| 1. |
- Grahn, Elin, et al.
(author)
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New crystal structures of human glutathione transferase A1-1 shed light on glutathione binding and the conformation of the C-terminal helix.
- 2006
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In: Acta Crystallogr D Biol Crystallogr. - 0907-4449. ; 62:Pt 2, s. 197-207
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Journal article (peer-reviewed)abstract
- Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 angstroms resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.
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| 2. |
- Ilina, Yulia, et al.
(author)
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Characterization of the DNA-binding motif of the arsenic-responsive transcription factor Yap8p.
- 2008
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In: The Biochemical journal. - 1470-8728. ; 415:3, s. 467-75
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Journal article (peer-reviewed)abstract
- Saccharomyces cerevisiae uses several mechanisms for arsenic detoxification including the arsenate reductase Acr2p and the arsenite efflux protein Acr3p. ACR2 and ACR3 are transcribed in opposite directions from the same promoter and expression of these genes is regulated by the AP-1 (activator protein 1)-like transcription factor Yap8p. Yap8p has been shown to permanently associate with this promoter and to stimulate ACR2/ACR3 expression in response to arsenic. In the present study we characterized the DNA sequence that is targeted by Yap8p. We show that Yap8p binds to a pseudo-palindromic TGATTAATAATCA sequence that is related to, but distinct from, the sequence recognized by other fungal AP-1 proteins. Probing the promoter by mutational analysis, we confirm the importance of the TTAATAA core element and pin-point nucleotides that flank this element as crucial for Yap8p binding and in vivo activation of ACR3 expression. A genome-wide search for this element combined with global gene expression analysis indicates that the principal function of Yap8p is to control expression of ACR2 and ACR3. We conclude that Yap8p and other yeast AP-1 proteins require distinct DNA-binding motifs to induce gene expression and propose that this fact contributed towards a separation of function between AP-1 proteins during evolution.
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| 3. |
- Lin, Xionghui, 1976-, et al.
(author)
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Ancient Cytokines, the Role of Astakines as Hematopoietic Growth Factors
- 2010
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:37, s. 28577-28586
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Journal article (peer-reviewed)abstract
- Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. Vertebrate prokineticins (PKs) are known to take part in this process, as are the invertebrate prokineticin domain proteins, astakines. In Pacifastacus leniusculus, astakine 1 is essential for the release of new hemocytes into the open circulatory system of these animals. In addition to astakine 1, we have now cloned a homologue of astakine 1 with an insert of 13 amino acids, named as astakine 2. Both crustacean astakines lack the N-terminal AVIT motif, which is present in vertebrate PKs, and hence receptor binding differs from that of vertebrate PKs. We have found astakine-like sequences in 19 different invertebrate species, and the sequences show that some motifs are conserved among invertebrate groups. Previously we showed that astakine 1 is directly involved in hematopoiesis, and now we show that astakine 1 and astakine 2 have different roles in hemocyte lineage differentiation. Astakine 1 can stimulate proliferation of hematopoietic tissue (Hpt) cells (precursor of hemocytes) as well as specifically induce differentiation of Hpt cells along the semigranular cell lineage, whereas astakine 2 plays a role in granular cell differentiation. Moreover, we discuss the impact of the putative structures of different astakines in comparison with the vertebrate prokineticins.
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| 4. |
- Lin, Xionghui, 1976-, et al.
(author)
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Invertebrate astakines - regulators of differentiation in hematopoietic tissues
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Other publication (other academic/artistic)abstract
- Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. Vertebrate prokineticins are known to take part in this process, as are the invertebrate prokineticin domain proteins, astakines. In Pacifastacus leniusculus astakine 1 is essential for the release of new hemocytes into the open circulatory system of these animals. In addition to astakine 1 we have now cloned a homologue of astakine 1 with an insert of 13 amino acids, named astakine 2. Common to both crustacean astakines is the lack of the N-terminal AVIT amino acids present in vertebrate PKs, and hence receptor binding differs from that of vertebrate PKs. Now we have found astakine-like sequences in 19 different invertebrate species and the sequences show that some motifs are conserved among invertebrate groups. Previously we showed that astakine 1 is directly involved in hematopoiesis and now we show that astakine 1 and astakine 2 have different roles in hemocyte lineage differentiation and that astakine 1 specifically induce differentiation along the semigranular cell lineage. Further we discuss the impact of the putative structure of different astakines in comparison with the vertebrate prokineticins.
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| 5. |
- Novotny, Marian, et al.
(author)
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A survey of left-handed helices in protein structures.
- 2005
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In: J Mol Biol. - 0022-2836. ; 347:2, s. 231-41
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Journal article (peer-reviewed)abstract
- All naturally occurring amino acids with the exception of glycine contain one or more chiral carbon atoms and can therefore occur in two different configurations, L (levo, left-handed) and D (dextro, right-handed). Proteins are almost exclusively built from L-amino acids. The stereochemical bias of nature is further reflected at the secondary structure level where right-handed helices are strongly preferred over left-handed helices. The handedness of helices has not received much attention in the past and is often overlooked during the analysis, description and deposition of experimentally solved protein structures. Therefore, an extensive survey of left-handed helices in the Protein Data Bank (PDB) was undertaken to analyse their frequency of occurrence, length, amino acid composition, conservation and possible structural or functional role. All left-handed helices (of four or more residues) in a non-redundant subset of the PDB, were identified using hydrogen-bonding analysis, comparison of related structures, and experimental electron density assessment to filter out likely spurious and artefactual hits. This analysis yielded 31 verified left-handed helices in a set of 7284 proteins. The phi angles of the residues in the left-handed helices lie between 30 degrees and 130 degrees and the psi angles lie between -50 degrees and 100 degrees . Most of the helices are short (four residues) and for 87% of them, it was possible to determine that they are important for the stability of the protein, for ligand binding, or as part of the active site. This suggests that, even though left-handed helices are rare, when they do occur, they are structurally or functionally significant. Four secondary structure assignment programs were tested for their ability to identify the handedness of the helices. Of these programs, only DSSP correctly assigns the handedness.
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| 6. |
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| 7. |
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| 8. |
- Novotny, Marian, 1979-
(author)
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Applications of Structural Bioinformatics for the Structural Genomics Era
- 2007
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Doctoral thesis (other academic/artistic)abstract
- Structural bioinformatics deals with the analysis, classification and prediction of three-dimensional structures of biomacromolecules. It is becoming increasingly important as the number of structures is growing rapidly. This thesis describes three studies concerned with protein-function prediction and two studies about protein structure validation.New protein structures are often compared to known structures to find out if they have a known fold, which may provide hints about their function. The functionality and performance of eleven fold-comparison servers were evaluated. None of the tested servers achieved perfect recall, so in practise a combination of servers should be used.If fold comparison does not provide any hints about the function of a protein, structural motif searches can be employed. A survey of left-handed helices in known protein structures was carried out. The results show that left-handed helices are rare motifs, but most of them occur in active or ligand-binding sites. Their identification can therefore help to pinpoint potentially important residues.Sometimes all available methods fail to provide hints about the function of a protein. Therefore, the potential of using docking techniques to predict which ligands are likely to bind to a particular protein has been investigated. Initial results show that it will be difficult to build a reliable automated docking protocol that will suit all proteins.The effect of various phenomena on the precision of accessible surface area calculations was also investigated. The results suggest that it is prudent to report such values with a precision of 50 to 100 Å2.Finally, a survey of register shifts in known protein structures was carried out. The identified potential register shifts were analysed and classified. A machine-learning approach ("rough sets") was used in an attempt to diagnose register errors in structures.
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| 9. |
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| 10. |
- Novotny, Marian, et al.
(author)
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Evaluation of protein fold comparison servers.
- 2004
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In: Proteins. - 1097-0134. ; 54:2, s. 260-70
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Journal article (peer-reviewed)abstract
- When a new protein structure has been determined, comparison with the database of known structures enables classification of its fold as new or belonging to a known class of proteins. This in turn may provide clues about the function of the protein. A large number of fold comparison programs have been developed, but they have never been subjected to a comprehensive and critical comparative analysis. Here we describe an evaluation of 11 publicly available, Web-based servers for automatic fold comparison. Both their functionality (e.g., user interface, presentation, and annotation of results) and their performance (i.e., how well established structural similarities are recognized) were assessed. The servers were subjected to a battery of performance tests covering a broad spectrum of folds as well as special cases, such as multidomain proteins, Calpha-only models, new folds, and NMR-based models. The CATH structural classification system was used as a reference. These tests revealed the strong and weak sides of each server. On the whole, CE, DALI, MATRAS, and VAST showed the best performance, but none of the servers achieved a 100% success rate. Where no structurally similar proteins are found by any individual server, it is recommended to try one or two other servers before any conclusions concerning the novelty of a fold are put on paper.
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