| 11. |
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| 12. |
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| 13. |
- Björklund, Emil, et al.
(författare)
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Analysis of protein-ligand interactions from titrations and nuclear magnetic resonance relaxation dispersions
- 2022
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 31:1, s. 301-307
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Tidskriftsartikel (refereegranskat)abstract
- We present PLIS, a publicly available, open-source software for the determination of protein-ligand dissociation constants that can be used to characterize biological processes or to shed light on biophysical aspects of interactions. PLIS can analyze data from titration experiments monitored by for instance fluorescence spectroscopy or from nuclear magnetic resonance relaxation dispersion experiments. In addition to analysis of experimental data, PLIS includes functionality for generation of synthetic data, useful for understanding how different parameters effect the data in order to better analyze experiments.
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| 14. |
- Brocca, Stefania, et al.
(författare)
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Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa
- 2000
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 9:5, s. 985-990
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Tidskriftsartikel (refereegranskat)abstract
- Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn35 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect' the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.
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| 15. |
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| 16. |
- Carey, Jannette, et al.
(författare)
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Protein reconstitution and three-dimensional domain swapping: Benefits and constraints of covalency
- 2007
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 16:11, s. 2317-2333
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Forskningsöversikt (refereegranskat)abstract
- The phenomena of protein reconstitution and three-dimensional domain swapping reveal that highly similar structures can be obtained whether a protein is comprised of one or more polypeptide chains. In this review, we use protein reconstitution as a lens through which to examine the range of protein tolerance to chain interruptions and the roles of the primary structure in related features of protein structure and folding, including circular permutation, natively unfolded proteins, allostery, and amyloid fibril formation. The results imply that noncovalent interactions in a protein are sufficient to specify its structure under the constraints imposed by the covalent backbone.
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| 17. |
- Carey, Jannette, et al.
(författare)
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WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H: quinone oxidoreductases
- 2007
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 16:10, s. 2301-2305
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of the flavodoxin-like protein WrbA with oxidized FMN bound reveals a close relationship to mammalian NAD(P) H:quinone oxidoreductase, Nqo1. Structural comparison of WrbA, flavodoxin, and Nqo1 indicates how the twisted open-sheet fold of flavodoxins is elaborated to form multimers that extend catalytic function from one-electron transfer between protein partners using FMN to two-electron reduction of xenobiotics using FAD. The structure suggests a novel physiological role for WrbA and Nqo1.
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| 18. |
- Carnrot, Cecilia, et al.
(författare)
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Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase
- 2008
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 17:9, s. 1486-1493
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus anthracis is well known in connection with biological warfare. The search for new drug targets and antibiotics is highly motivated because of upcoming multiresistant strains. Thymidylate kinase is an ideal target since this enzyme is at the junction of the de novo and salvage synthesis of dTTP, an essential precursor for DNA synthesis. Here the expression and characterization of thymidylate kinase from B. anthracis (Ba-TMPK) is presented. The enzyme phosphorylated deoxythymidine-5'-monophosphate (dTMP) efficiently with K-m and V-max values of 33 mu M and 48 mu mol mg(-1) min(-1), respectively. The efficiency of deoxyuridine-5'-monophosphate phosphorylation was; similar to 10% of that of dTMP. Several dTMP analogs were tested, and D-FMAUMP (2'-fluoroarabinosyl-5-methyldeoxyuridine-5'- monophosphate) was selectively phosphorylated with an efficiency of 172% of that of D-dTMP, but L-FMAUMP was a poor substrate as were 5-fluorodeoxyuridine-5'-monophosphate (5FdUMP) and 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4TMP). No activity could be detected with 3'-azidothymidine-5'-monophosphate (AZTMP). The corresponding nucleosides known as efficient anticancer and antiviral compounds were also tested, and D-FMAU was a strong inhibitor with an IC50 value of 10 mu M, while other nucleosides-L-FMAU, dThd, 5-FdUrd, d4T, and AZT, and 2'-arabinosylthymidine-were poor inhibitors. A structure model was built for Ba-TMPK based on the Staphylococcus aureus TMPK structure. Docking with various substrates suggested mechanisms explaining the differences in substrate selectivity of the human and the bacterial TMPKs. These results may serve as a start point for development of new antibacterial agents.
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| 19. |
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| 20. |
- Casbarra, A, et al.
(författare)
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Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis
- 2004
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 13:5, s. 1322-1330
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Tidskriftsartikel (refereegranskat)abstract
- A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the P-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of a-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.
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