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  • 51.
    Amlinger, Lina
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    The type I-E CRISPR-Cas system: Biology and applications of an adaptive immune system in bacteria2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    CRISPR-Cas systems are adaptive immune systems in bacteria and archaea, consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) array and CRISPR associated (Cas) proteins. In this work, the type I-E CRISPR-Cas system of Escherichia coli was studied.

    CRISPR-Cas immunity is divided into three stages. In the first stage, adaptation, Cas1 and Cas2 store memory of invaders in the CRISPR array as short intervening sequences, called spacers. During the expression stage, the array is transcribed, and subsequently processed into small CRISPR RNAs (crRNA), each consisting of one spacer and one repeat. The crRNAs are bound by the Cascade multi-protein complex. During the interference step, Cascade searches for DNA molecules complementary to the crRNA spacer. When a match is found, the target DNA is degraded by the recruited Cas3 nuclease.

    Host factors required for integration of new spacers into the CRISPR array were first investigated. Deleting recD, involved in DNA repair, abolished memory formation by reducing the concentration of the Cas1-Cas2 expression plasmid, leading to decreased amounts of Cas1 to levels likely insufficient for spacer integration. Deletion of RecD has an indirect effect on adaptation. To facilitate detection of adaptation, a sensitive fluorescent reporter was developed where an out-of-frame yfp reporter gene is moved into frame when a new spacer is integrated, enabling fluorescent detection of adaptation. Integration can be detected in single cells by a variety of fluorescence-based methods. A second aspect of this thesis aimed at investigating spacer elements affecting target interference. Spacers with predicted secondary structures in the crRNA impaired the ability of the CRISPR-Cas system to prevent transformation of targeted plasmids. Lastly, in absence of Cas3, Cascade was successfully used to inhibit transcription of specific genes by preventing RNA polymerase access to the promoter.

    The CRISPR-Cas field has seen rapid development since the first demonstration of immunity almost ten years ago. However, much research remains to fully understand these interesting adaptive immune systems and the research presented here increases our understanding of the type I-E CRISPR-Cas system. 

    Delarbeten
    1. Deletion of recD indirectly reduce adaptation in the type I-E CRISPR-Cas system
    Öppna denna publikation i ny flik eller fönster >>Deletion of recD indirectly reduce adaptation in the type I-E CRISPR-Cas system
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Nationell ämneskategori
    Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-312230 (URN)
    Tillgänglig från: 2017-01-08 Skapad: 2017-01-08 Senast uppdaterad: 2017-01-09
    2. Quantification of CRISPR-Cas spacer integration using a fluorescent reporter
    Öppna denna publikation i ny flik eller fönster >>Quantification of CRISPR-Cas spacer integration using a fluorescent reporter
    Visa övriga...
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Nationell ämneskategori
    Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-312231 (URN)
    Tillgänglig från: 2017-01-08 Skapad: 2017-01-08 Senast uppdaterad: 2017-01-09
    3. Effect of spacer sequence on efficiency of Type I-E CRISPR-Cas systems
    Öppna denna publikation i ny flik eller fönster >>Effect of spacer sequence on efficiency of Type I-E CRISPR-Cas systems
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Nationell ämneskategori
    Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-312233 (URN)
    Tillgänglig från: 2017-01-08 Skapad: 2017-01-08 Senast uppdaterad: 2017-01-09
    4. Efficient programmable gene silencing by Cascade
    Öppna denna publikation i ny flik eller fönster >>Efficient programmable gene silencing by Cascade
    Visa övriga...
    2015 (Engelska)Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, nr 1, s. 237-246Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Methods that permit controlled changes in the expression of genes are important tools for biological and medical research, and for biotechnological applications. Conventional methods are directed at individually changing each gene, its regulatory elements or its mRNA's translation rate. We demonstrate that the CRISPR-associated DNA-binding Cascade complex can be used for efficient, long-lasting and programmable gene silencing. When Cascade is targeted to a promoter sequence the transcription of the downstream gene is inhibited, resulting in dramatically reduced expression. The specificity of Cascade binding is provided by the integral crRNA component, which is easily designed to target virtually any stretch of DNA. Cascade targeted to the ORF sequence of the gene can also silence expression, albeit at lower efficiency. The system can be used to silence plasmid and chromosome targets, simultaneously target several genes and is active in different bacterial species and strains. The findings described here are an addition to the expanding range of CRISPR-based technologies and may be adapted to additional organisms and cell systems.

    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-249042 (URN)10.1093/nar/gku1257 (DOI)000350207100026 ()25435544 (PubMedID)
    Tillgänglig från: 2015-04-23 Skapad: 2015-04-10 Senast uppdaterad: 2018-02-28Bibliografiskt granskad
  • 52.
    Amlinger, Lina
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, E. Gerhart H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Koskiniemi, Sanna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Quantification of CRISPR-Cas spacer integration using a fluorescent reporterManuskript (preprint) (Övrigt vetenskapligt)
  • 53.
    Amlinger, Lina
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Koskiniemi, Sanna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 10392Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

  • 54.
    Amlinger, Lina
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Koskiniemi, Sanna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 10392Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

  • 55.
    Amlinger, Lina
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Larsson, Martin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Koskiniemi, Sanna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Deletion of recD indirectly reduce adaptation in the type I-E CRISPR-Cas systemManuskript (preprint) (Övrigt vetenskapligt)
  • 56.
    Amlinger, Lina
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Saunders, Sita J.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Backofen, Rolf
    Effect of spacer sequence on efficiency of Type I-E CRISPR-Cas systemsManuskript (preprint) (Övrigt vetenskapligt)
  • 57.
    Amrein, Beat A.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Bauer, Paul
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Duarte, Fernanda
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Janfalk Carlsson, Åsa
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Naworyta, Agata
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Mowbray, Sherry L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Kamerlin, Shina C. L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Expanding the catalytic triad in epoxide hydrolases and related enzymes2015Ingår i: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, nr 10, s. 5702-5713Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

  • 58.
    Amrein, Beat Anton
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Extending the Reach of Computational Approaches to Model Enzyme Catalysis2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Recent years have seen tremendous developments in methods for computational modeling of (bio-) molecular systems. Ever larger reactive systems are being studied with high accuracy approaches, and high-level QM/MM calculations are being routinely performed. However, applying high-accuracy methods to large biological systems is computationally expensive and becomes problematic when conformational sampling is needed. To address this challenge, classical force field based approaches such as free energy perturbation (FEP) and empirical valence bond calculations (EVB) have been employed in this work. Specifically:

    1. Force-field independent metal parameters have been developed for a range of alkaline earth and transition metal ions, which successfully reproduce experimental solvation free energies, metal-oxygen distances, and coordination numbers. These are valuable for the computational study of biological systems.

    2. Experimental studies have shown that the epoxide hydrolase from Solanum tuberosum (StEH1) is not only an enantioselective enzyme, but for smaller substrates, displays enantioconvergent behavior. For StEH1, two detailed studies, involving combined experimental and computational efforts have been performed: We first used trans-stilbene oxide to establish the basic reaction mechanism of this enzyme. Importantly, a highly conserved and earlier ignored histidine was identified to be important for catalysis. Following from this, EVB and experiment have been used to investigate the enantioconvergence of the StEH1-catalyzed hydrolysis of styrene oxide. This combined approach involved wildtype StEH1 and an engineered enzyme variant, and established a molecular understanding of enantioconvergent behavior of StEH1.

    3. A novel framework was developed for the Computer-Aided Directed Evolution of Enzymes (CADEE), in order to be able to quickly prepare, simulate, and analyze hundreds of enzyme variants. CADEE’s easy applicability is demonstrated in the form of an educational example.

    In conclusion, classical approaches are a computationally economical means to achieve extensive conformational sampling. Using the EVB approach has enabled me to obtain a molecular understanding of complex enzymatic systems. I have also increased the reach of the EVB approach, through the implementation of CADEE, which enables efficient and highly parallel in silico testing of hundreds-to-thousands of individual enzyme variants.

    Delarbeten
    1. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Öppna denna publikation i ny flik eller fönster >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Visa övriga...
    2014 (Engelska)Ingår i: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, nr 16, s. 4351-4362Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

    Nationell ämneskategori
    Fysikalisk kemi
    Identifikatorer
    urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
    Forskningsfinansiär
    Swedish National Infrastructure for Computing (SNIC), 2013/26-1
    Tillgänglig från: 2014-06-23 Skapad: 2014-06-04 Senast uppdaterad: 2018-12-03Bibliografiskt granskad
    2. Expanding the catalytic triad in epoxide hydrolases and related enzymes
    Öppna denna publikation i ny flik eller fönster >>Expanding the catalytic triad in epoxide hydrolases and related enzymes
    Visa övriga...
    2015 (Engelska)Ingår i: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, nr 10, s. 5702-5713Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Forskningsämne
    Biokemi
    Identifikatorer
    urn:nbn:se:uu:diva-260232 (URN)10.1021/acscatal.5b01639 (DOI)000362391500006 ()
    Forskningsfinansiär
    EU, FP7, Sjunde ramprogrammet, 306474Vetenskapsrådet, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12
    Tillgänglig från: 2015-08-18 Skapad: 2015-08-18 Senast uppdaterad: 2017-12-04Bibliografiskt granskad
    3. Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
    Öppna denna publikation i ny flik eller fönster >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
    Visa övriga...
    2016 (Engelska)Ingår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, nr 24, s. 5639-5651Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
    Forskningsfinansiär
    Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, Europeiska forskningsrådet, 306474;283570Vetenskapsrådet, 621-2011-6055Carl Tryggers stiftelse för vetenskaplig forskning , CTS13:104
    Tillgänglig från: 2016-04-01 Skapad: 2016-04-01 Senast uppdaterad: 2017-11-30Bibliografiskt granskad
    4. CADEE: Computer-Aided Directed Evolution of Enzymes
    Öppna denna publikation i ny flik eller fönster >>CADEE: Computer-Aided Directed Evolution of Enzymes
    Visa övriga...
    2017 (Engelska)Ingår i: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 4, nr 1, s. 50-64Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

    Nyckelord
    computational directed evolution, computational enzyme design, distributed computing, empirical valence bond, triosephosphate isomerase
    Nationell ämneskategori
    Strukturbiologi Bioinformatik (beräkningsbiologi) Teoretisk kemi
    Identifikatorer
    urn:nbn:se:uu:diva-314218 (URN)10.1107/S2052252516018017 (DOI)000392925800007 ()
    Forskningsfinansiär
    EU, FP7, Sjunde ramprogrammet, 306474Knut och Alice Wallenbergs StiftelseKungliga VetenskapsakademienVetenskapsrådet, 2015-04928Swedish National Infrastructure for Computing (SNIC), 2015/16-12
    Tillgänglig från: 2017-01-31 Skapad: 2017-01-31 Senast uppdaterad: 2018-01-13Bibliografiskt granskad
  • 59.
    Amrein, Beat Anton
    et al.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Steffen-Munsberg, Fabian
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Szeler, Ireneusz
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Purg, Miha
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Kulkarni, Yashraj
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Kamerlin, Shina Caroline Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    CADEE: Computer-Aided Directed Evolution of Enzymes2017Ingår i: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 4, nr 1, s. 50-64Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

  • 60.
    Amselem, Elias
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär systembiologi.
    Marklund, Emil
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär systembiologi.
    Kipper, Kalle
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Johansson, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär systembiologi.
    Deindl, Sebastian
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär systembiologi.
    Elf, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär systembiologi.
    Real- Time Single Protein Tracking with Polarization Readout using a Confocal Microscope2017Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, nr 3, s. 295A-295AArtikel i tidskrift (Övrigt vetenskapligt)
  • 61.
    Andaloussi, Mounir
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Henriksson, Lena M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Wieckowska, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Lindh, Martin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Björkelid, Christofer
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Larsson, Anna M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Suresh, Surisetti
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Iyer, Harini
    Srinivasa, Bachally R.
    Bergfors, Terese
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Unge, Torsten
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Mowbray, Sherry L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Larhed, Mats
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Jones, T. Alwyn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Karlén, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase2011Ingår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, nr 14, s. 4964-4976Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. alpha-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 mu M on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 mu g/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

  • 62.
    Andaloussi, Mounir
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Lindh, Martin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Björkelid, Christofer
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Suresh, Surisetti
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Wieckowska, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Iyer, Harini
    Karlén, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Larhed, Mats
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Substitution of the phosphonic acid and hydroxamic acid functionalities of the DXR inhibitor FR900098: An attempt to improve the activity against Mycobacterium tuberculosis2011Ingår i: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 21, nr 18, s. 5403-5407Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Two series of FR900098/fosmidomycin analogs were synthesized and evaluated for MtDXR inhibition and Mycobacterium tuberculosis whole-cell activity. The design rationale of these compounds involved the exchange of either the phosphonic acid or the hydroxamic acid part for alternative acidic and metal-coordinating functionalities. The best inhibitors provided IC(50) values in the micromolar range, with a best value of 41 mu M.

  • 63. Anders, Alfjorden
    et al.
    Astvaldsson, Asgeir
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Eva, Jansson
    Svärd, Staffan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Experimental challenge of Atlantic salmon (Salmo salar) with the diplomonad parasite Spironucleus salmonicida to characterize the infection cycleManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Experimental infections were performed of Atlantic salmon (Salmo salar) from the Baltic Sea region with the Diplomonad fish parasite Spironucleus salmonicida in order to define the infection cycle, specifically the time-line and putative routes of transmission. An oral infection protocol using axenic parasites was developed, as were new diagnostic tools using PCR and specific antibodies. We also produced firefly luciferase expressing S. salmonicida parasites that could be identified in the infected fish using in vivo and ex vivo imaging. The new tools made it possible to follow the S. salmonicida infection cycle in detail. Three different stages of the infection were identified: one initial intestinal stage, followed by a blood stage and a final tissue stage. Parasites intubated into the intestine attached to the intestinal surface and were identified in the blood after 1-3 weeks. Skin lesions and infections of the muscles, internal organs and eyes were seen 4-10 weeks after initiation of infection. Several morphologically different forms of S. salmonicida cells were detected in ex vivo cell-cultures of biopsies from skin lesions. By this infection trial we have been able to show that S. salmonicida may use several alternative routes of transmission. One alternative is the fecal-oral route, similar to other Diplomonad parasites but the parasites can also be excreted directly into the surrounding water from the mucous layer of the skin or from an ulcerated skin lesion. This information can be used to prevent the transmission of the parasite in fish farms.

  • 64. Anderson, Frank E.
    et al.
    Córdoba, Alonso J.
    Thollesson, Mikael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Centrum för bioinformatik.
    Bilaterian phylogeny based on analyses of a region of the sodium-potassium ATPase alpha-subunit gene2004Ingår i: Journal of Molecular Evolution, ISSN 0022-2844, E-ISSN 1432-1432, Vol. 58, nr 3, s. 252-268Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Molecular investigations of deep-level relationships within and among the animal phyla have been hampered by a lack of slowly evolving genes that are amenable to study by molecular systematists. To provide new data for use in deep-level metazoan phylogenetic studies, primers were developed to amplify a 1.3-kb region of the subunit of the nuclear-encoded sodium–potassium ATPase gene from 31 bilaterians representing several phyla. Maximum parsimony, maximum likelihood, and Bayesian analyses of these sequences (combined with ATPase sequences for 23 taxa downloaded from GenBank) yield congruent trees that corroborate recent findings based on analyses of other data sets (e.g., the 18S ribosomal RNA gene). The ATPase-based trees support monophyly for several clades (including Lophotrochozoa, a form of Ecdysozoa, Vertebrata, Mollusca, Bivalvia, Gastropoda, Arachnida, Hexapoda, Coleoptera, and Diptera) but do not support monophyly for Deuterostomia, Arthropoda, or Nemertea. Parametric bootstrapping tests reject monophyly for Arthropoda and Nemertea but are unable to reject deuterostome monophyly. Overall, the sodium–potassium ATPase -subunit gene appears to be useful for deep-level studies of metazoan phylogeny.

  • 65.
    Andersson, Anders F.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och evolution. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi.
    Pelve, Erik A.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution.
    Lindeberg, Stefan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Nilsson, Peter
    Bernander, Rolf
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution.
    Replication-biased genome organisation in the crenarchaeon Sulfolobus2010Ingår i: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, s. 454-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Species of the crenarchaeon Sulfolobus harbour three replication origins in their single circular chromosome that are synchronously initiated during replication. Results: We demonstrate that global gene expression in two Sulfolobus species is highly biased, such that early replicating genome regions are more highly expressed at all three origins. The bias by far exceeds what would be anticipated by gene dosage effects alone. In addition, early replicating regions are denser in archaeal core genes (enriched in essential functions), display lower intergenic distances, and are devoid of mobile genetic elements. Conclusion: The strong replication-biased structuring of the Sulfolobus chromosome implies that the multiple replication origins serve purposes other than simply shortening the time required for replication. The higher-level chromosomal organisation could be of importance for minimizing the impact of DNA damage, and may also be linked to transcriptional regulation.

  • 66.
    Andersson, C. Evalena
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Structure-Function Studies of Enzymes from Ribose Metabolism2004Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In the pentose phosphate pathway, carbohydrates such as glucose and ribose are degraded with production of reductive power and energy. Another important function is to produce essential pentoses, such as ribose 5-phosphate, which later can be used in biosynthesis of nucleic acids and cofactors.

    This thesis presents structural and functional studies on three enzymes involved in ribose metabolism in Escherichia coli.

    Ribokinase is an enzyme that phosphorylates ribose in the presence of ATP and magnesium, as the first step of exogenous ribose metabolism. Two important aspects of ribokinase function, not previously known, have been elucidated. Ribokinase was shown to be activated by monovalent cations, specifically potassium. Structural analysis of the monovalent ion binding site indicates that the ion has a structural rather than catalytic role; a mode of activation involving a conformational change has been suggested. Product inhibition studies suggest that ATP is the first substrate to bind the enzyme. Independent Kd measurements with the ATP analogue AMP-PCP support this. The results presented here will have implications for several enzymes in the protein family to which ribokinase belongs, in particular the medically interesting enzyme adenosine kinase.

    Ribose 5-phosphate isomerases convert ribose 5-phosphate into ribulose 5-phosphate or vice versa. Structural studies on the two genetically distinct isomerases in E. coli have shown them to be fundamentally different in many aspects, including active site architecture. However, a kinetic study has demonstrated both enzymes to be efficient in terms of catalysis. Sequence searches of completed genomes show ribose 5-phosphate isomerase B to be the sole isomerase in many bacteria, although ribose 5-phosphate isomerase A is a nearly universal enzyme. All genomes contain at least one of the two enzymes. These results confirm that both enzymes must be independently capable of supporting ribose metabolism, a fact that had not previously been established.

    Delarbeten
    1. Activation of Ribokinase by Monovalent Cations
    Öppna denna publikation i ny flik eller fönster >>Activation of Ribokinase by Monovalent Cations
    2002 (Engelska)Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 315, nr 3, s. 409-419Artikel i tidskrift (Refereegranskat) Published
    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-91364 (URN)10.1006/jmbi.2001.5248 (DOI)
    Tillgänglig från: 2004-02-12 Skapad: 2004-02-12 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
    2. Structure of Escherichia coli Ribose 5-phosphate Isomerase: A Ubiquitous Enzyme of the Pentose Phosphate Pathway and the Calvin Cycle
    Öppna denna publikation i ny flik eller fönster >>Structure of Escherichia coli Ribose 5-phosphate Isomerase: A Ubiquitous Enzyme of the Pentose Phosphate Pathway and the Calvin Cycle
    Visa övriga...
    2003 Ingår i: Structure, Vol. 11, nr 1, s. 31-42Artikel i tidskrift (Refereegranskat) Published
    Identifikatorer
    urn:nbn:se:uu:diva-91365 (URN)
    Tillgänglig från: 2004-02-12 Skapad: 2004-02-12Bibliografiskt granskad
    3. The 2.2 Å Resolution Structure of RpiB/AlsB from Escherichia coli Illutrates a New Approach to the Ribose 5-phosphate Isomerase Reaction
    Öppna denna publikation i ny flik eller fönster >>The 2.2 Å Resolution Structure of RpiB/AlsB from Escherichia coli Illutrates a New Approach to the Ribose 5-phosphate Isomerase Reaction
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    2003 Ingår i: Journal of Molecular Biology, Vol. 332, nr 5, s. 1083-1094Artikel i tidskrift (Refereegranskat) Published
    Identifikatorer
    urn:nbn:se:uu:diva-91366 (URN)
    Tillgänglig från: 2004-02-12 Skapad: 2004-02-12Bibliografiskt granskad
    4. Specificity and Activity of Escherichia coli Ribokinase
    Öppna denna publikation i ny flik eller fönster >>Specificity and Activity of Escherichia coli Ribokinase
    Manuskript (Övrigt vetenskapligt)
    Identifikatorer
    urn:nbn:se:uu:diva-91367 (URN)
    Tillgänglig från: 2004-02-12 Skapad: 2004-02-12 Senast uppdaterad: 2016-05-09Bibliografiskt granskad
    5. Mycobacterium tuberculosis Ribose 5-phosphate Isomerase has a Known Fold, but a Novel Active Site
    Öppna denna publikation i ny flik eller fönster >>Mycobacterium tuberculosis Ribose 5-phosphate Isomerase has a Known Fold, but a Novel Active Site
    Visa övriga...
    2004 Ingår i: Journal of Molecular Biology, Vol. 335, nr 3, s. 799-809Artikel i tidskrift (Refereegranskat) Published
    Identifikatorer
    urn:nbn:se:uu:diva-91368 (URN)
    Tillgänglig från: 2004-02-12 Skapad: 2004-02-12Bibliografiskt granskad
  • 67.
    Andersson, C. Evalena
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Mowbray, Sherry L.
    Activation of Ribokinase by Monovalent Cations2002Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 315, nr 3, s. 409-419Artikel i tidskrift (Refereegranskat)
  • 68.
    Andersson, C. Evalena
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Simon, Jill A.
    Cameron, Alexander D.
    Mowbray, Sherry L.
    Specificity and Activity of Escherichia coli RibokinaseManuskript (Övrigt vetenskapligt)
  • 69.
    Andersson, C. Evalena
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Simon, Jill A.
    Cameron, Alexander D.
    Mowbray, Sherry L.
    Specificity and Activity of Escherichia coli RibokinaseManuskript (Övrigt vetenskapligt)
  • 70. Andersson, Charlotta S.
    et al.
    Högbom, Martin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    A Mycobacterium tuberculosis ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold2009Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, nr 14, s. 5633-5638Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chlamydia trachomatis R2c is the prototype for a recently discovered group of ribonucleotide reductase R2 proteins that use a heterodinuclear Mn/Fe redox cofactor for radical generation and storage. Here, we show that the Mycobacterium tuberculosis protein Rv0233, an R2 homologue and a potential virulence factor, contains the heterodinuclear manganese/iron-carboxylate cofactor but displays a drastic remodeling of the R2 protein scaffold into a ligand-binding oxidase. The first structural characterization of the heterodinuclear cofactor shows that the site is highly specific for manganese and iron in their respective positions despite a symmetric arrangement of coordinating residues. In this protein scaffold, the Mn/Fe cofactor supports potent 2-electron oxidations as revealed by an unprecedented tyrosine-valine crosslink in the active site. This wolf in sheep's clothing defines a distinct functional group among R2 homologues and may represent a structural and functional counterpart of the evolutionary ancestor of R2s and bacterial multicomponent monooxygenases.

  • 71.
    Andersson, Claes
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Centrum för bioinformatik.
    Fusing Domain Knowledge with Data: Applications in Bioinformatics2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Massively parallel measurement techniques can be used for generating hypotheses about the molecular underpinnings of a biological systems. This thesis investigates how domain knowledge can be fused to data from different sources in order to generate more sophisticated hypotheses and improved analyses. We find our applications in the related fields of cell cycle regulation and cancer chemotherapy. In our cell cycle studies we design a detector of periodic expression and use it to generate hypotheses about transcriptional regulation during the course of the cell cycle in synchronized yeast cultures as well as investigate if domain knowledge about gene function can explain whether a gene is periodically expressed or not. We then generate hypotheses that suggest how periodic expression that depends on how the cells were perturbed into synchrony are regulated. The hypotheses suggest where and which transcription factors bind upstreams of genes that are regulated by the cell cycle. In our cancer chemotherapy investigations we first study how a method for identifiyng co-regulated genes associated with chemoresponse to drugs in cell lines is affected by domain knowledge about the genetic relationships between the cell lines. We then turn our attention to problems that arise in microarray based predictive medicine, were there typically are few samples available for learning the predictor and study two different means of alleviating the inherent trade-off betweeen allocation of design and test samples. First we investigate whether independent tests on the design data can be used for improving estimates of a predictors performance without inflicting a bias in the estimate. Then, motivated by recent developments in microarray based predictive medicine, we propose an algorithm that can use unlabeled data for selecting features and consequently improve predictor performance without wasting valuable labeled data.

    Delarbeten
    1. In vitro drug sensitivity-gene expression correlations involve a tissue of origin dependency
    Öppna denna publikation i ny flik eller fönster >>In vitro drug sensitivity-gene expression correlations involve a tissue of origin dependency
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    2007 (Engelska)Ingår i: Journal of chemical information and modeling, ISSN 1549-9596, Vol. 47, nr 1, s. 239-248Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    A major concern of chemogenomics is to associate drug activity with biological variables. Several reports have clustered cell line drug activity profiles as well as drug activity-gene expression correlation profiles and noted that the resulting groupings differ but still reflect mechanism of action. The present paper shows that these discrepancies can be viewed as a weighting of drug-drug distances, the weights depending on which cell lines the two drugs differ in.

    Nyckelord
    computers in chemistry, computer program
    Nationell ämneskategori
    Medicin och hälsovetenskap Signalbehandling
    Forskningsämne
    Elektroteknik med inriktning mot signalbehandling
    Identifikatorer
    urn:nbn:se:uu:diva-20891 (URN)10.1021/ci060073n (DOI)000243577400029 ()17238270 (PubMedID)
    Tillgänglig från: 2007-10-28 Skapad: 2007-10-28 Senast uppdaterad: 2016-09-25Bibliografiskt granskad
    2. Bayesian detection of periodic mRNA time profiles withouth use of training examples
    Öppna denna publikation i ny flik eller fönster >>Bayesian detection of periodic mRNA time profiles withouth use of training examples
    2006 (Engelska)Ingår i: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 7, s. 63-Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    BACKGROUND: Detection of periodically expressed genes from microarray data without use of known periodic and non-periodic training examples is an important problem, e.g. for identifying genes regulated by the cell-cycle in poorly characterised organisms. Commonly the investigator is only interested in genes expressed at a particular frequency that characterizes the process under study but this frequency is seldom exactly known. Previously proposed detector designs require access to labelled training examples and do not allow systematic incorporation of diffuse prior knowledge available about the period time. RESULTS: A learning-free Bayesian detector that does not rely on labelled training examples and allows incorporation of prior knowledge about the period time is introduced. It is shown to outperform two recently proposed alternative learning-free detectors on simulated data generated with models that are different from the one used for detector design. Results from applying the detector to mRNA expression time profiles from S. cerevisiae showsthat the genes detected as periodically expressed only contain a small fraction of the cell-cycle genes inferred from mutant phenotype. For example, when the probability of false alarm was equal to 7%, only 12% of the cell-cycle genes were detected. The genes detected as periodically expressed were found to have a statistically significant overrepresentation of known cell-cycle regulated sequence motifs. One known sequence motif and 18 putative motifs, previously not associated with periodic expression, were also over represented. CONCLUSION: In comparison with recently proposed alternative learning-free detectors for periodic gene expression, Bayesian inference allows systematic incorporation of diffuse a priori knowledge about, e.g. the period time. This results in relative performance improvements due to increased robustness against errors in the underlying assumptions. Results from applying the detector to mRNA expression time profiles from S. cerevisiae include several new findings that deserve further experimental studies.

    Nationell ämneskategori
    Medicin och hälsovetenskap Teknik och teknologier
    Identifikatorer
    urn:nbn:se:uu:diva-96785 (URN)10.1186/1471-2105-7-63 (DOI)16469110 (PubMedID)
    Tillgänglig från: 2008-02-20 Skapad: 2008-02-20 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
    3. Revealing cell cycle control by combining model-based detection of periodic expression with novel cis-regulatory descriptors
    Öppna denna publikation i ny flik eller fönster >>Revealing cell cycle control by combining model-based detection of periodic expression with novel cis-regulatory descriptors
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    2007 (Engelska)Ingår i: BMC Systems Biology, ISSN 1752-0509, Vol. 1, s. 45-Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Background: We address the issue of explaining the presence or absence of phase-specific transcription in budding yeast cultures under different conditions. To this end we use a model-based detector of gene expression periodicity to divide genes into classes depending on their behavior in experiments using different synchronization methods. While computational inference of gene regulatory circuits typically relies on expression similarity (clustering) in order to find classes of potentially co-regulated genes, this method instead takes advantage of known time profile signatures related to the studied process. Results: We explain the regulatory mechanisms of the inferred periodic classes with cis-regulatory descriptors that combine upstream sequence motifs with experimentally determined binding of transcription factors. By systematic statistical analysis we show that periodic classes are best explained by combinations of descriptors rather than single descriptors, and that different combinations correspond to periodic expression in different classes. We also find evidence for additive regulation in that the combinations of cis-regulatory descriptors associated with genes periodically expressed in fewer conditions are frequently subsets of combinations associated with genes periodically expression in more conditions. Finally, we demonstrate that our approach retrieves combinations that are more specific towards known cell-cycle related regulators than the frequently used clustering approach. Conclusion: The results illustrate how a model-based approach to expression analysis may be particularly well suited to detect biologically relevant mechanisms. Our new approach makes it possible to provide more refined hypotheses about regulatory mechanisms of the cell cycle and it can easily be adjusted to reveal regulation of other, non-periodic, cellular processes.

    Nationell ämneskategori
    Biologiska vetenskaper Signalbehandling
    Forskningsämne
    Elektroteknik med inriktning mot signalbehandling
    Identifikatorer
    urn:nbn:se:uu:diva-96786 (URN)10.1186/1752-0509-1-45 (DOI)000252363100001 ()17939860 (PubMedID)
    Tillgänglig från: 2008-02-20 Skapad: 2008-02-20 Senast uppdaterad: 2016-09-25Bibliografiskt granskad
    4. Improving Bayesian credibility intervals for classifier error rates using maximum entropy empirical priors
    Öppna denna publikation i ny flik eller fönster >>Improving Bayesian credibility intervals for classifier error rates using maximum entropy empirical priors
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    2010 (Engelska)Ingår i: Artificial Intelligence in Medicine, ISSN 0933-3657, E-ISSN 1873-2860, Vol. 49, nr 2, s. 93-104Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Objective:

    Successful use of classifiers that learn to make decisions from a set of patient examples require robust methods for performance estimation. Recently many promising approaches for determination of an upper bound for the error rate of a single classifier have been reported but the Bayesian credibility interval (Cl) obtained from a conventional holdout test still delivers one of the tightest bounds. The conventional Bayesian CI becomes unacceptably large in real world applications where the test set sizes are less than a few hundred. The source of this problem is that fact that the Cl is determined exclusively by the result on the test examples. In other words, there is no information at all provided by the uniform prior density distribution employed which reflects complete lack of prior knowledge about the unknown error rate. Therefore, the aim of the study reported here was to study a maximum entropy (ME) based approach to improved prior knowledge and Bayesian CIs, demonstrating its relevance for biomedical research and clinical practice.

    Method and material:

    It is demonstrated how a refined non-uniform prior density distribution can be obtained by means of the ME principle using empirical results from a few designs and tests using non-overlapping sets of examples.

    Results:

    Experimental results show that ME based priors improve the CIs when employed to four quite different simulated and two real world data sets.

    Conclusions:

    An empirically derived ME prior seems promising for improving the Bayesian Cl for the unknown error rate of a designed classifier.

    Nationell ämneskategori
    Medicin och hälsovetenskap Data- och informationsvetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-96787 (URN)10.1016/j.artmed.2010.02.004 (DOI)000279172200003 ()
    Tillgänglig från: 2008-02-20 Skapad: 2008-02-20 Senast uppdaterad: 2018-01-13
    5. Feature Selection using Classification of Unlabeled Data
    Öppna denna publikation i ny flik eller fönster >>Feature Selection using Classification of Unlabeled Data
    <