protein. Three potential sites for grafting the catalytic His-His pair were identified, and the corresponding N62H/H64, F131H/V135H and L198H/P202H mutants were constructed. The most efficient variant, F131H/V135H, has a maximum kcat/KM value of approximately 14 000 M-1 s-1, with a kcat value that is increased by a factor of 3 relative to that of the wild-type HCAII, and by a factor of over 13 relative to the H64A mutant. The results show that an esterase can be designed in a stepwise way by a combination of substrate design and grafting of a designed catalytic motif into a well-defined substrate binding site.

  • 24.
    Kondori, N
    et al.
    Gothenburg University.
    Baltzer, Lars
    Uppsala University.
    Dolphin, Gunnar
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Mattsby-Baltzer, I
    Gothenburg University.
    Fungicidal activity of human lactoferrin-derived peptides based on the antimicrobial alpha beta region2011In: INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS, ISSN 0924-8579, Vol. 37, no 1, p. 51-57Article in journal (Refereed)
    Abstract [en]

    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 andgt;= 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.

  • 25. Nilsson, J
    et al.
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Reactive-site design in folded-polypeptide catalysts - The leaving group pK(a) of reactive esters sets the stage for cooperativity in nucleophilic and general-acid catalysis2000In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 6, no 12, p. 2214-2220Article in journal (Refereed)
    Abstract [en]

    The second-order rate constants for the hydrolysis of nitrophenyl esters catalysed by a number of folded designed polypeptides have been determined. and 1900-fold rate enhancements over those of the 4-methylimidazole-catalysed reactions have been observed. The rate enhancements are much larger than those expected from the pK(a) depression of the nucleophilic His residues alone. Kinetic solvent isotope effects were observed at pn values lower than the pK(a), values of the leaving groups and suggests that general-acid catalysis contributes in the pH range where the leaving group is predominantly protonated. In contrast, no isotope effects were observed at pH values above the pK(a) of the leaving group. A Hammett rho value of 1.4 has been determined fur the peptide-catalysed hydrolysis reaction by variation of the substituents of the leaving phenol. The corresponding values For the imidazole-catalysed reaction is 0.8 and For phenol dissociation is 2.2. There is therefore, very approximately, half a negative charge localised on the phenolate oxygen in the transition stair in agreement with the conclusion that transition-state hydrogen-bond formation may contribute to the observed catalysis. The elucidation at a molecular level of the principles that control cooperativity in the biocatalysed ester-hydrolysis reaction represents the first step towards a level of understanding of the concept of cooperativity that may eventually allow us to design tailor-made enzymes for chemical reactions not catalysed by nature.

  • 26.
    Nilsson, Peter
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Rydberg, Johan
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Inganäs, Olle
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Self-assembly of synthetic peptides control conformation and optical properties of a zwitterionic polythiophene derivative2003In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 18, p. 10170-10174Article in journal (Refereed)
    Abstract [en]

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

  • 27.
    Nilsson, Peter
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Rydberg, Johan
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Inganäs, Olle
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Twisting macromolecular chains: self-assembly of a chiral supermolecule from nonchiral polythiophene polyanions and random-coil synthetic peptides2004In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, no 31, p. 11197-11202Article in journal (Refereed)
    Abstract [en]

    The self-assembly of a negatively charged conjugated polythiophene derivative and a positively charged synthetic peptide will create a chiral, well ordered supermolecule. This supermolecule has the three-dimensional ordered structure of a biomolecule and the electronic properties of a conjugated polymer. The molecular complex being formed clearly affects the conformation of the polymer backbone. A main-chain chirality, such as a predominantly one-handed helical structure induced by the acid–base complexation between the conjugated polymer and the synthetic peptide, is seen. The alteration of the polymer backbone influences the optical properties of the polymer, seen as changes in the absorption, emission, and Raman spectra of the polymer. The complexation of the polythiophene and the synthetic peptide also induce a change from random-coil to helical structure of the synthetic peptide. The supermolecule described in this article may be used in a wide range of applications such as biomolecular devices, artificial enzymes, and biosensors.

  • 28.
    Rossi, P.
    et al.
    University of Padova, Department of Chemical Sciences, ITM-CNR Padova Section, Via Marzolo, 1, 35131 Padova, Italy.
    Tecilla, P.
    Dipartimento di Scienze Chimiche, Università di Trieste, Via Giorgieri 1, 34127 Trieste, Italy.
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Scrimin, P.
    University of Padova, Department of Chemical Sciences, ITM-CNR Padova Section, Via Marzolo, 1, 35131 Padova, Italy.
    De novo metallonucleases based on helix-loop-helix motifs2004In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 10, no 17, p. 4163-4170Article in journal (Refereed)
    Abstract [en]

    Three new 42-mer peptides (PRI-III) designed to fold into a hairpin helix-loop-helix motif have been prepared. In the peptide sequence two (PRII-III) or four (PRI) copies of an unnatural amino acid bearing a triazacyclononane metal-ion binding site (ATANP) have been inserted in appropriate positions to allow the ligand subunits to face each other either within the same helix or between the two helices of the hairpin motif. Circular dichroism (C(d) studies in solution have shown that the apopeptides adopt a well-defined helix-loop-helix tertiary structure that dimerizes in solution at concentrations above 2001 µM to form a four-helix bundle. However, the helical content is strongly dependent on pH and metal-ion binding. Both protonation of the amines of the triazacyclononane units present in the ATANP lateral arm and complexation with ZnII ions cause a significant decrease of the helical content of the sequences. The ZnII complexes of the three peptides catalyze the transesterification of the RNA model substrate 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) with different efficiency. The best catalyst appears to be PRI-4ZnII. that is, the peptide incorporating four ATANP units. Michaelis-Menten saturation kinetics allowed us to estimate that substrate fully bound to the catalyst reacts 380 times faster than in its absence. The kinetic evidence suggests cooperativity between (at least two) metal ions: one activating the nucleophilic species (directly or indirectly) and the other facilitating nucleophilic attack by coordination of the phosphate.

  • 29.
    Rydberg, Johan
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
    Vijayalekshmi, S.
    Baltzer, Lars
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Intrinsically unstructured proteins by design: electrostatic interactions can control binding, folding and function of a helix-loop-helix heterodimer2006Article in journal (Refereed)
  • 30. Vijayalekshmi, S.
    et al.
    George, S.K.
    Department of Chemistry, Umeå University, 412 527 Umeå, Sweden.
    Andersson, L.K.
    Department of Chemistry, Organic Chemistry, Göteborg University, 412 96 Göteborg, Sweden.
    Kihlberg, J.
    Department of Chemistry, Umeå University, 412 527 Umeå, Sweden.
    Baltzer, Lars
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    A surface exposed O-linked galactose residue destabilises the structure of a folded helix-loop-helix dimer2003In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 1, no 14, p. 2455-2460Article in journal (Refereed)
    Abstract [en]

    A 42-residue glycopeptide Tn-15 and the corresponding reference polypeptide Thr-15 were designed and synthesized to provide a model system for the study of how glycosylation affects the stability of a molten globule-like protein. Tn-15 and Thr-15 fold into hairpin helix-loop-helix motifs that dimerise to form four-helix bundles and the only difference between the sequences is that Tn-15 carries an O-linked N-acetylgalactosamine residue at the side chain of threonine-15 whereas the sequence Thr-15 is unglycosylated. An analysis of the mean residue ellipticities at 222 nm of the two polypeptides and of the a-H chemical shift deviations from random coil values showed that glycosylation reduced the helical content of the polypeptides and increased the dissociation constant of the helix-loop-helix dimer to form monomers. The pH dependencies of the helical content of Tn-15 and Thr-15 differed as that of Thr-15 was largely unaffected by pH in the range from pH 4 to pH 10, whereas Tn-15 lost almost half of the helical content at pH 4 upon raising the pH to 10. No single amino acid residue was found to ionize in a way that could explain the observed pH dependence of Tn-15. The temperature dependence of the mean residue ellipticity of Tn-15 revealed a surprising decrease in helicity at 278 K in comparison with that at 293 K, reminiscent of cold denaturation, that was not observed for the reference four-helix bundle Thr-15.

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