| 1. |
- Berntsson, Ronnie P. -A., et al.
(författare)
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Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:4, s. 2725-2735
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Tidskriftsartikel (refereegranskat)abstract
- The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 angstrom resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.
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| 2. |
- Eldridge, Bill, et al.
(författare)
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An in vitro selection strategy for conferring protease resistance to ligand binding peptides
- 2009
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 22:11, s. 691-698
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Tidskriftsartikel (refereegranskat)abstract
- One drawback to the use of peptides as therapeutics has been their susceptibility to proteolysis. Here, we have used an in vitro display technology, CIS display, to enhance the proteolytic resistance of ligand-binding peptides by selection of protecting motifs from a large peptide library. The premise to this selection was that certain linear peptides within a library could form structures capable of preventing the access of proteases to defined cleavage sites without affecting ligand binding. A diverse 12-mer peptide library was inserted between a FLAG epitope motif and a thrombin cleavage site and this construct was fused to the bacterial initiator protein RepA for CIS display selection. After five rounds of selection, protection motifs were isolated that were capable of preventing proteolytic cleavage of the adjacent thrombin site. Some of the selected peptides were also resistant to more promiscuous proteases, such as chymotrypsin and trypsin, which were not used in the selection. The observed resistance to thrombin, trypsin and chymotrypsin translated into increased resistance to plasma proteases in vitro and to an increase in circulating half-lives in rats. This method can be applied to enhancing the in vivo stability of therapeutic peptides.
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| 3. |
- Frykholm, Karolin, 1977, et al.
(författare)
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DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels
- 2016
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 44:15, s. 7219-7227
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Tidskriftsartikel (refereegranskat)abstract
- The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA-Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and W Phi affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and W Phi Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions.
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| 4. |
- Gustafsson, Robert, et al.
(författare)
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Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster
- 2017
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 591:22, s. 3781-3792
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Tidskriftsartikel (refereegranskat)abstract
- Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 Å, respectively. We show that they belong to the TULIP protein superfamily, which are often involved in lipid binding. OrfX1 and OrfX2 were both found to bind phosphatidylinositol lipids.
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| 5. |
- Ljunggren, Maria, et al.
(författare)
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Interfaculty differences in collaboration : A study of a multi faculty university
- 2013
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Ingår i: Studies in Regional Science. - : Japan Section of the Regional Science Association International. - 1880-6465 .- 0287-6256. ; 43:1, s. 47-60
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Tidskriftsartikel (refereegranskat)abstract
- Prominent research has focused on collaboration and its change through the last decades, from international comparative approaches to relationships between public research organisations and the surrounding society. Within academia, research on collaboration has mostly concerned natural sciences, engineering and technology. Researchers from such disciplines generally possess a more positive attitude to collaboration activities. We aim to add to the research on collaboration with a focus on external and the nature of the internal social capital. This is done by quantitatively analysing a large number of researchers' attitude to collaboration and activity level through a smaller set of questions. The empirical design is exploratory and can be used as a base for the development of further research. The faculties of interest are humanities, social science, and natural science within a multi faculty university. The results demonstrate that professors and researchers from natural science have a more positive attitude towards collaboration. However, researchers from social science are significantly more engaged in collaboration activities than natural scientist. We conclude that the attitude to collaboration and actual participation in collaboration activities not necessarily correlates.
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| 6. |
- Nilsson, Hanna, 1984-, et al.
(författare)
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Interactions between the regulatory repressors of phage P2 and host proteins, a puzzling story
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Bacteriophage P2 belongs to a group of P2-like phages that have been classified as non-inducible. This is based on the fact that they are not induced by UV light, and upon inactivation of the repressor the bacteria will die but no progeny phage is produced. When the prophage is derepressed it is replicated in situ, but unable to excise due to a lack of integrase. The transcriptional switch of phage P2 contains two repressors, the immunity repressor C and the Cox repressor. The C gene is transcribed from the Pc promoter that also controls the integrase gene, and the C repressor controls the early Pe promoter. The cox gen is transcribed from the Pe promoter and the Cox repressor controls the Pc promoter, making the two promoters mutually exclusive. Thus, the integrase cannot be expressed at the same time as Cox and both proteins are required for phage excision. To try to resolve this paradox, a two-hybrid screen has been performed to find possible host proteins that interact with C or Cox that could control the transcriptional switch. Eight E. coli proteins showed interactions with C and three with Cox, out of which all also interacted with C. One of the candidate genes is known to be a "sticky" protein, and was not analysed further. Using a plasmid containing the transcriptional switch, we found that deletions of two of the candidate genes encoding proteins interacting with C or Cox gave a reduced percentage of plasmids choosing the lysogenic pathway; the E. coli yeeD and yqjG genes. YeeD interacts with C as well as Cox, and it is a conserved 8 kD hypothetical proteins with a SirA motif, and YqjG is a predicted glutathione S-transferase. More studies are required to clarify their involvement of these genes in regulating the transcriptional switch.
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| 7. |
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| 8. |
- Odegrip, Richard, 1968-
(författare)
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Initiation of bacteriophage P2 rolling circle replication
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Viruses are very simple entities. In its most simple form, the virus consists of a protein shell containing genetic material. To propagate the virus needs a host to parasitize, in which it can replicate its genetic material and assembly new virus particles. Viruses that parasitize bacteria are termed bacteriophages. In this study the DNA replication of bacteriophage P2 has been investigated. Bacteriophage P2 replicates by a rolling circle mechanism, which is a replication strategy used by many replicons, ranging from bacterial plasmids to animal viruses. Two phage encoded proteins are necessary for P2 rolling circle replication; A and B. The A protein initiates rolling circle replication by a single-stranded, site-specific nick at the origin of replication. This exposes a free 3' end which serves as a primer for DNA polymerisation. Upon nicking, P2 A links itself covalently to the 5' end of the DNA through one of its two active-site tyrosine residues. The A protein can also catalyse the reverse reaction. This event releases the displaced strand simultaneously as a new round of replication is initiated. The B protein has been shown to be required for lagging strand synthesis and has been suggested to be an E. coli DnaB helicase loader.In this work, the interplay between the two tyrosine residues located in the presumed catalytic site of P2 A has been closely examined. We found that the two tyrosine residues, 450 and 454, play non-equivalent functional roles in their catalytic activity. We propose that tyrosine 454 initiates replication and that tyrosine 450 only cleaves when tyrosine 454 is covalently joined to DNA, thereby reinitiating replication. Also, data from the catalytic activity of P2 A, involving different divalent cations, suggests that conformational changes occur within the protein upon binding DNA. Another intriguing phenomenon of P2 A is that it acts in cis in vivo, i.e. it can only act upon the molecule from which it was transcribed. Here, we show that the cis-activity is retained in vitro during coupled transcription and translation in a S30 extract and that the information required for the cis-activity in vivo and in vitro is contained entirely in the coding region for the A protein. An elegant model for the mechanism of the P2 A cis-activity is also presented. Further, in this work in vivo and in vitro evidence for a physical association between P2 B and E. coli DnaB are provided supporting the view that P2 B is a helicase loader. Presumably, the B protein plays an important role as a molecular matchmaker for the whole P2 replisome assembly. Here, also presented are the enigma of the inverted palindrome nature of the P2 origin, the P2 A localisation within the bacterial cell, other RCR initiating enzymes, a compilation of all known cis-acting proteins, evolutionary aspects of cis-action, the outline for exploiting P2 A as a biotechnology tool and much much more.
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| 9. |
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| 10. |
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| 11. |
- Skaar, Karin, et al.
(författare)
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Crystal structure of the bacteriophage P2 integrase catalytic domain
- 2015
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 589:23, s. 3556-3563
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Tidskriftsartikel (refereegranskat)abstract
- Bacteriophage P2 is a temperate phage capable of integrating its DNA into the host genome by site-specific recombination upon lysogenization. Integration and excision of the phage genome requires P2 integrase, which performs recognition, cleavage and joining of DNA during these processes. This work presents the high-resolution crystal structure of the catalytic domain of P2 integrase, and analysis of several non-functional P2 integrase mutants. The DNA binding area is characterized by a large positively charged patch, harbouring key residues. The structure reveals potential for large dimer flexibility, likely essential for rearrangement of DNA strands upon integration and excision.
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