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  • 1. Aaldering, L. J.
    et al.
    Poongavanam, V.
    Langkjær, N.
    Natarajan Arul, Murugan
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Jørgensen, P. T.
    Wengel, J.
    Veedu, R. N.
    Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule2017In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 8, p. 755-763Article in journal (Refereed)
    Abstract [en]

    The thrombin-binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G-quadruplex-forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three-carbon spacer (spacer-C3), unlocked nucleic acid (UNA) and 3′-amino-modified UNA (amino-UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G-quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer-C3 introduction at the T7 loop position (TBA-SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA-SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  • 2.
    Aboye, Teshome L.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Strömstedt, Adam A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Gunasekera, Sunithi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Bruhn, Jan G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    El-Seedi, Hesham
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Rosengren, K. Johan
    Göransson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    A Cactus-Derived Toxin-Like Cystine Knot Peptide with Selective Antimicrobial Activity2015In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 7, p. 1068-1077Article in journal (Refereed)
    Abstract [en]

    Naturally occurring cystine knot peptides show a wide range of biological activity, and as they have inherent stability they represent potential scaffolds for peptide-based drug design and biomolecular engineering. Here we report the discovery, sequencing, chemical synthesis, three-dimensional solution structure determination and bioactivity of the first cystine knot peptide from Cactaceae (cactus) family: Ep-AMP1 from Echinopsis pachanoi. The structure of Ep-AMP1 (35 amino acids) conforms to that of the inhibitor cystine knot (or knottin) family but represents a novel diverse sequence; its activity was more than 500 times higher against bacterial than against eukaryotic cells. Rapid bactericidal action and liposome leakage implicate membrane permeabilisation as the mechanism of action. Sequence homology places Ec-AMP1 in the plant C6-type of antimicrobial peptides, but the three dimensional structure is highly similar to that of a spider neurotoxin.

  • 3.
    Aisenbrey, Christopher
    et al.
    Institut de Chimie Universit0 Louis Pasteur Strasbourg—CNRS, UMR 7177 4, Rue Blaise Pascal, 67000 Strasbourg (France); Max-Planck-Institut f>r Biochemie Am Klopferspitz 18A, 82152 Martinsried (Germany).
    Cusan, Monica
    Lambotte, Stephan
    Jasperse, Pieter
    Georgescu, Julia
    Harzer, Ulrike
    Bechinger, Burkhard
    Specific Isotope Labeling of Colicin E1 and B Channel Domains For Membrane Topological Analysis by Oriented Solid-State NMR Spectroscopy2008In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, no 6, p. 944-951Article in journal (Refereed)
    Abstract [en]

    An approach is presented to selectively label the methionines of the colicin E1 and B channel domains, each about 200 residues in size, and use them for oriented solid-state NMR investigations. By combining site-directed mutagenesis, bacterial overexpression in a methionine auxotroph E. coli strain and biochemical purification, quantitative amounts of the proteins for NMR structural investigations were obtained. The proteins were selectively labeled with 15N at only one, or at a few, selected sites. Multidimensional heteronuclear correlation high-resolution NMR spectroscopy and mass spectrometry were used to monitor the quality of isotopic labeling. Thereafter the proteins were reconstituted into oriented phospholipid bilayers and investigated by proton-decoupled 15N solid-state NMR spectroscopy. The colicin E1 thermolytic fragment that carries a single 15N methionine within its hydrophobic helix 9 region exhibited 15N resonances that are characteristic of helices that are oriented predominantly parallel to the membrane surface at low temperature, and a variety of alignments and conformations at room temperature. This suggests that the protein can adopt both umbrella and pen-knife conformations.

  • 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. 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.

  • 6.
    Balliu, Aleksandra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Baltzer, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Conjugation of a Dipicolyl Chelate to Polypeptide Conjugates Increases Binding Affinities for Human Serum Albumin and Survival Times in Human Serum2017In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 14, p. 1408-1414Article in journal (Refereed)
    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.

  • 7.
    Balliu, Aleksandra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Baltzer, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions2017In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 14, p. 1396-1407Article in journal (Refereed)
    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.

  • 8.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hydrolases in Organic Synthesis: Regio- and Stereoselective Biotransformation: By Uwe T. Bornscheuer and Romas J. Kazlauskas2006In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 7, no 8, p. 1280-Article, book review (Other academic)
  • 9.
    Bergquist, Helen
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Nikravesh, Abbas
    Fernández, Raquel Domingo
    Larsson, Veronica
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Nguyen, Chi-Hung
    Good, Liam
    Zain, Rula
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Structure-Specific Recognition of Friedreich’s Ataxia (GAA)n Repeats by Benzoquinoquinoxaline Derivatives2009In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 10, no 16, p. 2629-2637Article in journal (Refereed)
    Abstract [en]

    Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreich's ataxia. (GAA)nrepeats form non-B-DNA structures, including triple helix H-DNA and higher-order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non-B-DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)nbinding by triplex-stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex-DNA-cleaving BQQ-1,10-phenanthroline (BQQ-OP) compounds. We also examined the ability of these compounds to act as structural probes for H-DNA formation within higher-order structures at pathological frataxin sequences in plasmids. DNA-complex-formation analyses with a gel-mobility-shift assay and sequence-specific probing of H-DNA-forming (GAA)nsequences by single-strand oligonucleotides and triplex-directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)nrepeats under physiologically relevant conditions.

  • 10. Bunyapaiboonsri, T.
    et al.
    Ramström, Olof
    KTH, Superseded Departments, Chemistry.
    Lohmann, S.
    Lehn, J. M.
    Peng, L.
    Goeldner, M.
    Dynamic deconvolution of a pre-equilibrated dynamic combinatorial library of acetylcholinesterase inhibitors2001In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 2, no 6, p. 438-444Article in journal (Refereed)
    Abstract [en]

    A dynamic combinatorial library composed of interconverting acylhydrazones has been generated and screened towards inhibition of acetylcholinesterase from the electric ray Torpedo marmorata. Starting from a small set (13) of initial hydrazide and aldehyde building blocks, a library containing possibly 66 different species was obtained in a single operation. Of all possible acylhydrazones formed, active compounds containing two terminal cationic recognition groups separated by an appropriate distance, permitting two-site binding, could be rapidly identified by using a dynamic deconvolution-screening procedure, based on the sequential removal of starting building blocks. A very potent bis-pyridinium inhibitor (K (i)= 1.09 nM, alphaK(i) = 2.80 nM) was selected from the process and the contribution of various structural features to inhibitory potency was evaluated.

  • 11. Cammenberg, Maria
    et al.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Park, Seongsoon
    Molecular basis for the enhanced lipase-catalyzed N-acylation of 1-phenylethanamine with methoxyacetate2006In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 7, no 11, p. 1745-1749Article in journal (Refereed)
    Abstract [en]

    One of the commercial methods for preparing enantiopure amines is lipase-catalyzed kinetic resolution, although lipases catalyze, aminolysis with only low activity. Interestingly, in 1997 Balkenhohl et al. used, ethyl methoxyacetate instead of ethyl butyrate as an acylation reagent for the aminolysis of 1-phenylethanamine and increased the reaction rate more than a 100-fold. This method has been applied to other aminolysis reactions, but the molecular basis for the enhanced rate is not understood. A moecular-modeling study of the transition-state analogue for the aminolysis showed that an interaction between the beta-oxygen atom in methoxyacetate and the amine nitrogen atom might be a key factor in the rate enhancement. Other acylation reagents, such as methyl 3-methoxypropionate and methyl 4-methoxybutyrate, were chosen to test the influence of this interaction because these molecules can be spatially arranged to have similar to that in the acylation with methyoxyacetate. The initial aminolysis rates were improved (11-fold and sixfold, respectively) compared to that with butyrate. In with 1-phenylethanol afforded the same rate with all acyl donors.

  • 12.
    Caraballo, Rémi
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Sakulsombat, Morakot
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Towards Dynamic Drug Design: Identification and Optimization of β-Galactosidase Inhibitors from a Dynamic Hemithioacetal System2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 11, p. 1600-1606Article in journal (Refereed)
    Abstract [en]

    A discovery strategy relying on the identification of fragments through resolution of a constitutional dynamic system, coupled to subsequent static ligand design and optimization, is demonstrated. The strategic design and synthesis of the best molecular fragments identified from a dynamic hemithioacetal system into static ligand structures yielded a range of -galactosidase inhibitors. Two series of structures mimicking the hemithioacetal motif were envisaged: thioglycosides and C-glycosides. Inhibition studies provided important structural information for the two groups, and 1-thiobenzyl--D-galactopyranoside demonstrated the best inhibitory effects.

  • 13. Carbajales, Carlos
    et al.
    Prado, Miguel Angel
    Gutierrez-de-Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Cores, Angel
    Azuaje, Jhonny
    Novio, Silvia
    Jesus Nunez, Maria
    Fernandez-Garcia, Belen
    Sotelo, Eddy
    Garcia-Mera, Xerardo
    Sanchez-Lazo, Pedro
    Freire-Garabal, Manuel
    Coelho, Alberto
    Structure-Based Design of New KSP-Eg5 Inhibitors Assisted by a Targeted Multicomponent Reaction2014In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 15, no 10, p. 1471-1480Article in journal (Refereed)
    Abstract [en]

    An integrated multidisciplinary approach that combined structure-based drug design, multicomponent reaction synthetic approaches and functional characterization in enzymatic and cell assays led to the discovery of new kinesin spindle protein (KSP) inhibitors with antiproliferative activity. A focused library of new benzimidazoles obtained by a Ugi + Boc removal/cyclization reaction sequence generated low-micromolar-range KSP inhibitors as promising anticancer prototypes. The design and functional studies of the new chemotypes were assessed by computational modeling and molecular biology techniques. The most active compounds-20 (IC50=1.49 mu m, EC50=3.63 mu m) and 22 (IC50=1.37 mu m, EC50=6.90 mu m)-were synthesized with high efficiency by taking advantage of the multicomponent reactions.

  • 14.
    Carlqvist, Peter
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Svedendahl, Maria
    KTH, School of Biotechnology (BIO), Biochemistry.
    Branneby, Cecilia
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, p. 331-336Article in journal (Refereed)
    Abstract [en]

    Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant locks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The k(cat) values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.

  • 15.
    Correia, Mario S. P.
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery.
    Rao, Menghua
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ballet, Caroline
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery.
    Globisch, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Coupled Enzymatic Treatment and Mass Spectrometric Analysis for Identification of Glucuronidated Metabolites in Human Samples2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, no 13, p. 1678-1683Article in journal (Refereed)
    Abstract [en]

    Glucuronidation is the most common phase II modification and plays an important role in human clearance metabolism. Glucuronidated metabolites have also been linked to disease development and microbiota-host co-metabolism. Although many of these compounds have been identified, the total number of unknown glucuronides and their impact on the human host's physiology can only be estimated. Herein, we describe the combination of an untargeted metabolomics analysis and enzymatic metabolic conversion for the selective detection of glucuronide conjugates by using UPLC-MS/MS in human urine samples. Our study demonstrates that this powerful strategy can be used for the selective identification of glucuronidated molecules and to discover unknown natural metabolites. In total, we identified 191 metabolites in a single sample including microbiota-derived compounds as well as previously unidentified molecules.

  • 16.
    Cuetos, Anibal
    et al.
    Univ York, York Struct Biol Lab, York YO10 5DD, N Yorkshire, England..
    Steffen-Munsberg, Fabian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanchez, Juan Mangas
    Univ York, York Struct Biol Lab, York YO10 5DD, N Yorkshire, England..
    Frese, Amina
    Univ York, York Struct Biol Lab, York YO10 5DD, N Yorkshire, England..
    Bornscheuer, Uwe T.
    Ernst Moritz Arndt Univ Greifswald, Inst Biochem, Felix Hausdorff Str 4, D-17487 Greifswald, Germany..
    Hoehne, Matthias
    Ernst Moritz Arndt Univ Greifswald, Inst Biochem, Felix Hausdorff Str 4, D-17487 Greifswald, Germany..
    Grogan, Gideon
    Univ York, York Struct Biol Lab, York YO10 5DD, N Yorkshire, England..
    Structural Basis for Phospholyase Activity of a Class III Transaminase Homologue2016In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 24, p. 2308-2311Article in journal (Refereed)
    Abstract [en]

    Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a remarkable diversity of chemical reactions in nature. A1RDF1 from Arthrobacter aurescens TC1 is a fold type I, PLP-dependent enzyme in the class III transaminase (TA) subgroup. Despite sharing 28% sequence identity with its closest structural homologues, including beta-alanine: pyruvate and gamma-aminobutyrate: alpha-ketoglutarate TAs, A1RDF1 displayed no TA activity. Activity screening revealed that the enzyme possesses phospholyase (E.C. 4.2.3.2) activity towards O-phosphoethanolamine (PEtN), an activity described previously for vertebrate enzymes such as human AGXT2L1, enzymes for which no structure has yet been reported. In order to shed light on the distinctive features of PLP-dependent phospholyases, structures of A1RDF1 in complex with PLP (internal aldimine) and PLP.PEtN (external aldimine) were determined, revealing the basis of substrate binding and the structural factors that distinguish the enzyme from class III homologues that display TA activity.

  • 17.
    Dorau, Robin
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Gorbe, Tamas
    Humble, Maria Svedendahl
    Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering2018In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 4, p. 338-346Article in journal (Refereed)
    Abstract [en]

    Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues for mutagenesis were found by combining available literature data with molecular modeling. SC variants were created by site-directed mutagenesis and were evaluated for a model transacylation reaction containing 1-phenylethanol in THF. Variants showing high E values (>100) were found. However, the conversions were still low. A second mutation was made, and both the E values and conversions were increased. Relative to that shown by the wild type, the most successful variant, G165L/M221F, showed increased conversion (up to 36%), enantioselectivity (E values up to 400), substrate scope, and stability in THF.

  • 18. Dorau, Robin
    et al.
    Görbe, Tamás
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Svedendahl Humble, Maria
    Improved Enantioselectivity of Subtilisin Carlsberg Towards Secondary Alcohols by Protein Engineering2018In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 4, p. 338-346Article in journal (Refereed)
    Abstract [en]

    Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues for mutagenesis were found by combining available literature data with molecular modeling. SC variants were created by site-directed mutagenesis and were evaluated for a model transacylation reaction containing 1-phenylethanol in THF. Variants showing high E values (>100) were found. However, the conversions were still low. A second mutation was made, and both the E values and conversions were increased. Relative to that shown by the wild type, the most successful variant, G165L/M221F, showed increased conversion (up to 36 %), enantioselectivity (E values up to 400), substrate scope, and stability in THF.

  • 19.
    Eildal, Jonas N. N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bach, Anders
    Dogan, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ye, Fei
    Zhang, Mingjie
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Stromgaard, Kristian
    Rigidified Clicked Dimeric Ligands for Studying the Dynamics of the PDZ1-2 Supramodule of PSD-952015In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 1, p. 64-69Article in journal (Refereed)
    Abstract [en]

    PSD-95 is a scaffolding protein of the MAGUK protein family, and engages in several vital protein-protein interactions in the brain with its PDZ domains. It has been suggested that PSD-95 is composed of two supramodules, one of which is the PDZ1-2 tandem domain. Here we have developed rigidified high-affinity dimeric ligands that target the PDZ1-2 supramodule, and established the biophysical parameters of the dynamic PDZ1-2/ligand interactions. By employing ITC, protein NMR, and stopped-flow kinetics this study provides a detailed insight into the overall conformational energetics of the interaction between dimeric ligands and tandem PDZ domains. Our findings expand our understanding of the dynamics of PSD-95 with potential relevance to its biological role in interacting with multivalent receptor complexes and development of novel drugs.

  • 20.
    Engfeldt, Torun
    et al.
    KTH, School of Biotechnology (BIO).
    Renberg, Björn
    KTH, School of Biotechnology (BIO).
    Brumer, Harry
    KTH, School of Biotechnology (BIO).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO).
    Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 6, p. 1043-1050Article in journal (Refereed)
    Abstract [en]

    Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical Synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-nophthalenesulfonic acid (EDANS) and 6(7-nitrobenzofurazon-4-yiamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA.

  • 21.
    Eriksson, Adam
    et al.
    KTH, School of Chemical Science and Engineering (CHE).
    Kürten, Charlotte
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling2017In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 23, p. 2301-2305Article in journal (Refereed)
    Abstract [en]

    Terpenes represent one of the most diversified classes of natural products with potent biological activities. The key to the myriad of polycyclic terpene skeletons with crucial functions in organisms from all kingdoms of life are terpene cyclase enzymes. These biocatalysts enable stereospecific cyclization of relatively simple, linear, prefolded polyisoprenes by highly complex, partially concerted, electrophilic cyclization cascades that remain incompletely understood. Herein, additional mechanistic light is shed on terpene biosynthesis by kinetic studies in mixed H2O/D2O buffers of a classII bacterial ent-copalyl diphosphate synthase. Mass spectrometry determination of the extent of deuterium incorporation in the bicyclic product, reminiscent of initial carbocation formation by protonation, resulted in a large kinetic isotope effect of up to seven. Kinetic analysis at different temperatures confirmed that the isotope effect was independent of temperature, which is consistent with hydrogen tunneling.

  • 22. Filipiak, Kamila
    et al.
    Kubinski, Konrad
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Ramos, Ana
    de Pascual-Teresa, Beatriz
    Human Protein Kinase CK2 Phosphorylates Matrix Metalloproteinase 2 and Inhibits its Activity2014In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 15, no 13, p. 1873-1876Article in journal (Refereed)
    Abstract [en]

    Matrix metalloproteinase 2 (MMP-2) is involved in cancer development and is overexpressed in a variety of malignant tumors. MMP-2 activity is controlled mainly by transcription, proteolytic activation, and inhibition by endogenous inhibitors. It had previously been demonstrated that MMP-2 activity is also regulated by phosphorylation at several sites by protein kinase C. Here we demonstrate, by means of bioinformatics and biochemical and cellular assays, that protein kinase CK2 also acts as a modulator of MMP-2 activity. CK2 down-regulates MMP-2 in vitro, and inhibition of CK2 in human fibrosarcoma cells results in up-regulation of MMP-2. The discovery of the crosstalk between MMP-2 and CK2 opens the possibility of new combined anticancer therapies.

  • 23. Gallardo, Rodrigo
    et al.
    Ivarsson, Ylva
    KU Leuven.
    Schymkowitz, Joost
    Rousseau, Frédéric
    Zimmermann, Pascale
    Structural diversity of PDZ-lipid interactions2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 4, p. 456-67Article in journal (Refereed)
    Abstract [en]

    PDZ domains are globular protein modules that are over-and-above appreciated for their interaction with short peptide motifs found in the cytosolic tail of membrane receptors, channels, and adhesion molecules. These domains predominate in scaffold molecules that control the assembly and the location of large signaling complexes. Studies have now emerged showing that PDZ domains can also interact with membrane lipids, and in particular with phosphoinositides. Phosphoinositides control various aspects of cell signaling, vesicular trafficking, and cytoskeleton remodeling. When investigated, lipid binding appears to be extremely relevant for PDZ protein functionality. Studies point to more than one mechanism for PDZ domains to associate with lipids. Few studies have been focused on the structural basis of PDZ-phosphoinositide interactions, and the biological consequences of such interactions. Using the current knowledge on syntenin-1, syntenin-2, PTP-Bas, PAR-3 and PICK1, we recapitulate our understanding of the structural and biochemical aspects of PDZ-lipid interactions and the consequences for peptide interactions.

  • 24.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lipids and Cellular Membranes in Amyloid Diseases: Edited by Raz Jelinek2011In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 12, no 17, p. 2699-2700Article, book review (Refereed)
  • 25.
    Gunasekera, Sunithi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.
    Muhammad, Taj
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.
    Strömstedt, Adam A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.
    Rosengren, K. Johan
    Univ Queensland, Sch Biomed Sci, Brisbane, Qld 4072, Australia.
    Göransson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.
    Alanine and Lysine Scans of the LL-37-Derived Peptide Fragment KR-12 Reveal Key Residues for Antimicrobial Activity2018In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 9, p. 931-939Article in journal (Refereed)
    Abstract [en]

    The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions. Residues 18-29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR-12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad-spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR-12 is a tuneable template for antibiotic development.

  • 26.
    Gurell, Ann
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Modification of substrate specificity resulted in an epoxide hydrolase with shifted enantiopreference for (2,3-epoxypropyl)benzene2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 10, p. 1422-1429Article in journal (Refereed)
    Abstract [en]

    Random mutagenesis targeted at hot spots of non-catalytic active-site residues of potato epoxide hydrolase StEH1 combined with an enzyme-activity screen allowed for isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the variant displaying most radical functional changes, showed a 15:1 preference for the R-enantiomer. This mutant had accumulated four substitutions distributed to two, out of four mutated, hot spots: W106L, L109Y, V141K and I151V. The underlying causes of the enantioselectivity were a decreased catalytic efficiency in the catalyzed hydrolysis of the S-enantiomer combined with retained activity with the R-enantiomer. The results demonstrate the feasibility to mold stereoselectivity in this biocatalytically relevant enzyme.

  • 27.
    Göransson, Ulf
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Herrmann, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Burman, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Haugaard-Jonsson, M
    Rosengren, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    The Conserved Glu in the Cyclotide Cycloviolacin O2 Has a Key Structural Role2009In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 10, no 14, p. 2354-2360Article in journal (Refereed)
    Abstract [en]

    Cyclotides are a large family of plant peptides that are characterised by a head-to-tail circular backbone and three disulfide bonds that are arranged in a cystine knot. This unique structural feature, which is referred to as a cyclic cystine knot, gives the cyclotides remarkable stability against chemical and biological degradation. In addition to their natural function as insecticides for plant defence, the cyclotides have a range of bioactivities with pharmaceutical relevance, including cytotoxicity against cancer cell lines. A glutamic acid residue, aside from the invariable disulfide array, is the most conserved feature throughout the cyclotide family, and it has recently been shown to be crucial for biological activity. Here we have used solution-state NMR spectroscopy to determine the three-dimensional structures of the potent cytotoxic cyclotide cycloviolacin O2, and an inactive analogue in which this conserved glutamic acid has been methylated. The structures of the peptides show that the glutamic acid has a key structural role in coordinating a set of hydrogen bonds in native cycloviolacin O2; this interaction is disrupted in the methylated analogue. The proposed mechanism of action of cyclotides is membrane disruption and these results suggest that the glutamic acid is linked to cyclotide function by stabilising the structure to allow efficient aggregation in membranes, rather than in a direct interaction with a target receptor.

  • 28. Göransson, Ulf
    et al.
    Herrmann, Anders
    Burman, Robert
    Haugaard-Kedström (published under the name Haugaard-Jönsson), Linda M.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Rosengren, K. Johan
    University of Kalmar, School of Pure and Applied Natural Sciences.
    The conserved Glu in the cyclotide cycloviolacin O2 has a key structural role2009In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 10, no 14, p. 2354-2360Article in journal (Refereed)
    Abstract [en]

    Cyclotides are a large family of plant peptides that are characterised by a head-to-tail circular backbone and three disulfide bonds that are arranged in a cystine knot. This unique structural feature, which is referred to as a cyclic cystine knot, gives the cyclotides remarkable stability against chemical and biological degradation. In addition to their natural function as insecticides for plant defence, the cyclotides have a range of bioactivities with pharmaceutical relevance, including cytotoxicity against cancer cell lines. A glutamic acid residue, aside from the invariable disulfide array, is the most conserved feature throughout the cyclotide family, and it has recently been shown to be crucial for biological activity. Here we have used solution-state NMR spectroscopy to determine the three-dimensional structures of the potent cytotoxic cyclotide cycloviolacin O2, and an inactive analogue in which this conserved glutamic acid has been methylated. The structures of the peptides show that the glutamic acid has a key structural role in coordinating a set of hydrogen bonds in native cycloviolacin O2; this interaction is disrupted in the methylated analogue. The proposed mechanism of action of cyclotides is membrane disruption and these results suggest that the glutamic acid is linked to cyclotide function by stabilising the structure to allow efficient aggregation in membranes, rather than in a direct interaction with a target receptor.

  • 29.
    Hampel, Sabrina
    et al.
    Albert Ludwigs Univ Freiburg, Inst Pharmazeut Wissensch, Albertstr 25, D-79104 Freiburg, Germany.
    Steitz, Jan-Patrick
    Albert Ludwigs Univ Freiburg, Inst Pharmazeut Wissensch, Albertstr 25, D-79104 Freiburg, Germany.
    Baierl, Anna
    Forschungszentrum Julich, IBG Biotechnol 1, Wilhelm Johnen Str, D-52425 Julich, Germany.
    Lehwald, Patrizia
    Albert Ludwigs Univ Freiburg, Inst Pharmazeut Wissensch, Albertstr 25, D-79104 Freiburg, Germany.
    Wiesli, Luzia
    Empa Swiss Fed Labs Mat Sci & Technol, Lab Biointerfaces, Lerchenfeldstr 5, CH-9014 St Gallen, Switzerland.
    Richter, Michael
    Empa Swiss Fed Labs Mat Sci & Technol, Lab Biointerfaces, Lerchenfeldstr 5, CH-9014 St Gallen, Switzerland;Fraunhofer Inst Interfacial Engn & Biotechnol IGB, Branch BioCat, Schulgasse 11a, D-94315 Straubing, Germany.
    Fries, Alexander
    Albert Ludwigs Univ Freiburg, Inst Pharmazeut Wissensch, Albertstr 25, D-79104 Freiburg, Germany.
    Pohl, Martina
    Forschungszentrum Julich, IBG Biotechnol 1, Wilhelm Johnen Str, D-52425 Julich, Germany.
    Schneider, Gunter
    Karolinska Inst, Dept Med Biochem & Biophys, Tomtebodavagen 6, S-17177 Stockholm, Sweden.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mueller, Michael
    Albert Ludwigs Univ Freiburg, Inst Pharmazeut Wissensch, Albertstr 25, D-79104 Freiburg, Germany.
    Structural and Mutagenesis Studies of the Thiamine-Dependent, Ketone-Accepting YerE from Pseudomonas protegens2018In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 21, p. 2283-2292Article in journal (Refereed)
    Abstract [en]

    A wide range of thiamine diphosphate (ThDP)-dependent enzymes catalyze the benzoin-type carboligation of pyruvate with aldehydes. A few ThDP-dependent enzymes, such as YerE from Yersinia pseudotuberculosis (YpYerE), are known to accept ketones as acceptor substrates. Catalysis by YpYerE gives access to chiral tertiary alcohols, a group of products difficult to obtain in an enantioenriched form by other means. Hence, knowledge of the three-dimensional structure of the enzyme is crucial to identify structure-activity relationships. However, YpYerE has yet to be crystallized, despite several attempts. Herein, we show that a homologue of YpYerE, namely, PpYerE from Pseudomonas protegens (59 % amino acid identity), displays similar catalytic activity: benzaldehyde and its derivatives as well as ketones are converted into chiral 2-hydroxy ketones by using pyruvate as a donor. To enable comparison of aldehyde- and ketone-accepting enzymes and to guide site-directed mutagenesis studies, PpYerE was crystallized and its structure was determined to a resolution of 1.55 angstrom.

  • 30. Hossain, M. Akhter
    et al.
    Bathgate, Ross A.D.
    Kong, Chze K.R.
    Shabanpoor, Fazel
    Zhang, Suode
    Haugaard-Kedström (published under the name Haugaard-Jönsson), Linda M.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Rosengren, Johan
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Tregear, Geoffrey W.
    Wade, John D.
    Synthesis, conformation, and activity of human insulin-like peptide 5 (INSL5)2008In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, no 11, p. 1816-1822Article in journal (Refereed)
    Abstract [en]

    Insulin-like peptide 5 (INSL5) was first identified through searches of the expressed sequence tags (EST) databases. Primary sequence analysis showed it to be a prepropeptide that was predicted to be processed in vivo to yield a two-chain sequence (A and B) that contained the insulin-like disulfide cross-links. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142) coupled with their apparent coevolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for RXFP4. Given that the primary function of the INSL5–RXFP4 pair remains unknown, an effective means of producing sufficient quantities of this peptide and its analogues is needed to systematically investigate its structural and biological properties. A combination of solid-phase peptide synthesis methods together with regioselective disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to standard synthesis protocols and required highly optimized conditions for their acquisition. In particular, the use of a strong tertiary amidine, DBU, as Nα-deprotection base was required for the successful assembly of the B chain; this highlights the need to consider incomplete deprotection rather than acylation as a cause of failed synthesis. Following sequential disulfide bond formation and chain combination, the resulting synthetic INSL5, which was obtained in good overall yield, was shown to possess a similar secondary structure to human relaxin-3 (H3 relaxin). The peptide was able to inhibit cAMP activity in SK-N-MC cells that expressed the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast, it had no activity on the human RXFP3 receptor. Synthetic INSL5 demonstrates equivalent activity to the recombinant-derived peptide, and will be an important tool for the determination of its biological function.

  • 31. Höst, Gunnar
    et al.
    Razkin, Jesus
    Baltzer, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    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 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.

  • 32.
    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.

  • 33. Ito, Mika
    et al.
    Shibata, Aya
    Zhang, Jie
    Hiroshima, Michio
    Sako, Yasushi
    Nakano, Yukiko
    Kojima-Aikawa, Kyoko
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Shuto, Satoshi
    Ito, Yoshihiro
    Morgenstern, Ralf
    Abe, Hiroshi
    Universal Caging Group for the in-Cell Detection of Glutathione Transferase Applied to 19F NMR and Bioluminogenic Probes2012In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, no 10, p. 1428-1432Article in journal (Refereed)
  • 34. Ito, Mika
    et al.
    Shibata, Aya
    Zhang, Jie
    Hiroshima, Michio
    Sako, Yasushi
    Nakano, Yukiko
    Kojima-Aikawa, Kyoko
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Shuto, Satoshi
    Ito, Yoshihiro
    Morgenstern, Ralf
    Abe, Hiroshi
    Universal Caging Group for the in-Cell Detection of Glutathione Transferase Applied to 19F NMR and Bioluminogenic Probes2012In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, no 10, p. 1428-1432Article in journal (Refereed)
  • 35.
    Janfalk Carlsson, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dobritzsch, Doreeen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Nilsson, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Kamerlin, S. C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis2016In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 18, p. 1693-1697Article in journal (Refereed)
    Abstract [en]

    Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)-3-phenylpropane-1,2-diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)-diol is not only due to changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)-benzyloxirane. To probe the structural origin of these differences in substrate selectivities and catalytic regiopreferences, we have solved the crystal structures for the in-vitro evolved StEH1 variants. We have additionally used these structures as a starting point for docking the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking calculations, the apparent preferred binding modes obtained from the docking appears to rationalize the experimentally determined regioselectivities. These calculations could also identify an active site residue (F33) as a putatively important interaction partner, a role that could explain the high degree of conservation of this residue during evolution. Overall, our combined experimental, structural and computational studies of this system provide snapshots into the evolution of enantioconvergence in StEH1 catalyzed epoxide hydrolysis.

  • 36.
    Johansson, Susanne M C
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wadell, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Kihlberg, Jan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Multivalent HSA conjugates of 3 '-siallyllactose are potent inhibitors of adenoviral cell attachment and infection2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 2, p. 358-364Article in journal (Refereed)
    Abstract [en]

    Adenoviruses of serotypes 8, 19 and 37 are the major cause of the severe eye infection EKC (epidemic keratoconjunctivitis). In general, all adenoviruses interact with their cellular receptors through the fibre proteins, which extend from the virus particle. Recently, adenovirus type 37 (Ad37) was found to bind and infect human corneal cells through attachment to carbohydrate structures that carry terminal alpha-(2-3)-linked sialic acids. Herein we present a synthetic route to a 3'-sialyllactose derivative and corresponding multivalent HSA conjugates with varying orders of valency. The potential of these compounds as inhibitors of EKC causing adenovirus of serotype Ad37, was studied with both binding assay and an infectivity assay. The results revealed that these compounds effectively prevent Ad37 from binding to and infecting human corneal epithelial (HCE) cells. Moreover, the inhibition is significantly increased with higher orders of multivalency.

  • 37. Kaspersen, Jørn D.
    et al.
    Pedersen, Jannik N.
    Hansted, Jon G.
    Nielsen, Søren B.
    Sakthivel, Srinivasan
    Wilhelm, Kristina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nemashkalova, Ekaterina L.
    Permyakov, Sergei E.
    Permyakov, Eugene A.
    Pinto Oliveira, Cristiano Luis
    Morozova-Roche, Ludmilla A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Otzen, Daniel E.
    Pedersen, Jan Skov
    Generic structures of cytotoxic liprotides: nano-sized complexes with oleic acid cores and shells of disordered proteins2014In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 15, no 18, p. 2693-2702Article in journal (Refereed)
    Abstract [en]

    The cytotoxic complex formed between alpha-lactalbumin and oleic acid (OA) has inspired many studies on protein-fatty acid complexes, but structural insight remains sparse. After having used small-angle X-ray scattering (SAXS) to obtain structural information, we present a new, generic structural model of cytotoxic protein-oleic acid complexes, which we have termed liprotides (lipids and partially denatured proteins). Twelve liprotides formed from seven structurally unrelated proteins and prepared by different procedures all displayed core-shell structures, each with a micellar OA core and a shell consisting of flexible, partially unfolded protein, which stabilizes the OA micelle. The common structure explains similar effects exerted on cells by different liprotides and is consistent with a cargo off-loading of the OA into cell membranes.

  • 38.
    Kasrayan, Alex
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Bocola, Marco
    Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany.
    Sandström, Anders G.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Lavén, Gaston
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Bäckvall, Jan-Erling
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Prediction of the Candida antarctica lipase A protein structure by comparative modeling and site-directed mutagenesis2007In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 8, no 12, p. 1409-1415Article in journal (Refereed)
    Abstract [en]

    A number of model structures of the CalA suggested by comparative modeling were tested by site-directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified mutant enzymes indicate a structure where the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. This model can be used for future targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability, and higher enantioselectivity.

  • 39.
    Klingstedt, Therése
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Blechschmidt, Cristiane
    Charite, Germany .
    Nogalska, Anna
    University of So Calif, CA USA .
    Prokop, Stefan
    Charite, Germany .
    Häggqvist, Bo
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Danielsson, Olof
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    King Engel, W
    University of So Calif, CA USA .
    Askanas, Valerie
    University of So Calif, CA USA .
    Heppner, Frank L.
    Charite, Germany .
    Nilsson, K Peter R
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Luminescent Conjugated Oligothiophenes for Sensitive Fluorescent Assignment of Protein Inclusion Bodies2013In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 14, no 5, p. 607-616Article in journal (Refereed)
    Abstract [en]

    Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands.

  • 40.
    Kourist, Robert
    et al.
    Univ Greifswald, Dept Biotechnol & Enzyme Catalysis.
    Bartsch, Sebastian
    Univ Greifswald, Dept Biotechnol & Enzyme Catalysis.
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bornscheuer, Uwe T.
    Univ Greifswald, Dept Biotechnol & Enzyme Catalysis.
    Understanding promiscuous amidase activity of an esterase from Bacillus subtilis2008In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, no 1, p. 67-69Article in journal (Refereed)
    Abstract [en]

    Water works. Bacillus subtilis esterase BS2 is a promiscuous esterase that shows amidase activity. This amidase activity was shown to depend on a hydrogen-bond network with the substrate amide hydrogen (indicated by arrow). When this stabilising hydrogen bond network was removed by a point mutation, the amide activity was significantly lowered in comparison with the esterase activity. (Figure Presented)

  • 41.
    Land, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth CBH, Dept Ind Biotechnol, AlbaNova Univ Ctr, S-10691 Stockholm, Sweden.
    Campillo-Brocal, Jonatan C.
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth CBH, Dept Ind Biotechnol, AlbaNova Univ Ctr, S-10691 Stockholm, Sweden.
    Humble, Maria Svedendahl
    Pharem Biotech AB, Biovat Pk,Forskargatan 20 J, S-15136 Sodertalje, Sweden.
    Berglund, Per
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth CBH, Dept Ind Biotechnol, AlbaNova Univ Ctr, S-10691 Stockholm, Sweden.
    B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase: Evaluation of the Proline Rule as a Method for Enzyme Stabilization2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, no 10, p. 1297-1304Article in journal (Refereed)
    Abstract [en]

    Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. Here we present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60 degrees C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3 degrees C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the proline rule was deemed successful at increasing the stability of this enzyme.

  • 42.
    Land, Henrik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Campillo-Brocal, Jonatan C.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl Humble, Maria
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase – An Evaluation of the Proline Rule as a Method for Enzyme Stabilization2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, no 10, p. 1297-1304Article in journal (Refereed)
    Abstract [en]

    Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. We hereby present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60°C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3°C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the Proline rule was deemed successful at increasing the stability of this enzyme.

  • 43.
    Larsen, Marianne Wittrup
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Zielinska, Dorota F.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hidalgo, Aurelio
    Jensen, Lars Juhl
    Bornscheuer, Uwe T.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Suppression of Water as a Nucleophile in Candida antarctica Lipase B Catalysis2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 6, p. 796-801Article in journal (Refereed)
    Abstract [en]

    A water tunnel in Candida antarctica lipase B that provides the active site with substrate water is hypothesized. A small, focused library created in order to prevent water from entering the active site through the tunnel was screened for increased transacylation over hydrolysis activity. A single mutant, S47L, in which the inner part of the tunnel was blocked, catalysed the transacylation of vinyl butyrate to 20 mm butanol 14 times faster than hydrolysis. The single mutant Q46A, which has a more open outer end of the tunnel, showed an increased hydrolysis rate and a decreased hydrolysis to transacylation ratio compared to the wild-type lipase. Mutants with a blocked, tunnel could be very useful in applications in which hydrolysis is unwanted, such as the acylation of highly hydrophilic compounds in the presence of water.

  • 44.
    Leta Aboye, Teshome
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Clark, Richard J
    Craik, David J
    Göransson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O22008In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, no 1, p. 103-113Article in journal (Refereed)
    Abstract [en]

    The cyclic cystine knot motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering. To date, however, the utilisation of this scaffold has been limited by the inability to synthesise members of the most diverse and biologically active subfamily, the bracelet cyclotides. This study describes the synthesis and first direct oxidative folding of a bracelet cyclotide-cycloviolacin O2-and thus provides an efficient method for exploring the most potent cyclic cystine knot peptides. The linear chain of cycloviolacin O2 was assembled by solid-phase Fmoc peptide synthesis and cyclised by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single-step reaction. The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure.

  • 45.
    Lindgren, Joel
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Ekblad, Caroline
    Abrahmsén, Lars
    Karlström, Amelie Eriksson
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    A Native Chemical Ligation Approach for Combinatorial Assembly of Affibody Molecules2012In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, no 7, p. 1024-1031Article in journal (Refereed)
    Abstract [en]

    Affinity molecules labeled with different reporter groups, such as fluorophores or radionuclides, are valuable research tools used in a variety of applications. One class of engineered affinity proteins is Affibody molecules, which are small (6.5 kDa) proteins that can be produced by solid phase peptide synthesis (SPPS), thereby allowing site-specific incorporation of reporter groups during synthesis. The Affibody molecules are triple-helix proteins composed of a variable part, which gives the protein its binding specificity, and a constant part, which is identical for all Affibody molecules. In the present study, native chemical ligation (NCL) has been applied for combinatorial assembly of Affibody molecules from peptide fragments produced by Fmoc SPPS. The concept is demonstrated for the synthesis of three different Affibody molecules. The cysteine residue introduced at the site of ligation can be used for directed immobilization and does not interfere with the function of the investigated proteins. This strategy combines a high-yield production method with facilitated preparation of proteins with different C-terminal modifications.

  • 46.
    Lindgren, Joel
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Intramolecular thioether cross-linking of therapeutic proteins to increase proteolytic stability2014In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 15, no 14, p. 2132-2138Article in journal (Other academic)
    Abstract [en]

    Protein-based pharmaceuticals typically display high selectivity and low toxicity, but are also characterized by low oral availability, mainly because of enzymatic degradation in the gastrointestinal tract and poor permeability across the intestinal wall. One way to increase the proteolytic stability of peptides and proteins is by intramolecular crosslinking, such as the introduction of disulfide bridges. However, disulfide bridges are at risk of thiol-disulfide exchange or reduction during production, purification, and/or therapeutic use, whereas thioether bridges are expected to be stable under the same conditions. In this study, thioether crosslinking was investigated for a 46 aa albumin-binding domain (ABD) derived from streptococcal protein G. ABD binds with high affinity to human serum albumin (HSA) and has been proposed as a fusion partner to increase the in vivo half-lives of therapeutic proteins. In the study, five ABD variants with single or double intramolecular thioether bridges were designed and synthesized. The binding affinity, secondary structure, and thermal stability of each protein was investigated by SPR-based biosensor analysis and CD spectroscopy. The proteolytic stability in the presence of the major intestinal proteases pepsin (found in the stomach) and trypsin in combination with chymotrypsin (found in pancreatin secreted to the duodenum by the pancreas) was also investigated. The most promising crosslinked variant, ABD_CL1, showed high thermal stability, retained high affinity in binding to HSA, and showed dramatically increased stability in the presence of pepsin and trypsin/chymotrypsin, compared to the ABD reference protein. This suggests that the intramolecular thioether crosslinking strategy can be used to increase the stability towards gastrointestinal enzymes.

  • 47.
    Lundström, Patrik
    et al.
    Lund University, Department of Biophysical Chemistry.
    Akke, Mikael
    Lund University, Department of Biophysical Chemistry.
    Microsecond protein dynamics measured by C-13(alpha) rotating-frame spin relaxation2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 9, p. 1685-1692Article in journal (Refereed)
    Abstract [en]

    NMR spin relaxation in the rotating frame (R-1p) is a unique method for atomic-resolution characterisation of conformational (chemical) exchange processes occuring on the microsecond timescale. We present a rotating frame C-13 relaxation dispersion experiment for measuring conformational dynamics in uniformly C-13-labeled proteins. The experiment was validated by using the E140Q mutant of the C-terminal fragment of calmodulin, which exhibits significant conformational exchange between two major conformations, as gauged from previous N-15 and H-1 relaxation studies. Consistent with previous work, the present C-13, R-1p experiment detects conformational-exchange dynamics throughout the protein. The average correlation time of <tau(ex)> = 25 +/- 8 mu s is in excellent agreement with those determined previously from H-1 and N-15 R-1p, relaxation data: <tau(ex)> = 19 +/- 7 and 21 +/- 3 mu s, respectively. The extracted chemical-shift differences between the exchanging states reveal significant fluctuations in dihedral angles within single regions of Ramachandran phi-psi space that were not identified from the H-1 and N-15 relaxation data. The present results underscore the advantage of using several types of nuclei to probe exchange dynamics in biomolecules.

  • 48.
    Léonard, Valérie
    et al.
    Univ Rochelle, FRE CNRS 2766, Lab Biotechnol & Chim Bioorgan.
    Fransson, Linda
    KTH, School of Biotechnology (BIO), Biochemistry.
    Lamare, Sylvain
    Univ Rochelle, FRE CNRS 2766, Lab Biotechnol & Chim Bioorgan.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Graber, Marianne
    Univ Rochelle, FRE CNRS 2766, Lab Biotechnol & Chim Bioorgan.
    A water molecule in the stereospecificity pocket of Candida antarctica lipase B enhances enantioselectivity towards pentan-2-ol2007In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 8, no 6, p. 662-667Article in journal (Refereed)
    Abstract [en]

    The effect of water activity on enzyme-catalyzed enantioselective transesterification was studied by using a solid/gas reactor. The experimental results were compared with predictions from molecular modelling. The system studied was the esterification of pentan-2-ol with methylpropanoate as acyl donor and lipase B from Candida antarctica as catalyst. The data showed a pronounced water-activity effect on both reaction rote and enantioselectivity. The enantioselectivity increased from 100, at water activity close to zero, to a maximum of 320, at a water activity of 0.2. Molecular modelling revealed how a water molecule could bind in the active site and obstruct the binding of the slowly reacting enantiomer. Measurements of enantioselectivity at different water-activity values and temperatures showed that the water molecule had a high affinity for the stereospecificity pocket of the active site with a binding energy of 9 kJ mol(-1), and that it lost all its degrees of rotation, corresponding to an entropic energy of 37 Jmol(-1)K(-1).

  • 49. Löfgren, J.
    et al.
    Görbe, T.
    Oschmann, M.
    Svedendahl, Maria
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Bäckvall, J. -E
    Transesterification of a Tertiary Alcohol by Engineered Candida antarctica Lipase A2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633Article in journal (Refereed)
    Abstract [en]

    Tertiary alcohols are known to be challenging substrates for applications in asymmetric synthesis due to their complexity and steric hinderance. The occurrence of tertiary alcohols and their esters in nature indicates the presence of natural biocatalytic synthetic routes for their preparation. Lipase A from Candida antarctica (CalA) is a hydrolase that has previously been shown to catalyze the transesterification of racemic 2-phenylbut-3-yn-2-ol at a low rate. In this work, the activity of that enzyme was improved by protein engineering through a semi-rational design strategy. An enzyme library was created and screened for transesterification activity towards racemic 2-phenylbut-3-yn-2-ol in an organic solvent. One successful enzyme variant (L367G) showed a tenfold increased reaction rate compared to the wild-type enzyme, while maintaining a high enantioselectivity.

  • 50.
    Löfgren, Johanna
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Görbe, Tamás
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Oschmann, Michael
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Svedendahl Humble, Maria
    Bäckvall, Jan-E
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Transesterification of a Tertiary Alcohol by Engineered Candida antarctica Lipase A2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, no 11, p. 1438-1443Article in journal (Refereed)
    Abstract [en]

    Tertiary alcohols are known to be challenging substrates for applications in asymmetric synthesis due to their complexity and steric hinderance. The occurrence of tertiary alcohols and their esters in nature indicates the presence of natural biocatalytic synthetic routes for their preparation. Lipase A from Candida antarctica (CalA) is a hydrolase that has previously been shown to catalyze the transesterification of racemic 2-phenylbut-3-yn-2-ol at a low rate. In this work, the activity of that enzyme was improved by protein engineering through a semi-rational design strategy. An enzyme library was created and screened for transesterification activity towards racemic 2-phenylbut-3-yn-2-ol in an organic solvent. One successful enzyme variant (L367G) showed a tenfold increased reaction rate compared to the wild-type enzyme, while maintaining a high enantioselectivity.

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