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  • 1. Aili, Daniel
    et al.
    Enander, Karin
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Kemiska institutionen.
    Liedberg, Bo
    Synthetic de novo designed polypeptides for control of nanoparticle assembly and biosensing2007Inngår i: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 35, nr 3, s. 532-534Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 2. Aili, Daniel
    et al.
    Enander, Karin
    Rydberg, Johan
    Lundström, Ingemar
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Liedberg, Bo
    Aggregation-Induced Folding of a de novo Designed Polypeptide Immobilized on Gold Nanoparticles2006Inngår i: J. Am. Chem. Soc., nr 128, s. 2194-2195Artikkel i tidsskrift (Fagfellevurdert)
  • 3. Aili, Daniel
    et al.
    Enander, Karin
    Rydberg, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Nesterenko, Irina
    Björefors, Fredrik
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Liedberg, Bo
    Folding Induced Assembly of Polypeptide Decorated Gold Nanoparticles2008Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 130, nr 17, s. 5780-5788Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

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

  • 4. Aili, Daniel
    et al.
    Gryko, Piotr
    Sepulveda, Borja
    Dick, John A. G.
    Kirby, Nigel
    Heenan, Richard
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi.
    Liedberg, Bo
    Ryan, Mary P.
    Stevens, Molly M.
    Polypeptide Folding-Mediated Tuning of the Optical and Structural Properties of Gold Nanoparticle Assemblies2011Inngår i: Nano letters (Print), ISSN 1530-6984, E-ISSN 1530-6992, Vol. 11, nr 12, s. 5564-5573Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 5. Andersson, Per Ola
    et al.
    Lundquist, Margaretha
    Tegler, Lotta
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi.
    Börjegren, Susanne
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi.
    Österlund, Lars
    A Novel ATR-FTIR Approach for Characterisation and Identification of Ex Situ Immobilised Species2007Inngår i: ChemPhysChem, ISSN 1439-4235, E-ISSN 1439-7641, Vol. 8, nr 5, s. 712-722Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 6. Andersson, Theresa
    et al.
    Lundquist, Martin
    Dolphin, Gunnar T.
    Enander, Karin
    Jonsson, Bengt-Harald
    Nilsson, Jonas W.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Cooperative binding of human Carbonic Anhydrase II by functionalized folded polypeptide receptors2005Inngår i: Chem. Biol., nr 12, s. 1245-1252Artikkel i tidsskrift (Fagfellevurdert)
  • 7.
    Balliu, Aleksandra
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi.
    Conjugation of a Dipicolyl Chelate to Polypeptide Conjugates Increases Binding Affinities for Human Serum Albumin and Survival Times in Human Serum2017Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, nr 14, s. 1408-1414Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 8.
    Balliu, Aleksandra
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi.
    Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions2017Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, nr 14, s. 1396-1407Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 9.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Polypeptide Conjugate Binders for Protein Recognition2007Inngår i: Topics in current chemistry, ISSN 0340-1022, E-ISSN 1436-5049, Vol. 277, s. 89-106Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    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.

  • 10.
    Baltzer, Lars
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Kemiska institutionen. Institutionen för biokemi och organisk kemi, Organisk kemi II.
    DeGrado, W. F.
    Engineering and design: Expanding the protein world2004Inngår i: Editorial overview Current Opinion of Structural Biology, Vol. 14, s. 455-457Artikkel, forskningsoversikt (Annet (populærvitenskap, debatt, mm))
  • 11.
    Baltzer, Lars
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Klinman, J.P.
    Hynes, J.T.
    Limbach, H-H.
    Acid base catalysis in designed polypeptides2006Inngår i: Handbook of Hydrogen Transfer, Wiley , 2006Kapittel i bok, del av antologi (Fagfellevurdert)
  • 12. Enander, Karin
    et al.
    Aili, D.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Lundström, I.
    Liedberg, B.
    Alpha helix-inducing dimerization of synthetic plypeptide scaffolds on gold2005Inngår i: Langmuir, nr 21, s. 2480-2487Artikkel i tidsskrift (Fagfellevurdert)
  • 13. Enander, Karin
    et al.
    Aili, D.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Kemiska institutionen. Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Lundström, I.
    Liedberg, B.
    Alpha helix-inducing dimerization of synthetic polypeptide scaffolds on gold2005Inngår i: Langmuir, nr 21, s. 2480-2487Artikkel i tidsskrift (Fagfellevurdert)
  • 14.
    Enander, Karin
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Kemiska institutionen. Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Dolphin, G. T.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Kemiska institutionen. Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Designed functionalized helix-loop-helix motifs that bind human Carbonic Anhydrase II- a new class of synthetic receptor molecules2004Inngår i: J. Am. Chem. Soc., nr 126, s. 4464-4465Artikkel i tidsskrift (Fagfellevurdert)
  • 15.
    Enander, Karin
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Kemiska institutionen. Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Dolphin, G.T.
    Liedberg, B.
    Lundström, I.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Kemiska institutionen. Institutionen för biokemi och organisk kemi, Organisk kemi II.
    A versatile polypeptide platform for intergrated recognition and reporting - affinity arrays for protein - ligand interaction analysis2004Inngår i: Chem. Eur. J., nr 10, s. 2375-2385Artikkel i tidsskrift (Fagfellevurdert)
  • 16. Enander, Karin
    et al.
    Dolphin, Gunnar T.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Designed, functionalized helix-loop-helix motifs that bind human Carbonic Anhydrase II - a new class of synthetic receptor molecules2004Inngår i: J. Am. Chem. Soc., nr 126, s. 4464-4465Artikkel i tidsskrift (Fagfellevurdert)
  • 17. Enander, Karin
    et al.
    Dolphin, Gunnar T.
    Liedberg, Bo
    Lundström, Ingemar
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    A versatile polypeptide platform for integrated recognition and reporting - affinity arrays for protein-ligand interaction2004Inngår i: Chem. Eur. J., nr 10, s. 2375-2385Artikkel i tidsskrift (Fagfellevurdert)
  • 18.
    Fromell, Karin
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Klinisk immunologi.
    Forsberg, Pontus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Mikrosystemteknik.
    Karlsson, Mikael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Mikrosystemteknik.
    Larsson, Karin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Oorganisk kemi.
    Nikolajeff, Fredrik
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Mikrosystemteknik.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Designed protein binders in combination with nanocrystalline diamond for use in high-sensitivity biosensors2012Inngår i: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 404, nr 6-7, s. 1643-1651Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A platform for diagnostic applications showing signal-to-noise ratios that by far surpass those of traditional bioanalytical test formats has been developed. It combines the properties of modified nanocrystalline diamond (NCD) surfaces and those of polyethylene oxide and polypropylene oxide based block copolymers for surface passivation and binder conjugation with a new class of synthetic binders for proteins. The NCD surfaces were fluorine-, hydrogen-, or oxygen-terminated prior to further biofunctionalization and the surface composition was characterized by X-ray photoelectron spectroscopy. In a proof of principle demonstration targeting the C-reactive protein, an ELISA carried out using an F-terminated diamond surface showed a signal-to-noise ratio of 3,900 which compares well to the signal-to-noise of 89 obtained in an antibody-based ELISA on a polystyrene microtiter plate, a standard test format used in most life science laboratories today. The increase in signal-to-noise ratio is to a large extent the result of extremely efficient passivation of the diamond surface. The results suggest that significant improvements can be obtained in standardized test formats using new materials in combination with new types of chemical coatings and receptor molecules.

  • 19.
    Giandomenico, Valeria
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Onkologisk endokrinologi.
    Modlin, Irvin M.
    Pontén, Fredrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylär och morfologisk patologi.
    Nilsson, Mats
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
    Bergquist, Jonas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi.
    Khan, Mohid S.
    Millar, Robert P.
    Långström, Bengt
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Borlak, Jurgen
    Eriksson, Barbro
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Endokrin tumörbiologi.
    Nielsen, Bengt
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Waterton, John C.
    Ahlström, Håkan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för radiologi.
    Öberg, Kjell
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Onkologisk endokrinologi.
    Improving the Diagnosis and Management of Neuroendocrine Tumors: Utilizing New Advances in Biomarker and Molecular Imaging Science2013Inngår i: Neuroendocrinology, ISSN 0028-3835, E-ISSN 1423-0194, Vol. 98, nr 1, s. 16-30Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Neuroendocrine tumors (NET) are malignant solid tumors that arise in hormone-secreting tissue of the diffuse neuroendocrine system or endocrine glands. Although traditionally understood to be a rare disease, the incidence and prevalence of NET have increased greatly in the past 3 decades. However, during this time, progress in diagnosis and outcome of NET has generally been modest. In order to achieve improved outcome in NET, a better understanding of NET biology combined with more reliable serum markers and better techniques to identify tumor localization and small lesions are needed. Although some NET biomarkers exist, sensitive and specific markers that predict tumor growth and behavior are generally lacking. In addition, the integration of new molecular imaging technologies in patient diagnosis and follow-up has the potential to enhance care. To discuss developments and issues required to improve diagnostics and management of NET patients, with specific focus on the latest advances in molecular imaging and biomarker science, 17 global leaders in the fields of NET, molecular imaging and biomarker technology gathered to participate in a 2-day meeting hosted by Prof. Kjell Oberg at the University of Uppsala in Sweden. During this time, findings were presented regarding methods with potential prognostic and treatment applications in NET or other types of cancers. This paper describes the symposium presentations and resulting discussions.

  • 20. Hederos, Sofia
    et al.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Nucleophile selectivity in the enzyme catalyzed acyl transfer reaction of a thiol ester2005Inngår i: Biopolymers, nr 79, s. 292-299Artikkel i tidsskrift (Fagfellevurdert)
  • 21. Hederos, Sofia
    et al.
    Broo, Kerstin
    Jakobsson, Emma
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II. Biokemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Kleywegt, Gerard J
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II. Biokemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Mannervik, Bengt
    Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II. Biokemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Baltzer, Lars
    Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II. Biokemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    A new enzyme by rational design - the incorporation of a single His residue enables efficient thioester hydrolysis by human glutathione transferase A1-12004Inngår i: Proc. Nat. Acad. Sci., Vol. 101, s. 13163-13167Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.

  • 22. Hederos, Sofia
    et al.
    Broo, Kerstin S
    Jakobsson, Emma
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Kemiska sektionen, Institutionen för biokemi och organisk kemi, Biokemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi. ICM.
    Kleywegt, Gerard J
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Kemiska sektionen, Institutionen för biokemi och organisk kemi, Biokemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi. ICM.
    Mannervik, Bengt
    Institutionen för naturvetenskaplig biokemi. Kemiska sektionen, Institutionen för biokemi och organisk kemi, Biokemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Baltzer, Lars
    Kemiska institutionen. Kemiska sektionen, Institutionen för biokemi och organisk kemi, Biokemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi.
    Incorporation of a single His residue by rational design enables thiol-ester hydrolysis by human glutathione transferase A1-1.2004Inngår i: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 101, nr 36, s. 13163-7Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.

  • 23. Höst, Gunnar
    et al.
    Razkin, Jesus
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi.
    Jonsson, Bengt-Harald
    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 substrate2007Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 8, nr 13, s. 1570-1576Artikkel i tidsskrift (Fagfellevurdert)
    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. Pengo, Paolo
    et al.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi.
    Pasquato, Lucia
    Scrimin, Paolo
    Substrate Modulation of the Activity of an Artificial Nanoesterase Made of Peptide-Functionalized Gold Nanoparticles2007Inngår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 46, nr 3, s. 400-404Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Nanozymes with a heart of gold: A functional artificial protein has been prepared by grafting a dodecapeptide onto the surface of gold nanoparticles (see picture). The system catalyzes the hydrolysis of carboxylate esters and features enzyme-like properties. (Figure Presented).

  • 25. Peter, K.
    et al.
    Nilsson, R.
    Rydberg, J.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Kemiska institutionen. Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Inganäs, O.
    Twisting macromolecular chains - self-assembly of a chiral supermolecule from non-chiral polythiophene polyanions and random coil synthetic peptides2004Inngår i: Proc. Nat. Acad.Sci, Vol. 101, s. 11197-11202Artikkel i tidsskrift (Fagfellevurdert)
  • 26. Peter, K.
    et al.
    Nilsson, R.
    Rydberg, Johan
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Inganäs, Olle
    Twisting macromolecular chains-self-assembly of a chiral supermolecule from non-chiral polythiophene polyanions and random coil synthetic peptides2004Inngår i: Proc. Nat. Acad. Sci., nr 101, s. 11197-11202Artikkel i tidsskrift (Fagfellevurdert)
  • 27. Ramapanicker, Ramesh
    et al.
    Sun, Xiaojiao
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Viljanen, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Powerful binders for the D-dimer by conjugation of the GPRP peptide to polypeptides from a designed set: illustrating a general route to new binders for proteins2013Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, nr 1, s. 17-25Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The synthetic tetrapeptide GPRP based on the amino-terminal GPR sequence of the fibrin α-chain, binds the D-dimer protein with a dissociation constant KD of 25 μM. The D-dimer protein, a well-known biomarker for thrombosis, contains two cross-linked D fragments from the fibrinogen protein formed upon degradation of the fibrin gel, the core component of blood clots. In order to develop a specific high-affinity binder for the D-dimer protein, GPRP was conjugated via an aliphatic spacer to each member of a set of sixteen polypeptides designed for the development of binder molecules for proteins in general. The binders were individually characterised and ranked using surface plasmon resonance (SPR) analysis. The dissociation constant of the complex formed from the D-dimer and 4-D15L8-GPRP labelled with fluorescein was determined by fluorescense titration and found to be 3 nM, an affinity four orders of magnitude higher than that of free GPRP. According to SPR analysis binding was completely inhibited by free GPRP at mM concentrations and the polypeptide conjugate was therefore shown to bind specifically to the binding site of GPRP. Affinities were further enhanced by dimerisation of the polypeptide conjugates via a bifunctional linker resulting in dissociation constants that were further decreased (affinities increased) by factors of 2-4. The results suggest an efficient route to specific binders for proteins based on short peptides with affinites that need only to be modest, thus shortening the time of binder development dramatically.

  • 28. Razkin, Jesus
    et al.
    Nilsson, Helena
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi.
    Catalysis of the Cleavage of Uridine 3‘-2,2,2-Trichloroethylphosphate by a Designed Helix−Loop−Helix Motif Peptide2007Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 129, nr 47, s. 14752-14758Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A 42-residue peptide that folds into a helix−loop−helix motif and dimerizes to form a four-helix bundle has been designed to catalyze the hydrolysis of phosphodiesters. The active site on the surface of the folded catalyst is composed of two histidine and four arginine residues, with the capacity to provide general acid, general base, and/or nucleophilic catalysis as well as transition state stabilization. Uridine 3‘-2,2,2 trichloroethylphosphate (2) is a mimic of RNA with a leaving group pKa of 12.3. Its hydrolysis is energetically less favorable than that of commonly used model substrates with p-nitrophenyl leaving groups and therefore a more realistic model for the design of catalysts capable of cleaving RNA. The second-order rate constant for the hydrolysis of 2 at pH 7.0 by the polypeptide catalyst was 418 × 10-6 M-1 s-1, and that of the imidazole catalyzed reaction was 1.66 × 10-6 M-1 s-1. The pH dependence suggested that catalysis is due to the unprotonated form of a residue with a pKa of around 5.3, and the observed kinetic solvent isotope effect of 1.9 showed that there is significant hydrogen bonding in the transition state, consistent with general acid−base catalysis. The rate constant ratio k2(Pep)/k2(Im) of 252 is probably due to a combination of nucleophilic and general acid−base catalysis, as well as transition state stabilization. Substrate binding was weak since no sign of saturation kinetics was observed for substrate concentrations in the range from 5 to 40 mM. The results provide a platform for the further development of catalysts for RNA cleavage with a potential role in the development of drugs.

     

  • 29. Rossi, Paola
    et al.
    Tecilla, Paolo
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Scrimin, Paolo
    De novo Metallonucleases based on Helix-loop-helix Motifs2004Inngår i: Chem. Eur. J., nr 10, s. 4163-4170Artikkel i tidsskrift (Fagfellevurdert)
  • 30. Rydberg, Johan
    et al.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Sarojini, Vijayalekshmi
    Intrinsically unstructured proteins by designelectrostatic interactions can control binding, folding, and function of a helix-loop-helix heterodimer2013Inngår i: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 19, nr 8, s. 461-469Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Intrinsically disordered proteins that exist as unordered monomeric structures in aqueous solution at pH7 but fold into four-helix bundles upon binding to recognized polypeptide targets have been designed. NMR and CD spectra of the monomeric polypeptides show the hallmarks of unordered structures, whereas in the bound state they are highly helical. Analytical ultracentrifugation data shows that the polypeptides bind to their targets to form exclusively heterodimers at neutral pH. To demonstrate the relationship between binding, folding, and function, a catalytic site for ester hydrolysis was introduced into an unordered and largely inactive monomer, but that was structured and catalytically active in the presence of a specific polypeptide target. Electrostatic interactions between surface-exposed residues inhibited the binding and folding of the monomers at pH7. Charge-charge repulsion between ionizable amino acids was thus found to be sufficient to disrupt binding between polypeptide chains despite their inherent propensities for structure formation and may be involved in the folding and function of inherently disordered proteins in biology. 

  • 31. Scrimin, P.
    et al.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Kemiska institutionen. Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Model Systems2005Inngår i: Editorial Overwiev, Current Opinion of Chemical Biology, nr 9, s. 620-621Artikkel, forskningsoversikt (Annet (populærvitenskap, debatt, mm))
  • 32. Scrimin, Paolo
    et al.
    Baltzer, LarsUppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Organisk kemi II.
    Model Systems2005Collection/Antologi (Annet vitenskapelig)
  • 33.
    Sun, Xiaojiao
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Yang, Jie
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Norberg, Thomas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    A synthetic polypeptide conjugate from a 42-residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity2012Inngår i: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 18, nr 12, s. 731-739Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Myeloperoxidase (MPO) is a 150 kD tetrameric heme protein consisting of two heavy chains and two light chains, which ispresent in neutrophils, white blood cells, at concentrations between 2% and 5% and plays an important role in the innateimmune system. The MPO concentration in serum or plasma has been shown to be linked to the risk for cardiovasculardiseases, and MPO is considered to be a high potential diagnostic biomarker. To develop a molecule that binds MPO,salicylhydroxamic acid (SHA), a substrate analog inhibitor of MPO with a KD=2uM, was conjugated to a designed set of42-residue polypeptide scaffolds via 9- and 11-carbon atom aliphatic spacers to form 20 different protein binder candidates,and their interactions with MPO were evaluated by surface plasmon resonance analysis. The polypeptide conjugate4C37L34C11SHA was found to bind to MPO with an affinity that could be estimated to have a dissociation constant of around400 pM, nearly four orders of magnitude higher than that of SHA. Inhibition of binding to MPO by free SHA was observed incompetition experiments demonstrating that the binding of the polypeptide conjugate is dominated by the interactions ofSHA with the heme cavity. Although still in the future, the discovery of these new synthetic binders for MPO suggests aroute to clinical diagnostic tests in vivo or in vitro, independent of antibodies.

  • 34. Wang, Yusong
    et al.
    Aili, Daniel
    Selegard, Robert
    Tay, Yeeyan
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Zhang, Hua
    Liedberg, Bo
    Specific functionalization of CTAB stabilized anisotropic gold nanoparticles with polypeptides for folding-mediated self-assembly2012Inngår i: Journal of Materials Chemistry, ISSN 0959-9428, E-ISSN 1364-5501, Vol. 22, nr 38, s. 20368-20373Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Anisotropic nanoparticles stabilized by cetyltrimethylammonium bromide (CTAB) are notoriously difficult to homogenously functionalize using conventional gold-thiol chemistry. Using surface assisted laser desorption time of flight mass spectroscopy and scanning transmission electron microscopy-energy dispersive X-ray spectroscopy, we demonstrate that silver species adsorbed on the particle surface prevent effective surface functionalization. When covered by a thin gold film, particle functionalization was drastically improved. A thiol-containing polypeptide was immobilized on arrowhead gold nanorods (NRs) and was subsequently able to selectively heteroassociate with a complementary polypeptide resulting in a folding-mediated bridging aggregation of the NRs. Despite using arrowhead NRs with a pronounced difference in surface arrangement on the {111} facets on the arrowheads compared to the {100} facets at the particle sides, the polypeptides were efficiently and homogeneously immobilized on the particles after gold film overgrowth.

  • 35.
    Yang, Jie
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi.
    Gustavsson, Anna-Lena
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, CBCS, Stockholm, Sweden..
    Haraldsson, Martin
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, CBCS, Stockholm, Sweden..
    Karlsson, Göran
    Gothenburg Univ, Swedish NMR Ctr, Gothenburg, Sweden..
    Norberg, Thomas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi.
    High-affinity recognition of the human C-reactive protein independent of phosphocholine2017Inngår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 15, nr 21, s. 4644-4654Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A high-affinity polypeptide conjugate 4-C25L22-DQ, has been developed for the molecular recognition of the human C-reactive protein, CRP, a well-known inflammation biomarker. CRP is one of the most frequently quantified targets in diagnostic applications and a target in drug development. With the exception of antibodies, most molecular constructs take advantage of the known affinity for CRP of phosphocholine that depends on Ca2+ for its ability to bind. 4-C25L22-DQ which is unrelated to phosphocholine binds in the absence of Ca2+ with a dissociation constant of 760 nM, an order of magnitude lower than that of phosphocholine, the KD of which is 5 μM. The small organic molecule 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) was designed based on the structural similarities between three hits from a set of compounds selected from a building block collection and evaluated with regards to affinity for CRP by NMR spectroscopy. 4-C25L22-DQ was shown in a competition experiment to bind CRP three orders of magnitude more strongly than DQ itself, and in a pull-down experiment 4-C25L22-DQ was shown to extract CRP from human serum. The development of a robust and phosphocholine-independent recognition element provides unprecedented opportunities in bioanalytical applications in vivo and in vitro under conditions where the concentration of Ca2+ ions is low, or where Ca2+ binding agents such as EDTA or heparin are needed to prevent blood coagulation. The identification from a compound library of a small organic molecule and its conjugation to a small set of polypeptides, none of which were previously known to bind CRP, illustrates a convenient and general route to selective high-affinity binders for proteins with dissociation constants in the μM to nM range for which no small molecule ligands are known.

  • 36.
    Yang, Jie
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi.
    Koruza, Katarina
    Fisher, Zoë
    Knecht, Wolfgang
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi.
    Improved molecular recognition of Carbonic Anhydrase IX by polypeptide conjugation to acetazolamide2017Inngår i: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, nr 20, s. 5838-5848Artikkel i tidsskrift (Fagfellevurdert)
  • 37.
    Ślósarczyk, Adam T.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Efficient formation of heterodimers from peptides and proteins using unsymmetrical polyfluorophenyl esters of dicarboxylic acids2012Inngår i: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 18, nr 4, s. 261-269Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An efficient method for the heteroconjugation of biomolecules carrying free amino groups was reported previously, where mixed polyfluorophenyl diesters of dicarboxylic acids with varied aliphatic chain length were shown to be efficient reagents for the conjugation of a variety of model biomolecules. The concept was based on the differential reactivity of the esters towards amines. The concept has now been further optimized, and a 2,6-difluorophenyl-pentafluorophenyl diester combination has been demonstrated to be the most efficient, both with respect to selectivity and to reaction rate. A pentafluorophenyl ester reacts faster with an amino group and requires a weaker base than a 2,6-difluorophenyl ester that requires a stronger base and longer reaction time. With the use of this combination of esters, we obtained considerably shortened reaction times compared with those reported previously, yet still retaining the desired selectivity in heteroconjugation. The increased reactivity of the bifunctional reagent allowed the construction of sophisticated peptide heteroconjugates from peptides, carbohydrates and proteins, showing a wide scope of applicability in the field of assembling functional bioconjugates.

  • 38.
    Ślósarczyk, Adam T.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi.
    The molecular recognition of phosphorylated proteins by designed polypeptides conjugated to a small molecule that binds phosphate2011Inngår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 9, nr 22, s. 7697-7704Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The conjugation of polypeptides from a designed set to the small molecule ligand 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, which in the presence of Zn2+ ions binds inorganic phosphate, has been shown to provide a polypeptide conjugate that binds α-casein, a multiply phosphorylated protein, with a dissociation constant KD of 17 nM. The measured affinity is more than three orders of magnitude higher than that of the small molecule ligand for phosphate and the binding of 500 nM of α-casein was not inhibited by 10 mM phosphate buffer, providing a 2000-fold excess of phosphate ion over protein. The selectivity for phosphoproteins was demonstrated by extraction of α-casein from solutions of various complexity, including milk and human serum spiked with α-casein. In addition to α-casein, β-casein was also recognized but not ovoalbumin. Conjugation of a polypeptide to the zinc chelating ligand was therefore shown to give rise to dramatically increased affinity and also increased selectivity. A set of polypeptide conjugates is expected to be able to capture a large number of phosphorylated proteins, perhaps all, and in combination with electrophoresis or mass spectrometry become a powerful tool for the monitoring of phosphorylation levels. The presented binder can easily be attached to various types of surfaces; here demonstrated for the case of polystyrene particles. The example of phosphoproteins was selected since posttranslational phosphorylation is of fundamental importance in cell biology due to its role in signaling and therefore of great interest in drug development. The reported concept for binder development is, however, quite general and high-affinity binders can conveniently be developed for a variety of proteins including those with posttranslational modifications for which small molecule recognition elements are available.

  • 39.
    Ślósarczyk, Adam T.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Ramapanicker, Ramesh
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Norberg, Thomas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Fysikalisk-organisk kemi.
    Mixed pentafluorophenyl and o-fluorophenyl esters of aliphatic dicarboxylic acids: efficient tools for peptide and protein conjugation2012Inngår i: RSC Advances, ISSN 2046-2069, Vol. 2, nr 3, s. 908-914Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An efficient methodology for the heteroconjugation of biomolecules with exposed free amino groups has been developed. Mixed pentafluorophenyl and o-fluorophenyl esters of aliphatic dicarboxylic acids with aliphatic chains of varying sizes have been prepared and used to conjugate a 42-residue polypeptides with short model peptides as well as a model dodecapeptide with the antigenic determinant of type B blood, a carbohydrate derivative, to form a glycopeptide. The concept is based on the difference in reactivity towards primary amino groups between phenyl esters with leaving groups of unlike pKa. The reactivities of several pentafluorophenyl and o-fluorophenyl esters towards amino groups were carefully determined under reaction conditions to identify leaving group combinations that would provide optimal differences in reactivity for maximum yields of heteroconjugate formation while keeping the reasonable reaction times. Pentafluorophenyl esters react faster with an amino group and require a weaker base, while an o-fluorophenyl ester requires a stronger base and longer reaction time. The method described is economic, quick and gives complete control over the conjugation reaction. The size of the spacer is conveniently varied by selection of the appropriate aliphatic dicarboxylic acid. While the presented examples describe conjugation reactions of polypeptides with a maximum of 42 residues it is envisioned that the bifunctional linkers reported here will find their most important applications in the heteroconjugation of proteins using lysine side chains, a reaction for which currently few alternatives exist, if access to spacers of variable size is required.

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