| 1. |
- 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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| 2. |
- 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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| 3. |
- Balliu, Aleksandra, et al.
(författare)
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Conjugation of a Dipicolyl Chelate to Polypeptide Conjugates Increases Binding Affinities for Human Serum Albumin and Survival Times in Human Serum
- 2017
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 18:14, s. 1408-1414
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Tidskriftsartikel (refereegranskat)abstract
- The affinity for human serum albumin (HSA) of a series of 2–5 kDa peptides covalently linked to 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, a dipicolyl chelator with micromolar affinity for Zn2+, was found by surface plasmon resonance to increase in the presence of 1 μm ZnCl2 at physiological pH. The dependence on polypeptide hydrophobicity was found to be minor, thus suggesting that the conjugates bound to the metal-binding site and not to the fatty-acid-binding site. The affinity of the conjugates increased strongly with the positive charge of the polypeptides, thus implicating the negatively charged protein surface surrounding the metal-binding site. The survival times of the peptides in human serum were extended as a consequence of stronger binding to HSA, thus suggesting that Zn2+-chelating agents might provide a general route to increased survival time of peptides in serum in therapeutic and diagnostic applications without significantly increasing their molecular weights.
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| 4. |
- Balliu, Aleksandra, et al.
(författare)
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Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces : Increasing Affinities in Peptide–Protein Interactions
- 2017
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 18:14, s. 1396-1407
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Tidskriftsartikel (refereegranskat)abstract
- A 42-residue polypeptide conjugated to a small-molecule organic ligand capable of targeting the phosphorylated side chain of Ser15 was shown to bind glycogen phosphorylase a (GPa) with a KD value of 280 nm. The replacement of hydrophobic amino acids by Ala reduced affinities, whereas the incorporation of l-2-aminooctanoic acid (Aoc) increased them. Replacing Nle5, Ile9 and Leu12 by Aoc reduced the KD value from 280 to 27 nm. “Downsizing” the 42-mer to an undecamer gave rise to an affinity for GPa an order of magnitude lower, but the undecamer in which Nle5, Ile9 and Leu12 were replaced by Aoc showed a KD value of 550 nm, comparable with that of the parent 42-mer. The use of Aoc residues offers a convenient route to increased affinity in protein recognition as well as a strategy for the “downsizing” of peptides essentially without loss of affinity. The results show that hydrophobic binding sites can be found on protein surfaces by comparing the affinities of polypeptide conjugates in which Aoc residues replace Nle, Ile, Leu or Phe with those of their unmodified counterparts. Polypeptide conjugates thus provide valuable opportunities for the optimization of peptides and small organic compounds in biotechnology and biomedicine.
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| 5. |
- Correia, Mario S. P., et al.
(författare)
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Coupled Enzymatic Treatment and Mass Spectrometric Analysis for Identification of Glucuronidated Metabolites in Human Samples
- 2019
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Ingår i: ChemBioChem. - : WILEY-V C H VERLAG GMBH. - 1439-4227 .- 1439-7633. ; 20:13, s. 1678-1683
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Cuetos, Anibal, et al.
(författare)
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Structural Basis for Phospholyase Activity of a Class III Transaminase Homologue
- 2016
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 17:24, s. 2308-2311
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Dorau, Robin, et al.
(författare)
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Improved Enantioselectivity of Subtilisin Carlsberg Towards Secondary Alcohols by Protein Engineering
- 2018
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 19:4, s. 338-346
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Eildal, Jonas N. N., et al.
(författare)
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Rigidified Clicked Dimeric Ligands for Studying the Dynamics of the PDZ1-2 Supramodule of PSD-95
- 2015
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 16:1, s. 64-69
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Eriksson, Adam, et al.
(författare)
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Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling
- 2017
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Ingår i: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 18:23, s. 2301-2305
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Gong, Haiyue, et al.
(författare)
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Boronic Acid Modified Polymer Nanoparticles for Enhanced Bacterial Deactivation
- 2019
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 20:24, s. 2991-2995
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Tidskriftsartikel (refereegranskat)abstract
- A new method has been developed to enhance the antibacterial efficiency of traditional antibiotics. Chloramphenicol‐imprinted polymer particles were decorated with boronic acid to improve their binding to both Gram‐negative and ‐positive bacteria. The polymer particles have a high antibiotic loading and provide a slow release of the antibiotic payload to deactivate the target bacteria. The boronic acid modified polymer particles not only contribute to enhanced antibacterial efficiency, but also have the potential to act as scavengers to remove unused antibiotic from the environment.
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