| 1. |
- Wellner, Eric, et al.
(författare)
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Synthesis of a C-glycoside analogue of β-D-galactosyl hydroxynorvaline and its use in immunological studies
- 2000
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Ingår i: ChemBioChem. - : John Wiley & Sons. - 1439-4227 .- 1439-7633. ; 1:4, s. 272-280
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Tidskriftsartikel (refereegranskat)abstract
- A C-linked isostere of β-d-galactosylated hydroxynorvaline has been prepared in eight steps from per-O-benzylated galactopyranolactone. Addition of a homoallylic Grignard reagent to the lactone, reduction of the resulting hemiacetal with triethylsilane, and a Wittig reaction with Garner's aldehyde were key steps in this synthesis. The C-linked building block was then incorporated at position 264 into the fragment CII(256–270) from type II collagen by solid-phase synthesis using a combination of the tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc) protective group strategies. Deprotection of the benzylated C-linked galactosyl moiety was achieved simultaneously with cleavage of the glycopeptide from the solid phase by using triethylsilyl trifluoromethanesulfonate in TFA. Helper T-cell hybridomas obtained in a mouse model for rheumatoid arthritis responded to the C-linked glycopeptide when presented by class II MHC molecules. However, 10- to 20-fold higher concentrations were required as compared to when O-linked β-d-galactosylated hydroxynorvaline or hydroxylysine (Hyl) were present at position 264 of CII(256–270). Thus, replacement of a single oxygen atom by a methylene group in the carbohydrate moiety of a glycopeptide antigen had a substantial influence on the T-cell response. This reveals that T cells are able to recognize the carbohydrate moiety of glycopeptide antigens with high specificity. Finally, the results suggest that structural modifications of β-d-Gal-Hyl264in CII(256–270) may give altered peptide ligands that can be used for induction of tolerance in autoimmune rheumatoid arthritis.
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| 2. |
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| 3. |
- Ramström, Olof, et al.
(författare)
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In situ generation and screening of a dynamic combinatorial carbohydrate library against concanavalin A
- 2000
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Ingår i: ChemBioChem. - 1439-4227 .- 1439-7633. ; 1:1, s. 41-48
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Tidskriftsartikel (refereegranskat)abstract
- Dynamic combinatorial chemistry (DCC) is a recently introduced approach that is based on the generation of combinatorial libraries by reversible interconversion of the library constitutents. In this study, the implementation of such libraries on carbohydrate-lectin interactions was examined. The dynamic carbohydrate libraries were generated from a small set (four or six compounds) of initial carbohydrate dimers through mild disulfide interchange, and selection was performed under two conditions defining either adaptive or pre-equilibrated libraries. Upon initiation, libraries were formed that contained comparable amounts of 10 or 21 individual dimeric species, dynamically interchanging during the scrambling process. They were probed with respect to binding to the plant lectin concanavalin A, either present during library generation or added after equilibration. The libraries could be generated easily both in the presence and absence of the receptor, and a bis-mannose structure was preferentially bound and selected from the mixture. Scrambling of the library in the presence of the receptor resulted in slightly higher yields than when the receptor was added after scrambling, indicating that the receptor to some extent acts as a thermodynamic trap during library generation. The present results illustrate the extention of the DCC approach to carbohydrate recognition groups, the generation of isoenergetic dynamic libraries, and the implementation of either adaptive or pre-equilibrated procedures.
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| 4. |
- Rotticci, D., et al.
(författare)
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Improved enantioselectivity of a lipase by rational protein engineering
- 2001
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Ingår i: ChemBioChem. - 1439-4227 .- 1439-7633. ; 2:10, s. 766-770
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Tidskriftsartikel (refereegranskat)abstract
- A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivity, 1-Halo-2-octanols were poorly resolved by Candida antarctica lipase B. Interactions between the substrates and the lipase were investigated with molecular modeling. Unfavorable interactions were found between the halogen moiety of the fast-reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mutated in this region and energy contour maps of some variants displayed better interactions for the target substrates. Four selected variants of the lipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutants gave rise to one variant with doubled enantioselectivity as well as one variant with annihilated enantioselectivity towards the target halohydrins. An increased volume of the stereoselectivity pocket caused a decrease in enantioselectivity, while changes in electrostatic potential increased enantioselectivity, The enantioselectivity of these new lipase variants towards other types of alcohols was also investigated. The changes in enantioselectivity caused by the mutations were well in agreement with the proposed model concerning the chiral recognition of alcohol enantiomers by this lipase.
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| 5. |
- Végvári, Ákos, et al.
(författare)
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High-resolution capillary zone and gel electrophoresis of structurally similar amphipathic glutathione conjugates based on interaction with beta-cyclodextrins
- 2002
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Ingår i: ChemBioChem. - 1439-4227 .- 1439-7633. ; 3:11, s. 1117-1125
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Tidskriftsartikel (refereegranskat)abstract
- The tripeptide glutathione is a prominent intracellular constituent that provides protection against genotoxic and carcinogenic electrophiles and is also a component of several biological signal substances. Glutathione conjugates, free glutathione, and glutathione disulfide contain charged amino acid residues, which contribute to solubility in aqueous media. However, the amphipathic nature of glutathione conjugates and the small differences that may distinguish the S substituents, pose analytical problems in their resolution. The present study demonstrates how homologous S-alkyl and S-benzyl conjugates of high structural similarity can be efficiently resolved by capillary electrophoresis. Inclusion of beta-cyclodextrins in the buffer or in a polyacrylamide gel affords baseline separation of the analytes. The separation methods described are applicable to enzyme assays in vitro and to the identification and quantification of glutathione conjugates of importance in toxicology and physiology. The contribution of beta-cyclodextrin to the separation is primarily based on interactions between its hydrophobic cavity and the S-alkyl and S-benzyl groups of the analytes.
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| 6. |
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| 7. |
- Aaldering, L. J., et al.
(författare)
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Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule
- 2017
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Ingår i: ChemBioChem. - : Wiley-VCH Verlag. - 1439-4227 .- 1439-7633. ; 18:8, s. 755-763
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Aboye, Teshome L., et al.
(författare)
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A Cactus-Derived Toxin-Like Cystine Knot Peptide with Selective Antimicrobial Activity
- 2015
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 16:7, s. 1068-1077
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Aisenbrey, Christopher, et al.
(författare)
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Specific Isotope Labeling of Colicin E1 and B Channel Domains For Membrane Topological Analysis by Oriented Solid-State NMR Spectroscopy
- 2008
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Ingår i: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 9:6, s. 944-951
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Allert, M., et al.
(författare)
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Noncovalent binding of a reaction intermediate by a designed helix-loop-helix motif - Implications for catalyst design
- 2003
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 4:4, s. 306-318
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Tidskriftsartikel (refereegranskat)abstract
- In our search for a catalyst for the transamination reaction of asparatic acid to form oxaloacetate, twenty-five forty-two-residue sequences were designed to fold into helix-loop-helix dimers and form binding sites for the key intermediate along the reaction pathway, the aldimine. This intermediate is formed from aspartic acid and the cofactor pyridoxal phosphate. The design of the binding sites followed a strategy in which exclusively noncovalent forces were used for binding the aldimine. Histidine residues were incorporated to catalyse the rate-limiting 1,3 proton transfer reactions that converts the aldimine into the ketimine, an intermediate that is subsequently hydrolysed to form oxaloacetate and pyridoxamine phosphate. The two most efficient catalysts, T-4 and T-16, selected from the pool of sequences by a simple screening procedure, were shown by CD and NMR spectroscopies to bind the aldimine intermediate with dissociation constants in the millimolar range. The mean residue ellipticity of T-4 in aqueous solution at pH 7.4 and a concentration of 0.75 mM was -18 500 deg cm-2 dmol-1. Upon addition of 6 mM L-aspartic acid and 1.5 mM pyridoxal phosphate to form the aldimine, the mean residue ellipticity changed to -19 900 deg cm2 dmol-1. The corresponding mean residue ellipticities of T-16 were -21 200 deg cm2 dmol-1 and -24 000 deg cm2 dmol-1. These result show that the helical content increased in the presence of the aldimine, and that the folded polypeptides bound the aldimine. The 1H NMR relaxation time of the imine CH proton of the aldimine was affected by the presence of T-4 as was the 31P NMR resonance linewidth. The catalytic efficienceis of T-4 and T-16 were compared to that of imidazole and found to be more than three orders of magnitude larger. The designed binding sites were thus shown to be capable of binding the aldimine in close proximity to His residues, by noncovalent forces, into conformations that proved to be catalytically active. The results show the first time the design of well-defined catalytic sites that bind a reaction intermediate with enzyme-like affinities under equilibrium conditions and represent an important advance in de novo catalyst design.
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