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  • 1.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Discovery and evaluation of direct acting antivirals against hepatitis C virus2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Until recently, the standard therapy for hepatitis C treatment has been interferon and ribavirin. Such treatment has only 50% efficacy and is not well tolerated. The emergence of new drugs has increased the treatment efficacy to 90%. Despite such an achievement, the success is limited since the virus mutates rapidly, causing the emergence of drug resistant forms. In addition, most new drugs were developed to treat genotype 1 infections. Thus, development of new potent antivirals is needed and drug discovery against hepatitis C is continued.

    In this thesis, a FRET-based protease assay was used to evaluate new pyrazinone based NS3 protease inhibitors that are structurally different to the newly approved and currently developing drugs. Several compounds in this series showed good potencies in the nanomolar range against NS3 proteases from genotype 1, 3, and the drug resistance variant R155K. We assume that these compounds can be further developed into drug candidates that possess activity against above mentioned enzyme variants.

    By using SPR technology, we analyzed interaction mechanisms and characteristics of allosteric inhibitors targeting NS5B polymerases from genotypes 1 and 3. The compounds exhibited different binding mechanisms and displayed a low affinity against NS5B from genotype 3.

    In order to evaluate the activity and inhibitors of the NS5B polymerase, we established an SPR based assay, which enables the monitoring of polymerization and its inhibition in real time. This assay can readily be implemented for the discovery of inhibitors targeting HCV.

    An SPR based fragment screening approach has also been established. A screen of a fragment library has been performed in order to identify novel scaffolds that can be used as a starting point for development of new allosteric inhibitors against NS5B polymerase. Selected fragments will be further elaborated to generate a new potent allosteric drug candidate.

    Alternative approaches have successfully been developed and implemented to the discovery of potential lead compounds targeting two important HCV drug targets.

    List of papers
    1. Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
    Open this publication in new window or tab >>Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
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    2016 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 24, no 12, p. 2603-2620Article in journal (Refereed) Published
    Abstract [en]

    Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wildtype and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance.

    Keywords
    Hepatitis C virus; Drug resistance; Pyrazinone; NS3 protease inhibitors; R155K
    National Category
    Organic Chemistry
    Research subject
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-243315 (URN)10.1016/j.bmc.2016.03.066 (DOI)000376727800002 ()27160057 (PubMedID)
    Funder
    Swedish Research Council, D0571301
    Available from: 2015-02-08 Created: 2015-02-08 Last updated: 2022-01-28Bibliographically approved
    2. Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotype 1a, 3a and the drug-resistant enzyme variant R155K
    Open this publication in new window or tab >>Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotype 1a, 3a and the drug-resistant enzyme variant R155K
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    (English)Manuscript (preprint) (Other academic)
    Abstract
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265295 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2022-01-25
    3. Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
    Open this publication in new window or tab >>Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
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    2013 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 97, no 3, p. 356-368Article in journal (Other academic) Published
    Abstract [en]

    Development of allosteric inhibitors into efficient drugs is hampered by their indirect mode-of-action and complex structure-kinetic relationships. To enablethe design of efficient allosteric drugs targeting the polymerase of hepatitis C virus(NS5B), the interaction characteristics of three non-nucleoside compounds (filibuvir, VX-222, and tegobuvir) inhibiting HCV replication via NS5B have been analyzed. Since there was no logical correlation between the anti-HCV replicative and enzyme inhibitory effects of the compounds, surface plasmon resonance biosensor technology was used to resolve the mechanistic, kinetic, thermodynamic and chemodynamic features of their interactions with their target and their effect on itsinteraction with RNA. Tegobuvir could not be seen to interact with NS5B at all while filibuvir interacted in a single reversible step (except at low temperatures) and VX-222 in two serial steps, interpreted as an induced fit mechanism. Both filibuvir and VX-222 interfered with the interaction between NS5B and RNA. They competed for binding to the enzyme, suggesting that they had a common inhibition mechanism and identical or overlapping binding sites. The greater anti-HCV replicative activityof VX-222 over filibuvir is hypothesized to be due to a greater allosteric conformational effect, resulting in the formation of a less catalytically competent complex. In addition, the induced fit mechanism of VX-222 gives it a kinetic advantage over filibuvir, exhibited as a longer residence time. These insights have important consequences for the selection and optimization of new allosteric NS5Binhibitors.

    Keywords
    HCV, NS5B, filibuvir, VX-222, tegobuvir, allosteric inhibitor, induced fit, kinetics, chemodynamics, thermodynamics
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry; Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-171996 (URN)10.1016/j.antiviral.2012.12.027 (DOI)000317709400018 ()
    Available from: 2012-04-03 Created: 2012-03-31 Last updated: 2017-12-07Bibliographically approved
    4. Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative study
    Open this publication in new window or tab >>Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative study
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265287 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
    5. A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discovery
    Open this publication in new window or tab >>A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discovery
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265290 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
    6. Fragment library screening addressing Hepatitis C protein NS5B from genotypes 1 and 3 using an SPR-based approach
    Open this publication in new window or tab >>Fragment library screening addressing Hepatitis C protein NS5B from genotypes 1 and 3 using an SPR-based approach
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265292 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
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  • 2.
    Abdurakhmanov, Eldar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Solbak, Sara
    Danielson, Helena
    Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative studyManuscript (preprint) (Other academic)
  • 3.
    Abdurakhmanov, Eldar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Solbak, Sara Oie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase2017In: Viruses, E-ISSN 1999-4915, Vol. 9, no 6, article id 151Article in journal (Refereed)
    Abstract [en]

    Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

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  • 4.
    Abramsson, Mia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Biophysical Characterization of Hit Compounds against a Structurally Dynamic Protein for Drug Discovery2020Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
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  • 5.
    Abramsson, Mia L.
    et al.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, S-17165 Stockholm, Sweden..
    Sahin, Cagla
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, S-17165 Stockholm, Sweden.;Univ Copenhagen, Linderstrom Lang Ctr Prot Sci, Dept Biol, DK-2200 Copenhagen, Denmark.;OMass Therapeut, SchrOdinger Bldg,Oxford Sci Pk, Oxford OX4 4GE, England..
    Hopper, Jonathan T. S.
    Univ Oxford, Dept Chem, Oxford OX1 3QZ, England..
    Branca, Rui M. M.
    Sci Life Lab, Dept Oncol Pathol, S-17165 Stockholm, Sweden.;Karolinska Inst, S-17165 Stockholm, Sweden..
    Danielsson, Jens
    Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden..
    Xu, Mingming
    Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden..
    Chandler, Shane A.
    Univ Oxford, Dept Chem, Oxford OX1 3QZ, England..
    Osterlund, Nicklas
    Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden..
    Ilag, Leopold L.
    Stockholm Univ, Dept Mat & Environm Chem, S-10691 Stockholm, Sweden..
    Leppert, Axel
    Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Costeira-Paulo, Joana
    Uppsala Univ, Dept Chem BMC, S-75123 Uppsala, Sweden..
    Lang, Lisa
    Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden..
    Teilum, Kaare
    Univ Copenhagen, Linderstrom Lang Ctr Prot Sci, Dept Biol, DK-2200 Copenhagen, Denmark..
    Laganowsky, Arthur
    Texas A&M Univ, Dept Chem, College Stn, TX 77843 USA..
    Benesch, Justin L. P.
    Univ Oxford, Dept Chem, Oxford OX1 3QZ, England..
    Oliveberg, Mikael
    Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden..
    Robinson, Carol, V
    Univ Oxford, Dept Chem, Oxford OX1 3QZ, England..
    Marklund, Erik
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Allison, Timothy M.
    Univ Canterbury, Biomol Interact Ctr, Sch Phys & Chem Sci, Christchurch 8140, New Zealand..
    Winther, Jakob R.
    Univ Copenhagen, Linderstrom Lang Ctr Prot Sci, Dept Biol, DK-2200 Copenhagen, Denmark..
    Landreh, Michael
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, S-17165 Stockholm, Sweden..
    Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry2021In: JACS Au, E-ISSN 2691-3704, Vol. 1, no 12, p. 2385-2393Article in journal (Refereed)
    Abstract [en]

    In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

  • 6. Abramsson, Mia L
    et al.
    Sahin, Cagla
    Hopper, Jonathan T S
    Branca, Rui M M
    Danielsson, Jens
    Xu, Mingming
    Chandler, Shane A
    Österlund, Nicklas
    Ilag, Leopold L
    Leppert, Axel
    Costeira-Paulo, Joana
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Lang, Lisa
    Teilum, Kaare
    Robinson, Carol V
    Laganowsky, Arthur
    Benesch, Justin L P
    Oliveberg, Mikael
    Marklund, Erik G
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Allison, Timothy M
    Winther, Jakob R
    Landreh, Michael
    Charge engineering reveals the roles of ionizable side chains in electrospray ionization mass spectrometryManuscript (preprint) (Other academic)
    Abstract [en]

    The role of ionizable side chains in the electrospray ionization mass spectrometry of intact proteins remains hotly debated but has not been conclusively addressed because multiple chargeable sites are present in virtually all proteins. Using engineered soluble proteins, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, whilst co-existing conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. We conclude that the sum of charges is governed solely by Coulombic terms, while their locations affect the stability of the protein in the gas phase.

  • 7. Akpiroro Peters, Marie Berit
    et al.
    Kassa, Eszter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Yau, Wai-Lok
    Lindqvist, Richard
    Nilsson, Emma
    Siljedahl, Michaela
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Lundmark, Richard
    Överby, Anna K.
    Tick-borne flaviviruses recruits the pro viral factor NUP153 to the replication siteManuscript (preprint) (Other academic)
  • 8.
    Aksoy, N. H.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Aksaray Univ, Dept Biochem, Aksaray, Turkey..
    Mannervik, B.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Inhibitory effects of ethacrynic acid on glutathione S-transferase A1-1 from Callithrix jacchus2015In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, p. 348-348Article in journal (Other academic)
  • 9.
    Al-Amin, Rasel A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Johansson, Lars
    Department of Medical Biochemistry and Biophysics, Chemical Biology Consortium Sweden (CBCS), Science for Life Laboratory, Karolinska Institutet.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical diabetology and metabolism. Center for Molecular Medicine, Department of Medicine (Solna), Science for Life Laboratory, Karolinska Institutet.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Arngården, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Blokzijl, Andries
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Svensson, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hammond, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lönn, Peter
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Haybaeck, Johannes
    Institute of Pathology, Neuropathology and Molecular Pathology, Medical University of Innsbruck; Diagnostic and Research Institute of Pathology, Medical University of Graz.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jenmalm Jensen, Annika
    Department of Medical Biochemistry and Biophysics, Chemical Biology Consortium Sweden (CBCS), Science for Life Laboratory, Karolinska Institutet.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundbäck, Thomas
    Department of Medical Biochemistry and Biophysics, Chemical Biology Consortium Sweden (CBCS), Science for Life Laboratory, Karolinska Institutet.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ2022In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, no 22, p. e129-e129Article in journal (Refereed)
    Abstract [en]

    Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues.

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  • 10.
    Al-Amin, Rasel A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab.
    Johansson, Lars
    Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet.
    Landegren, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Autoimmunity. Uppsala University, Science for Life Laboratory, SciLifeLab. Department of Medicine (Solna), Karolinska University Hospital, Karolinska Institutet.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Blokzijl, Andries
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Svensson, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lönn, Peter
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Dept. Of Immunology, Genetics and Pathology,.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundbäck, Thomas
    Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in SituManuscript (preprint) (Other academic)
    Abstract [en]

    It is important to determine the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimens. This is necessary to evaluate drug candidates from early stages of drug development to clinical evaluation of mutations potentially causing resistance to targeted therapy. We describe a technology where oligonucleotide-conjugated drug molecules are used to visualize and measure target engagement in situ via rolling-circle amplification (RCA) of circularized oligonucleotide probes (padlock probes). We established this target engagement-mediated amplification (TEMA) technique using kinase inhibitor precursor compounds, and we applied the assay to investigate target interactions by microscopy in pathology tissue sections and using flow cytometry for blood samples from patients, as well as in commercial arrays including almost half of all human proteins.  In the variant proxTEMAtechnique, in situ proximity ligation assays were performed by combining drug-DNA conjugates with antibody-DNA conjugates to specifically reveal drug binding to particular on- or off-targets in pathological tissues sections. In conclusion, the TEMA methods successfully visualize drug-target interaction by experimental and clinically approved kinase inhibitors in situ and with kinases among a large collection of arrayed proteins. 

  • 11.
    Al-Amin, Rasel Abdullah
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Muthelo, Phathutshedzo M.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vincke, Cecile
    Structural Biology Research Center, Vrije Universiteit Brussel, Belgium..
    Muyldermans, Serge
    Structural Biology Research Center, Vrije Universiteit Brussel, Belgium.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobody reagents2022In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 98, no 28, p. 10054-10061Article in journal (Refereed)
    Abstract [en]

    High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

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  • 12.
    Ali, Muhammad
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Identification of SLiMs: Mapping and characterizing motif-based protein interactions2020Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    During the last twenty years it has become evident that about 35-40% of amino acids in the proteome are in regions that have evolved to remain unstructured. These intrinsically disordered regions contain short linear motifs (SLiMs), which serve as docking sites for protein-protein interactions. SLiMs often mediate low-to-medium affinity interactions that are transient in their nature. The characteristics of SLiM-based interactions make them difficult to be captured using conventional approaches like affinity-purification coupled to mass spectrometry or yeast-two-hybrid. We therefore used and developed a dedicated method for large-scale screening of SLiM-based interactions termed proteomic peptide phage display (ProP-PD).

    Using ProP-PD, We identified large sets of ligands, for the binding pocket of shank1 PDZ domain, containing C-terminal or internal binding motifs and established the consensus motifs to be xTxL/F-COOH and xTxFx respectively. We further validated interactions using biophysical affinity determinations and pulldown experiments. Using X-ray crystallization, we uncovered that shank1 PDZ binds to internal xTxFx motifs using a binding mode similar to that for C-terminal peptides.

    Adding a level of complexity, we explored interactions of the multiple binding pocket containing FERM domains from four closely related proteins: ezrin, radixin, moesin and merlin. We found hundreds of FERM ligands, which contained binding motifs of at least four different classes. By combining docking simulations with experiments, we established ligands binding to different pockets, and uncovered a complex interplay between distinct pockets.

    We further developed an optimized version of a phage library that displays intrinsically disordered regions of the human proteome. We benchmarked the library using a set of protein domains and reported better recovery of known SLiM-based interactions. Furthermore, we highlighted the functional aspects of identified SLiMs, in the case of nuclear localization signals, found for binding to importin-subunit alpha-3. Finally, we validated predicted binding of SLiMs in the Sars-CoV-2 host receptor ACE2, which illustrates the importance of fundamental knowledge for SLiMs and their binding partners.

    This work, taken together, contributes with method development for expansion of motifs based interactomes and provide insights into the plastic yet selective nature of peptide binding proteins.

    List of papers
    1. Integrated analysis of Shank1 PDZ interactions with C-terminal andinternal binding motifs
    Open this publication in new window or tab >>Integrated analysis of Shank1 PDZ interactions with C-terminal andinternal binding motifs
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-424026 (URN)
    Available from: 2020-11-02 Created: 2020-11-02 Last updated: 2020-11-04
    2. Defining binding motifs and dynamics of the multi-pocket FERM domain from the ezrin, radixin, moesin and merlin
    Open this publication in new window or tab >>Defining binding motifs and dynamics of the multi-pocket FERM domain from the ezrin, radixin, moesin and merlin
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-424027 (URN)
    Available from: 2020-11-02 Created: 2020-11-02 Last updated: 2020-11-04
    3. Cytoplasmic short linear motifs in ACE2 and integrin b3link SARS-CoV-2 host cell receptors to endocytosis andautophagy
    Open this publication in new window or tab >>Cytoplasmic short linear motifs in ACE2 and integrin b3link SARS-CoV-2 host cell receptors to endocytosis andautophagy
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-424028 (URN)
    Available from: 2020-11-02 Created: 2020-11-02 Last updated: 2020-11-04
    4. Proteome-scale amino-acid resolution foot printing of protein-bindingregions in intrinsically disordered regions
    Open this publication in new window or tab >>Proteome-scale amino-acid resolution foot printing of protein-bindingregions in intrinsically disordered regions
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-424029 (URN)
    Available from: 2020-11-02 Created: 2020-11-02 Last updated: 2020-11-04
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  • 13.
    Ali, Muhammad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    High-throughput discovery of functional disordered regions2018In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 14, no 5, article id e8377Article in journal (Other academic)
    Abstract [en]

    Partially or fully intrinsically disordered proteins are widespread in eukaryotic proteomes and play important biological functions. With the recognition that well defined protein structure is not a fundamental requirement for function come novel challenges, such as assigning function to disordered regions. In their recent work, Babu and colleagues (Ravarani etal,) took on this challenge by developing IDR-Screen, a robust high-throughput approach for identifying functions of disordered regions.

  • 14.
    Ali, Muhammad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Khramushin, Alisa
    Hebrew Univ Jerusalem, Inst Med Res Israel Canada, Fac Med, Dept Microbiol & Mol Genet, IL-9112102 Jerusalem, Israel.
    Yadav, Vikash K.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Derby Univ, Coll Sci & Engn, Derby, England.
    Schueler-Furman, Ora
    Hebrew Univ Jerusalem, Inst Med Res Israel Canada, Fac Med, Dept Microbiol & Mol Genet, IL-9112102 Jerusalem, Israel.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Elucidation of Short Linear Motif-Based Interactions of the FERM Domains of Ezrin, Radixin, Moesin, and Merlin2023In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 62, no 11, p. 1594-1607Article in journal (Refereed)
    Abstract [en]

    The ERM (ezrin, radixin,and moesin) family of proteins and therelated protein merlin participate in scaffolding and signaling eventsat the cell cortex. The proteins share an N-terminal FERM [band four-point-one(4.1) ERM] domain composed of three subdomains (F1, F2, and F3) withbinding sites for short linear peptide motifs. By screening the FERMdomains of the ERMs and merlin against a phage library that displayspeptides representing the intrinsically disordered regions of thehuman proteome, we identified a large number of novel ligands. Wedetermined the affinities for the ERM and merlin FERM domains interactingwith 18 peptides and validated interactions with full-length proteinsthrough pull-down experiments. The majority of the peptides containedan apparent Yx-[FILV] motif; others show alternative motifs. We defineddistinct binding sites for two types of similar but distinct bindingmotifs (YxV and FYDF) using a combination of Rosetta FlexPepDock computationalpeptide docking protocols and mutational analysis. We provide a detailedmolecular understanding of how the two types of peptides with distinctmotifs bind to different sites on the moesin FERM phosphotyrosinebinding-like subdomain and uncover interdependencies between the differenttypes of ligands. The study expands the motif-based interactomes ofthe ERMs and merlin and suggests that the FERM domain acts as a switchableinteraction hub.

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  • 15.
    Ali, Muhammad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    McAuley, Mishal Mariam
    Goethe Univ Frankfurt, Inst Pharmaceut Chem, Max von Laue Str 9, D-60438 Frankfurt, Germany.;Buchmann Inst Mol Life Sci BMLS, Struct Genom Consortium SGC, Max von Laue Str 15, D-60438 Frankfurt, Germany..
    Lüchow, Susanne
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Knapp, Stefan
    Goethe Univ Frankfurt, Inst Pharmaceut Chem, Max von Laue Str 9, D-60438 Frankfurt, Germany.;Buchmann Inst Mol Life Sci BMLS, Struct Genom Consortium SGC, Max von Laue Str 15, D-60438 Frankfurt, Germany..
    Joerger, Andreas C.
    Goethe Univ Frankfurt, Inst Pharmaceut Chem, Max von Laue Str 9, D-60438 Frankfurt, Germany.;Buchmann Inst Mol Life Sci BMLS, Struct Genom Consortium SGC, Max von Laue Str 15, D-60438 Frankfurt, Germany..
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Integrated analysis of Shank1 PDZ interactions with C-terminal and internal binding motifs2021In: Current Research in Structural Biology, E-ISSN 2665-928X, Vol. 3, p. 41-50Article in journal (Refereed)
    Abstract [en]

    PDZ domains constitute a large family of modular domains that are well-known for binding C-terminal motifs of target proteins. Some of them also bind to internal PDZ binding motifs (PDZbms), but this aspect of the PDZ interactome is poorly studied. Here we explored internal PDZbm-mediated interactions using the PDZ domain of Shank1 as a model. We identified a series of human Shank1 ligands with C-terminal or internal PDZbms using proteomic peptide-phage display, and established that while the consensus sequence of C-terminal ligands is x-T-x-(L/F)-COOH, the consensus of internal PDZbm is exclusively x-T-x-F-x, where x is any amino acid. We found that the affinities of PDZbm interactions are in the low micromolar range. The crystal structure of the complex between Shank1 PDZ and an internal PDZbm revealed that the binding mode of internal PDZbms was similar to that of C-terminal ligands. Pull-down experiments confirmed that both C-terminal and internal PDZbm interactions can occur in the context of full-length proteins. Our study expands the interactome of Shank1 and hints at a largely unexplored interaction space of PDZ domains.

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  • 16.
    Allison, Timothy M.
    et al.
    Biomolecular Interaction Centre, School of Physical and Chemical Sciences, University of Canterbury, Christchurch, New Zealand.
    Degiacomi, Matteo T.
    Department of Physics, Durham University, Durham, UK.
    Marklund, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Jovine, Luca
    Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.
    Elofsson, Arne
    Science for Life Laboratory and Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden.
    Benesch, Justin L. P.
    Department of Chemistry, University of Oxford, Oxford, UK.
    Landreh, Michael
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet – Biomedicum, Stockholm, Sweden.
    Complementing machine learning‐based structure predictions with native mass spectrometry2022In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 31, no 6, article id e4333Article in journal (Refereed)
    Abstract [en]

    The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.

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  • 17.
    Al-Smadi, Derar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry. An Najah Natl Univ, Dept Chem, Fac Sci, Nablus, Palestine.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kessler, Vadim
    Swedish Univ Agr Sci, Dept Mol Sci, Box 7015, SE-75007 Uppsala, Sweden.
    Mhasal, Anil Rhanu
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Norberg, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Chemical and Biochemical Approaches for the Synthesis of Substituted Dihydroxybutanones and Di-, and Tri-Hydroxypentanones2019In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 84, no 11, p. 6982-6991Article in journal (Refereed)
    Abstract [en]

    Polyhydroxylated compounds are building blocks for the synthesis of carbohydrates and other natural products. Their synthesis is mainly achieved by different synthetic versions of aldol-coupling reactions, catalyzed either by organocatalysts, enzymes or metal-organic catalysts. We have investigated the formation of 1,4-substituted 2,3-dihydroxybutan-1-one derivatives from para- and meta-substituted phenylacetaldehydes by three distinctly different strategies. The first involved a direct aldol reaction with hydroxyacetone, dihydroxyacetone or 2-hydroxyacetophenone, catalyzed by the cinchona derivative cinchonine. The second was reductive cross-coupling with methyl or phenyl glyoxal promoted by SmI2 resulting in either 5-substituted 3,4-dihydroxypentan-2-ones or 1,4 bis-phenyl substituted butanones, respectively. Finally, in the third case, aldolase catalysis was employed for synthesis of the corresponding 1,3,4-trihydroxylated pentan-2-one derivatives. The organocatalytic route with cinchonine generated distereomerically enriched syn products (de = 60−99 %), with moderate enantiomeric excesses (ee = 43−56%), but did not produce aldols with either hydroxyacetone or dihydroxyacetone as donor ketones. The SmI2-promoted reductive cross-coupling generated product mixtures with diastereomeric and enantiomeric ratios close to unity. This route allowed for the production of both 1-methyl- and 1-phenylsubstituted 2,3-dihydroxybutanones, at yields between 40−60%. Finally, the biocatalytic approach resulted in enantiopure syn (3R,4S) 1,3,4-trihydroxypentan-2-ones.

  • 18.
    al-smadi, Derar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Norberg, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Kessler, Vadim
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    A Comparison of Synthetic Approaches to Derivatives of 1,4-Substituted 2,3 DihydroxybutanonesManuscript (preprint) (Other academic)
  • 19.
    Al-Smadi, Derar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Norberg, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Synthesis of substrates for aldolase-catalyzed reactions: A comparison of methods for the synthesis of substituted phenylacetaldehydes2018In: Synlett: Accounts and Rapid Communications in Synthetic Organic Chemistry, ISSN 0936-5214, E-ISSN 1437-2096, Vol. 29, no 9, p. 1187-1190Article in journal (Refereed)
    Abstract [en]

    Methods for the synthesis of phenylacetaldehydes (oxidation, one-carbon chain extension) were compared by using the synthesis of 4-methoxyphenylacetaldehyde as a model example. Oxidations of 4-methoxyphenylethanol with activated DMSO (Swern oxidation) or manganese dioxide gave unsatisfactory results; whereas oxidation with 2-iodoxybenzoic add (IBX) produced 4-methoxyphenylacetaldehyde in reasonable (75%) yield. However, Wittig-type one-carbon chain extension with methoxymethylene-triphenylphosphine followed by hydrolysis gave an excellent (81% overall) yield of 4-methoxyphenylacetaldehyde from 4-methoxybenzaldehyde (a cheap starting material). This approach was subsequently used to synthesise a set of 10 substituted phenylacetaldehydes in good to excellent yields.

  • 20.
    Amrein, Beat A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Duarte, Fernanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Naworyta, Agata
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C. L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Expanding the catalytic triad in epoxide hydrolases and related enzymes2015In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed)
    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.

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  • 21.
    Amrein, Beat Anton
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Runthala, Ashish
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    In Silico-Directed Evolution Using CADEE2018In: Computational Methods in Protein Evolution / [ed] T. Tobias Sikosek, Springer Science+Business Media, LLC, part of Springer Nature , 2018, p. 381-415Chapter in book (Other academic)
  • 22.
    Aronsson, Hanna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Biophysical Analysis of Novel Viral-Host Protein-Protein Interactions Identified by Proteomic Peptide Phage Display2020Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
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    Hanna_Aronsson_Master_Thesis
  • 23. Bagchi, Basabi
    et al.
    Corbel, Quentin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Animal ecology. University of Valencia.
    Khan, Imroze
    Payne, Ellen
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Animal ecology.
    Banerji, Devshuvam
    Liljestrand-Rönn, Johanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Animal ecology.
    Martinossi-Allibert, Ivain
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Animal ecology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology.
    Baur, Julian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Animal ecology.
    Sayadi, Ahmed
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Animal ecology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Immonen, Elina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology.
    Arnqvist, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Animal ecology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Berger, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Animal ecology.
    Sexual conflict drives micro- and macroevolution of sexual dimorphism in immunity2021In: BMC Biology, E-ISSN 1741-7007, Vol. 19, no 1, article id 114Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Sexual dimorphism in immunity is believed to reflect sex differences in reproductive strategies and trade-offs between competing life history demands. Sexual selection can have major effects on mating rates and sex-specific costs of mating and may thereby influence sex differences in immunity as well as associated host-pathogen dynamics. Yet, experimental evidence linking the mating system to evolved sexual dimorphism in immunity are scarce and the direct effects of mating rate on immunity are not well established. Here, we use transcriptomic analyses, experimental evolution and phylogenetic comparative methods to study the association between the mating system and sexual dimorphism in immunity in seed beetles, where mating causes internal injuries in females.

    RESULTS: We demonstrate that female phenoloxidase (PO) activity, involved in wound healing and defence against parasitic infections, is elevated relative to males. This difference is accompanied by concomitant sex differences in the expression of genes in the prophenoloxidase activating cascade. We document substantial phenotypic plasticity in female PO activity in response to mating and show that experimental evolution under enforced monogamy (resulting in low remating rates and reduced sexual conflict relative to natural polygamy) rapidly decreases female (but not male) PO activity. Moreover, monogamous females had evolved increased tolerance to bacterial infection unrelated to mating, implying that female responses to costly mating may trade off with other aspects of immune defence, an hypothesis which broadly accords with the documented sex differences in gene expression. Finally, female (but not male) PO activity shows correlated evolution with the perceived harmfulness of male genitalia across 12 species of seed beetles, suggesting that sexual conflict has a significant influence on sexual dimorphisms in immunity in this group of insects.

    CONCLUSIONS: Our study provides insights into the links between sexual conflict and sexual dimorphism in immunity and suggests that selection pressures moulded by mating interactions can lead to a sex-specific mosaic of immune responses with important implications for host-pathogen dynamics in sexually reproducing organisms.

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  • 24. Barreca, Maria Letizia
    et al.
    Manfroni, Giuseppe
    Leyssen, Pieter
    Winquist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Kaushik-Basu, Neerja
    Paeshuyse, Jan
    Krishnan, Ramalingam
    Iraci, Nunzio
    Sabatini, Stefano
    Tabarrini, Oriana
    Basu, Amartya
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Neyts, Johan
    Cecchetti, Violetta
    Structure-Based Discovery of Pyrazolobenzothiazine Derivatives As Inhibitors of Hepatitis C Virus Replication2013In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 56, no 6, p. 2270-2282Article in journal (Refereed)
    Abstract [en]

    The NS5B RNA-dependent RNA polymerase is an attractive target for the development of novel and selective inhibitors of hepatitis C virus replication. To identify novel structural hits as anti-HCV agents, we performed structure based virtual screening of our in-house library followed by rational drug design, organic synthesis, and biological testing. These studies led to the identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel anti-HCV agents targeting the NS5B polymerase. The best compound of this series was the meta-fluoro-N-1-phenyl pyrazolobenzothiazine derivative 4a, which exhibited an EC50 = 3.6 mu M, EC90 = 25.6 mu M, and CC50 > 180 mu M in the Huh 9-13 replicon system, thus providing a good starting point for further hit evolution.

  • 25.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Computational modelling of enzyme selectivity2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enantioselective reactions are one of the ways to produce pure chiral compounds. Understanding the basis of this selectivity makes it possible to guide enzyme design towards more efficient catalysts. One approach to study enzymes involved in chiral chemistry is through the use of computational models that are able to simulate the chemical reaction taking place. The potato epoxide hydrolase is one enzyme that is known to be both highly enantioselective, while still being robust upon mutation of residues to change substrate scope. The enzyme was used to investigate the epoxide hydrolysis mechanism for a number of different substrates, using the EVB approach to the reaction both in solution and in several enzyme variants. In addition to this, work has been performed on new ways of performing simulations of divalent transition metals, as well as development of new simulation software.

    List of papers
    1. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Open this publication in new window or tab >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
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    2014 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 16, p. 4351-4362Article in journal (Refereed) 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.

    National Category
    Physical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
    Funder
    Swedish National Infrastructure for Computing (SNIC), 2013/26-1
    Available from: 2014-06-23 Created: 2014-06-04 Last updated: 2018-12-03Bibliographically approved
    2. Expanding the catalytic triad in epoxide hydrolases and related enzymes
    Open this publication in new window or tab >>Expanding the catalytic triad in epoxide hydrolases and related enzymes
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    2015 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed) 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.

    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-260232 (URN)10.1021/acscatal.5b01639 (DOI)000362391500006 ()
    Funder
    EU, FP7, Seventh Framework Programme, 306474Swedish Research Council, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12
    Available from: 2015-08-18 Created: 2015-08-18 Last updated: 2017-12-04Bibliographically approved
    3. Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
    Open this publication in new window or tab >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
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    2016 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed) 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.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
    Funder
    Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, European Research Council, 306474;283570Swedish Research Council, 621-2011-6055Carl Tryggers foundation , CTS13:104
    Available from: 2016-04-01 Created: 2016-04-01 Last updated: 2017-11-30Bibliographically approved
    4. Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis
    Open this publication in new window or tab >>Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis
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    2016 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 18, p. 1693-1697Article in journal (Refereed) Published
    Abstract [en]

    Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)-3-phenylpropane-1,2-diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)-diol is not only due to changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)-benzyloxirane. To probe the structural origin of these differences in substrate selectivities and catalytic regiopreferences, we have solved the crystal structures for the in-vitro evolved StEH1 variants. We have additionally used these structures as a starting point for docking the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking calculations, the apparent preferred binding modes obtained from the docking appears to rationalize the experimentally determined regioselectivities. These calculations could also identify an active site residue (F33) as a putatively important interaction partner, a role that could explain the high degree of conservation of this residue during evolution. Overall, our combined experimental, structural and computational studies of this system provide snapshots into the evolution of enantioconvergence in StEH1 catalyzed epoxide hydrolysis.

    Keywords
    enantioselectivity; epoxide hydrolysis; evolutionary snapshots; laboratory evolution; protein engineering
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-298675 (URN)10.1002/cbic.201600330 (DOI)000384425400004 ()27383542 (PubMedID)
    Funder
    Swedish Research CouncilEU, European Research Council, 306474Swedish National Infrastructure for Computing (SNIC), SNIC2015-16-12EU, FP7, Seventh Framework Programme, 283570
    Available from: 2016-07-06 Created: 2016-07-06 Last updated: 2022-01-29Bibliographically approved
    5. Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
    Open this publication in new window or tab >>Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
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    2018 (English)In: IUCrJ, E-ISSN 2052-2525, Vol. 5, no 3, p. 269-282Article in journal (Refereed) Published
    Abstract [en]

    The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenyl­propane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure–activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.

    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-343750 (URN)10.1107/S2052252518003573 (DOI)000431151300004 ()29755743 (PubMedID)
    Funder
    Swedish Research CouncilEU, FP7, Seventh Framework Programme
    Available from: 2018-03-01 Created: 2018-03-01 Last updated: 2022-09-28Bibliographically approved
    6. Q6: A comprehensive toolkit for empirical valence bond and related free energy calculations
    Open this publication in new window or tab >>Q6: A comprehensive toolkit for empirical valence bond and related free energy calculations
    Show others...
    2018 (English)In: SoftwareX, E-ISSN 2352-7110, Vol. 7, p. 388-395Article in journal (Refereed) Published
    Abstract [en]

    Atomistic simulations have become one of the main approaches to study the chemistry and dynamicsof biomolecular systems in solution. Chemical modelling is a powerful way to understand biochemistry,with a number of different programs available to perform specialized calculations. We present here Q6, anew version of the Q software package, which is a generalized package for empirical valence bond, linearinteraction energy, and other free energy calculations. In addition to general technical improvements, Q6extends the reach of the EVB implementation to fast approximations of quantum effects, extended solventdescriptions and quick estimation of the contributions of individual residues to changes in the activationfree energy of reactions.

    Place, publisher, year, edition, pages
    Elsevier, 2018
    National Category
    Software Engineering
    Identifiers
    urn:nbn:se:uu:diva-360517 (URN)10.1016/j.softx.2017.12.001 (DOI)000457139300064 ()
    Funder
    Swedish Research Council, 2014-3688Swedish Research Council, 2014-2118Swedish Research Council, 2015-04928
    Note

    Title in the list of papers of Paul Bauer's thesis: Q Version 6, a comprehensive toolkit for empirical valence bond and related free energy calculations

    Available from: 2018-09-14 Created: 2018-09-14 Last updated: 2024-06-12Bibliographically approved
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    errata
  • 26.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Directed Evolution of ADH-A from Rhodococcus ruber DSM 445412014Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Download full text (pdf)
    fulltext
  • 27.
    Bauer, Paul
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Barrozo, Alexandre
    Department of Chemistry, University of Southern California, SGM 418, 3620 McClintock Ave., Los Angeles, CA 90089-1062, United StatesDepartment of Chemistry, University of Southern California, SGM 418, 3620 McClintock Ave., Los Angeles, CA 90089-1062, United States.
    Purg, Miha
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Amrein, Beat Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Esguerra, Mauricio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Wilson, Philippe Barrie
    Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, UK.
    Major, Dan Thomas
    Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Q6: A comprehensive toolkit for empirical valence bond and related free energy calculations2018In: SoftwareX, E-ISSN 2352-7110, Vol. 7, p. 388-395Article in journal (Refereed)
    Abstract [en]

    Atomistic simulations have become one of the main approaches to study the chemistry and dynamicsof biomolecular systems in solution. Chemical modelling is a powerful way to understand biochemistry,with a number of different programs available to perform specialized calculations. We present here Q6, anew version of the Q software package, which is a generalized package for empirical valence bond, linearinteraction energy, and other free energy calculations. In addition to general technical improvements, Q6extends the reach of the EVB implementation to fast approximations of quantum effects, extended solventdescriptions and quick estimation of the contributions of individual residues to changes in the activationfree energy of reactions.

    Download full text (pdf)
    fulltext
  • 28.
    Bauer, Paul
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Amrein, Beat A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, S. C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 12016In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed)
    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.

    Download full text (pdf)
    fulltext
  • 29.
    Belfrage, Anna Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Åkerblom, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Alogheli, Hiba
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Neyts, Johan
    Rega Institute, Department of Microbiology and Immunology, University of Leuven, B-3000 Leuven, Belgium.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Johansson, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Pan-NS3 protease inhibitors of hepatitis C virus based on an R3-elongated pyrazinone scaffold2018In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 148, p. 453-464Article in journal (Refereed)
    Abstract [en]

    Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors and show that elongated R-3 urea substituents were associated with increased inhibitory potencies over several NS3 protein variants. The inhibitors are believed to rely on beta-sheet mimicking hydrogen bonds which are similar over different genotypes and current drug resistant variants and correspond to the beta-sheet interactions of the natural peptide substrate. Inhibitor 36, for example, with a urea substituent including a cyclic imide showed balanced nanomolar inhibitory potencies against genotype la, both wild-type (K-i=30 nM) and R155K (K-i=2 nM), and genotype 3a (K-i=5 nM).

  • 30.
    Belfrage, Anna Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Åkerblom, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Oshalim, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Skogh, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Neyts, Johan
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Sandström, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease2016In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 24, no 12, p. 2603-2620Article in journal (Refereed)
    Abstract [en]

    Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wildtype and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance.

  • 31.
    Ben-David, Moshe
    et al.
    Weizmann Inst Sci, Rehovot, Israel; Ukko Ltd, HaMada 12, Rehovot, Israel.
    Soskine, Misha
    Weizmann Inst Sci, Rehovot, Israel; DNA Script, 29 Rue Faubourg St Jacques, Paris, France.
    Dubovetskyi, Artem
    Weizmann Inst Sci, Rehovot, Israel.
    Cherukuri, Kesava-Phaneendra
    Weizmann Inst Sci, Rehovot, Israel.
    Dym, Orly
    Weizmann Inst Sci, Rehovot, Israel.
    Sussman, Joel L
    Weizmann Inst Sci, Rehovot, Israel.
    Liao, Qinghua
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Szeler, Klaudia
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C. Lynn
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Tawfik, Dan S
    Weizmann Inst Sci, Rehovot, Israel.
    Enzyme Evolution An Epistatic Ratchet versus a Smooth Reversible Transition2020In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 37, no 4, p. 1133-1147Article in journal (Refereed)
    Abstract [en]

    Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.

  • 32.
    Benz, Caroline
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Diving into short linear motifs: Large-scale identification of endogenous and host-pathogen protein-protein interactions and further characterized by deep mutational scanning2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Short linear motifs (SLiMs) are protein-protein interaction sites that play an essential role in distinct cellular processes. Those interactions are challenging to capture by common high-throughput methods. Therefore, we established an improved version of Proteomic Peptide Phage Display (ProP-PD) as a dedicated method to identify SLiM-based interactions. ProP-PD libraries were created for the discovery of endogenous and host-pathogen protein-protein interactions. The M13 bacteriophage libraries present 16 amino acid long peptides from the intrinsically disordered regions (IDRs) of the human (HD2) proteome or the proteomes of RNA viruses (RiboVD). Through benchmarking of the approach using 35 well-known SLiMs binding domains and the HD2 library, we defined parameters for assigning confidence levels to the results. The selections against the HD2 library revealed >2000 SLiMs-based interaction pairs. Regarding host-pathogen interactions, we focused on interactions mediated by coronavirus proteins, exploring how human proteins bind to viral peptides and how viral proteins bind to human SLiMs. By screening more than 130 human bait proteins against the RiboVD, we revealed several host proteins potentially being targeted by SARS-CoV-2 proteins. Viral hijacking of human G3BP1/2 by the N-protein from SARS-CoV-2 impacted stress granule formation, and inhibition of the interaction was found to have an antiviral effect. Using SARS-CoV-2 proteins in selections with our HD2 library, we found that viral proteins may bind host SLiMs. Selected interactions were validated via affinity measurements revealing a wide range of affinities. Finally, we uncovered that a peptide binding to the NSP9 has an antiviral effect. It is not always possible to establish binding determinants directly from ProP-PD derived peptides. Therefore, we developed a deep mutational scanning (DMS) by phage display protocol. To test the approach, we designed libraries in which all amino acid positions of binding peptides were individually mutated, and the effect on binding was investigated through peptide phage selection. The approach was validated against well-studied interactions and applied to SLiM-based interactions between human proteins and SARS-CoV-2 proteins. Based on the DMS by phage display data we could create a higher affinity binder for NSP9 with increased antiviral effects. The research presented in this thesis has established a platform for large-scale interaction screening through phage display. The results contribute to a deeper understanding of the SLiMs binding and function and also pinpoint novel potential targets for the development of antiviral agents.

    List of papers
    1. Proteome-scale mapping of binding sites in the unstructured regions of the human proteome
    Open this publication in new window or tab >>Proteome-scale mapping of binding sites in the unstructured regions of the human proteome
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    2022 (English)In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 18, no 1, article id e10584Article in journal (Refereed) Published
    Abstract [en]

    Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type-specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide-phage display library that tiles all disordered regions of the human proteome and allows the screening of similar to 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)-binding domains and confirmed the quality of the produced data by complementary biophysical or cell-based assays. Finally, we show how the amino acid resolution-binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteomewide discovery of SLiM-based interactions.

    Place, publisher, year, edition, pages
    EMBO PressEMBO, 2022
    Keywords
    intrinsically disordered regions, peptides, phage display, protein-protein interactions, short linear motifs
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-467370 (URN)10.15252/msb.202110584 (DOI)000749669200001 ()35044719 (PubMedID)
    Funder
    Swedish Foundation for Strategic Research , SB16-0039Swedish Research Council, 2016-04965Swedish Research Council, 2020-03380Swedish Research Council, 2016-04134Swedish Research Council, 2020-04395Knut and Alice Wallenberg Foundation
    Note

    De tre första författarna delar förstaförfattarskapet.

    Available from: 2022-02-14 Created: 2022-02-14 Last updated: 2024-01-15Bibliographically approved
    2. Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities
    Open this publication in new window or tab >>Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities
    Show others...
    2021 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 12, no 1, article id 6761Article in journal (Refereed) Published
    Abstract [en]

    Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, most current large-scale methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a viral peptide discovery approach covering 23 coronavirus strains that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an ΦxFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its ΦxFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction dampened SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.

    Place, publisher, year, edition, pages
    Springer Nature, 2021
    Keywords
    General Physics and Astronomy, General Biochemistry, Genetics and Molecular Biology, General Chemistry
    National Category
    Infectious Medicine Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-460518 (URN)10.1038/s41467-021-26498-z (DOI)000720682600011 ()34799561 (PubMedID)
    Funder
    Swedish Research Council, 2020-03380Swedish Research Council, 2020-04395Swedish Research Council, 2017-02438Swedish Research Council, 2018-05851Swedish Foundation for Strategic Research , SB16-0039
    Available from: 2021-12-07 Created: 2021-12-07 Last updated: 2024-01-15Bibliographically approved
    3. Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets
    Open this publication in new window or tab >>Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets
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    2023 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1, article id 5636Article in journal (Refereed) Published
    Abstract [en]

    The virus life cycle depends on host-virus protein-protein interactions, which often involve a disordered protein region binding to a folded protein domain. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind 281 peptides from human proteins, and affinities of 31 interactions involving eight SARS-CoV-2 protein domains were determined (KD ∼ 7-300 μM). Key specificity residues of the peptides were established for six of the interactions. Two of the peptides, binding Nsp9 and Nsp16, respectively, inhibited viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously identify potential host-virus interactions and peptides with antiviral properties. Furthermore, the high number of low-affinity interactions suggest that overexpression of viral proteins during infection may perturb multiple cellular pathways.

    Place, publisher, year, edition, pages
    Springer Nature, 2023
    Keywords
    SARS-CoV-2, large scale discovery, phage display, Short linear motifs
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Medical Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-486758 (URN)10.1038/s41467-023-41312-8 (DOI)001087583700014 ()37704626 (PubMedID)
    Funder
    Swedish Foundation for Strategic Research, SB16-0039Swedish Research Council, 2020-03380Swedish Research Council, 2020-04395Swedish Research Council, 2018-05851Knut and Alice Wallenberg Foundation, KAW 2020.0241Knut and Alice Wallenberg Foundation, V-2020-0699Uppsala UniversityUmeå University
    Available from: 2022-10-16 Created: 2022-10-16 Last updated: 2023-11-22Bibliographically approved
    4. Parallel exploration of short linear motif-based interactions using deep mutational scanning by phage display
    Open this publication in new window or tab >>Parallel exploration of short linear motif-based interactions using deep mutational scanning by phage display
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-500375 (URN)
    Available from: 2023-04-15 Created: 2023-04-15 Last updated: 2023-04-26
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  • 33.
    Benz, Caroline
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ali, Muhammad
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Krystkowiak, Izabella
    Inst Canc Res, Div Canc Biol, London, England..
    Simonetti, Leandro
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Sayadi, Ahmed
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Mihalic, Filip
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kliche, Johanna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Andersson, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Davey, Norman E.
    Inst Canc Res, Div Canc Biol, London, England..
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Proteome-scale mapping of binding sites in the unstructured regions of the human proteome2022In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 18, no 1, article id e10584Article in journal (Refereed)
    Abstract [en]

    Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type-specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide-phage display library that tiles all disordered regions of the human proteome and allows the screening of similar to 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)-binding domains and confirmed the quality of the produced data by complementary biophysical or cell-based assays. Finally, we show how the amino acid resolution-binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteomewide discovery of SLiM-based interactions.

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  • 34.
    Berglin, Lennart
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Kjellander, Marcus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    In situ generation of iminodiacetic acid groups on nanoporous alumina for the reversible immobilization of enzymes and other biomolecules2014In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 36, no 9, p. 1819-1825Article in journal (Refereed)
    Abstract [en]

    Nanoporous alumina membranes were silanized with aminopropylsilane and iminodiacetic acid (IDA) groups were generated in situ by reaction with iodoacetate. The membranes were mounted in standard filter holders, connected to a HPLC system and saturated with selected metal ions. Cu(II) allowed the capture of chicken muscle lactate dehydrogenase with such stability, repeatability and reproducibility that Michaelis-Menten kinetics could be studied. The IDA surface was stable for months and could be depleted and regenerated with metal ions multiple times without appreciable loss of capacity. The binding of lactate dehydrogenase influenced the backpressure to the extent that could be expected for a monolayer according to Poiseuilles law.

  • 35.
    Bhaduri, Anindya
    et al.
    Johns Hopkins Univ, Dept Civil Engn, Baltimore, MD 21218 USA.
    Gardner, Jasmine
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Abrams, Cameron F.
    Drexel Univ, Dept Chem & Biol Engn, Philadelphia, PA 19104 USA.
    Graham-Brady, Lori
    Johns Hopkins Univ, Dept Civil Engn, Baltimore, MD 21218 USA.
    Free energy calculation using space filled design and weighted reconstruction: a modified single sweep approach2020In: Molecular Simulation, ISSN 0892-7022, E-ISSN 1029-0435, Vol. 46, no 3, p. 193-206Article in journal (Refereed)
    Abstract [en]

    A modified single sweep approach is proposed for generating free energy landscapes. The approach replaces the use of temperature-accelerated molecular dynamics (TAMD) to generate centres in collective variable (CV) space at which mean forces are computed using restrained molecular dynamics (MD) simulations with a sequential space-filling design. This approach also modifies the radial basis function reconstruction step of the traditional single sweep approach and proposes a weighted reconstruction of the free energy surface using the previously generated mean forces. The modified approach is compared to the traditional single sweep (SS) approach on the (phi, psi) dihedral free-energy map of solvated alanine dipeptide (AD). It is found that the new approach results in a more accurate reconstructed free energy than does the traditional approach when compared to the directly-computed reference free energy landscape. It is shown that the increased accuracy of the overall map stems from the improved 1-dimensional space filling (projective) property of the proposed design compared to that of the TAMD generated centres. A further enhancement in the accuracy of the crucial lower energy regions is enabled by the introduction of weights in the reconstruction step that give more importance to lower energy-valued regions.

  • 36.
    Biler, Michal
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Crean, Rory M.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Schweiger, Anna K.
    Graz Univ Technol, Inst Mol Biotechnol, A-8010 Graz, Austria..
    Kourist, Robert
    Graz Univ Technol, Inst Mol Biotechnol, A-8010 Graz, Austria..
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ground-State Destabilization by Active-Site Hydrophobicity Controls the Selectivity of a Cofactor-Free Decarboxylase2020In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 142, no 47, p. 20216-20231Article in journal (Refereed)
    Abstract [en]

    Bacterial arylmalonate decarboxylase (AMDase) and evolved variants have become a valuable tool with which to access both enantiomers of a broad range of chiral arylaliphatic acids with high optical purity. Yet, the molecular principles responsible for the substrate scope, activity, and selectivity of this enzyme are only poorly understood to date, greatly hampering the predictability and design of improved enzyme variants for specific applications. In this work, empirical valence bond and metadynamics simulations were performed on wild-type AMDase and variants thereof to obtain a better understanding of the underlying molecular processes determining reaction outcome. Our results clearly reproduce the experimentally observed substrate scope and support a mechanism driven by ground-state destabilization of the carboxylate group being cleaved by the enzyme. In addition, our results indicate that, in the case of the nonconverted or poorly converted substrates studied in this work, increased solvent exposure of the active site upon binding of these substrates can disturb the vulnerable network of interactions responsible for facilitating the AMDase-catalyzed cleavage of CO2. Finally, our results indicate a switch from preferential cleavage of the pro-(R) to the pro-(S) carboxylate group in the CLG-IPL variant of AMDase for all substrates studied. This appears to be due to the emergence of a new hydrophobic pocket generated by the insertion of the six amino acid substitutions, into which the pro-(S) carboxylate binds. Our results allow insight into the tight interaction network determining AMDase selectivity, which in turn provides guidance for the identification of target residues for future enzyme engineering.

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  • 37.
    Billinger, Erika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kinetic studies of serine protease inhibitors in simple and rapid 'active barrier' model systems: Diffusion through an inhibitor barrier2018In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 546, p. 43-49Article in journal (Refereed)
    Abstract [en]

    A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top "protective" layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer.

  • 38.
    Billinger, Erika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Viljanen, Johan V.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Lind Bergström, Sara
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Inhibition properties of free and conjugated leupeptin analogues2020In: Vol. 10, no 12Article, review/survey (Other academic)
    Abstract [en]

    Leupeptin is a naturally occurring inhibitor of various proteases, in particular serine proteases. Following its discovery, the inhibitory properties of several other peptidyl argininals have been studied. The specificity of leupeptin is most likely due to the Leu-Leu-Argininal sequence, and its C-terminal aldehyde group has been suggested to enhance the binding efficiency and to be essential for function. The terminal aldehyde group makes the structure less vulnerable to carboxypeptidases. Here, we investigated whether the inhibitory function of leupeptin toward serine proteases is retained after oxidation or reduction of the aldehyde group. The oxidized form, which corresponds to the natural precursor, was shown to be superior to the reduced form in terms of inhibitory properties. However, the original leupeptin possessed enhanced inhibitory properties as compared with the oxidized form. Based on these results, new synthetic leupeptin analogues, 6-aminohexanoic acid (Ahx)-Phe-Leu-Arg-COOH and Ahx-Leu-Leu-Arg-COOH, were prepared by solid-phase peptide synthesis using the Fmoc strategy. In these analogues, the N-terminal capping acetyl group was replaced with a 6-aminohexanoyl group to allow conjugation. The structures of the modified leupeptin and the synthetic peptides were confirmed by mass spectrometry. Determination of the inhibitory properties against trypsin (IEC 3.4.21.4, Chymotrypsin IEC 3.4.21.1) revealed that these further modified tripeptides were tight binding inhibitors to their target enzyme, similar to the naturally occurring leupeptin, with Ki values generally in the micromolar range. The Ahx-Phe-Leu-Arg-COOH analogue was selected for conjugation to inorganic oxide nanoparticles and agarose gel beads. All conjugates exhibited inhibitory activity in the same range as for the free peptides. 

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  • 39.
    Billinger, Erika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Zuo, Shusheng
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Characterization of Serine Protease Inhibitor from Solanum tuberosum Conjugated to Soluble Dextran and Particle Carriers2019In: ACS Omega, E-ISSN 2470-1343, Vol. 4, no 19, p. 18456-18464Article, review/survey (Refereed)
    Abstract [en]

    A serine protease inhibitor was extracted from potato tubers. The inhibitor was conjugated to soluble, prefractionated dextran and titanium dioxide and zinc oxide nanoparticles. Conjugation to dextran was achieved by periodate oxidation of the dextran, followed by Schiff base coupling to inhibitor amino groups, and finally reduction, whereas the conjugation to the oxide particles was carried out by aminosilanization and carbonyldiimidazole activation. The inhibitory effect of the conjugated inhibitor was compared to that of free inhibitor in solution and with gelatin gel as a direct substrate. A certain degree of inhibitory activity was retained for both the dextran-conjugated and particle-conjugated inhibitors. In particular, the apparent Ki value of the dextran-conjugated inhibitor was found to be in the same range as that for free inhibitor. The dextran conjugate retained a higher activity than the free inhibitor after 1 month of storage at room temperature.

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  • 40.
    Billinger, Erika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Zuo, Shusheng
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Lundmark, Kristoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Light scattering determination of the stoichiometry for protease-potato serine protease inhibitor complexes2019In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 582, article id 113357Article in journal (Refereed)
    Abstract [en]

    The interaction between pancreatic proteases and a serine protease inhibitor purified from potato tubers was investigated by chromatography-coupled light scattering measurements. The molar mass distribution in the chromatogram was compared to theoretical values calculated for the different possible combinations of complexes and free components by three different approaches, namely section analyses of the chromatograms, full mass average determination and mass distribution analysis. This revealed that the inhibitor was able to bind trypsin in a 2:1 complex, whereas the data for chymotrypsin clearly showed a limitation to 1:1 complex regardless of the molar ratio in the injected samples. The same experiment carried out with elastase and the potato inhibitor gave only weak indications of complex formation under the conditions used.

  • 41.
    Binti Shamsudin, Yasmin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Department of Chemistry, Stanford University, Stanford, CA, 94305, USA.
    Walker, Alice R.
    Jones, Chey M.
    Martínez, Todd J.
    Boxer, Steven G.
    Simulation-guided engineering of split GFPs with efficient β-strand photodissociation2023In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1, article id 7401Article in journal (Refereed)
    Abstract [en]

    Green fluorescent proteins (GFPs) are ubiquitous for protein tagging and live-cell imaging. Split-GFPs are widely used to study protein-protein interactions by fusing proteins of interest to split GFP fragments that create a fluorophore upon typically irreversible complementation. Thus, controlled dissociation of the fragments is desirable. Although we have found that split strands can be photodissociated, the quantum efficiency of light-induced photodissociation of split GFPs is low. Traditional protein engineering approaches to increase efficiency, including extensive mutagenesis and screening, have proved difficult to implement. To reduce the search space, key states in the dissociation process are modeled by combining classical and enhanced sampling molecular dynamics with QM/MM calculations, enabling the rational design and engineering of split GFPs with up to 20-fold faster photodissociation rates using non-intuitive amino acid changes. This demonstrates the feasibility of modeling complex molecular processes using state-of-the-art computational methods, and the potential of integrating computational methods to increase the success rate in protein engineering projects.

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  • 42. Blankenship, Connor M.
    et al.
    Xie, Jinshan
    Benz, Caroline
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Wang, Ao
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Jiang, Jiaoyang
    Motif-dependent binding on the intervening domain regulates O-GlcNAc transferase2023In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 19, no 11, p. 1423-1431Article in journal (Refereed)
    Abstract [en]

    The modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) moieties is a highly dynamic process that spatiotemporally regulates nearly every important cellular program. Despite its significance, little is known about the substrate recognition and regulation modes of O-GlcNAc transferase (OGT), the primary enzyme responsible for O-GlcNAc addition. In this study, we identified the intervening domain (Int-D), a poorly understood protein fold found only in metazoan OGTs, as a specific regulator of OGT protein–protein interactions and substrate modification. Using proteomic peptide phage display (ProP-PD) coupled with structural, biochemical and cellular characterizations, we discovered a strongly enriched peptide motif, employed by the Int-D to facilitate specific O-GlcNAcylation. We further show that disruption of Int-D binding dysregulates important cellular programs, including response to nutrient deprivation and glucose metabolism. These findings illustrate a mode of OGT substrate recognition and offer key insights into the biological roles of this unique domain.

  • 43.
    Blazic, Marija
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Gautier, Candice
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Norberg, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    High-throughput selection of (new) enzymes: phage display-mediated isolation of alkyl halide hydrolases from a library of active-site mutated epoxide hydrolases2024In: Faraday discussions, ISSN 1359-6640, E-ISSN 1364-5498Article in journal (Refereed)
    Abstract [en]

    Epoxide hydrolase StEH1, from potato, is similar in overall structural fold and catalytic mechanism to haloalkane dehalogenase DhlA from Xanthobacter autotrophicus. StEH1 displays low (promiscuous) hydrolytic activity with (2-chloro)- and (2-bromo)ethanebenzene producing 2-phenylethanol. To investigate possibilities to amplify these very low dehalogenase activities, StEH1 was subjected to targeted randomized mutagenesis at five active-site amino acid residues and the resulting protein library was challenged for reactivity towards a bait chloride substrate. Enzymes catalyzing the first half-reaction of a hydrolytic cycle were isolated following monovalent phage display of the mutated proteins. Several StEH1 derived enzymes were identified with enhanced dehalogenase activities.

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  • 44.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dahlström, Kärthe M.
    Åbo Akademi.
    Salminen, Tiina A.
    Åbo Akademi.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Stereoselective oxidation of aryl-substituted vicinal diols into chiral α-hydroxy aldehydes by re-engineered propanediol oxidoreductase2013In: ACS Catalysis, E-ISSN 2155-5435, Vol. 3, no 12, p. 3016-3025Article in journal (Refereed)
    Abstract [en]

    α-Hydroxy aldehydes are chiral building blocks used in synthesis of natural products and synthetic drugs. One route to their production is by regioselective oxidation of vicinal diols and, in this work, we aimed to perform the oxidation of 3-phenyl-1,2-propanediol into the corresponding α‑hydroxy aldehyde applying enzyme catalysis. Propanediol oxidoreductase from E. coli efficiently catalyzes the stereoselective oxidation of S-1,2-propanediol into S-lactaldehyde. The enzyme, however, shows no detectable activity with aryl-substituted or other bulky alcohols. We conducted ISM-driven directed evolution on FucO and were able to isolate several mutants that were active with S-3-phenyl-1,2-propanediol. The most efficient variant displayed a kcat/KM of 40 s-1M-1 and the most enantioselective variant an E-value (S/R) of 80. Furthermore, other isolated variants showed up to 4400-fold increased activity with another bulky substrate, phenylacetaldehyde. The results with engineered propanediol oxidoreductases identified amino acids important for substrate selectivity and asymmetric synthesis of aryl-substituted α-hydroxy aldehydes. In conclusion, our study demonstrates the feasibility of tailoring the catalytic properties of propanediol oxidoreductase for biocatalytic properties.

  • 45.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dahlström, Käthe
    Salminen, Tiina
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Substrate scope and selectivity in offspring to an enzyme subjected to directed evolution2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 10, p. 2387-2398Article in journal (Refereed)
    Abstract [en]

    We have analyzed the effects of mutations inserted during directed evolution of a specialized enzyme, Escherichia coli S-1,2-propanediol oxidoreductase (FucO). The kinetic properties of evolved variants have been determined and the observed differences have been rationalized by modeling the tertiary structures of isolated variants and the wild-type enzyme. The native substrate, S-1,2-propanediol, as well as phenylacetaldehyde and 2S-3-phenylpropane-1,2-diol, which are new substrates accepted by isolated variants, were docked into the active sites. The study provides a comprehensive picture of how acquired catalytic properties have arisen via an intermediate generalist enzyme, which had acquired a single mutation (L259V) in the active site. Further mutagenesis of this generalist resulted in a new specialist catalyst. We have also been able to relate the native enzyme activities to the evolved ones and linked the differences to individual amino acid residues important for activity and selectivity. F254 plays a dual role in the enzyme function. First, mutation of F254 into an isoleucine weakens the interactions with the coenzyme thereby increasing its dissociation rate from the active site and resulting in a four-fold increase in turnover number with S-1,2-propanediol. Second, F254 is directly involved in binding of aryl-substituted substrates via π–π interactions. On the other hand, N151 is critical in determining the substrate scope since the side chain amide group stabilizes binding of 1,2-substituted diols and is apparently necessary for enzymatic activity with these substrates. Moreover, the side chain of N151 introduces steric hindrance, which prevents high activity with phenylacetaldehyde. Additionally, the hydroxyl group of T149 is required to maintain the catalytically important hydrogen bonding network.

  • 46.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    High-throughput methods for identification of protein-protein interactions involving short linear motifs2015In: Cell Communication and Signaling, E-ISSN 1478-811X, Vol. 13, article id 38Article, review/survey (Refereed)
    Abstract [en]

    Interactions between modular domains and short linear motifs (3-10 amino acids peptide stretches) are crucial for cell signaling. The motifs typically reside in the disordered regions of the proteome and the interactions are often transient, allowing for rapid changes in response to changing stimuli. The properties that make domain-motif interactions suitable for cell signaling also make them difficult to capture experimentally and they are therefore largely underrepresented in the known protein-protein interaction networks. Most of the knowledge on domain-motif interactions is derived from low-throughput studies, although there exist dedicated high-throughput methods for the identification of domain-motif interactions. The methods include arrays of peptides or proteins, display of peptides on phage or yeast, and yeast-two-hybrid experiments. We here provide a survey of scalable methods for domain-motif interaction profiling. These methods have frequently been applied to a limited number of ubiquitous domain families. It is now time to apply them to a broader set of peptide binding proteins, to provide a comprehensive picture of the linear motifs in the human proteome and to link them to their potential binding partners. Despite the plethora of methods, it is still a challenge for most approaches to identify interactions that rely on post-translational modification or context dependent or conditional interactions, suggesting directions for further method development.

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  • 47.
    Bonagas, Nadilly
    et al.
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Gustafsson, Nina M. S.
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Henriksson, Martin
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Marttila, Petra
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Gustafsson, Robert
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden..
    Wiita, Elisee
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Borhade, Sanjay
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Green, Alanna C.
    Univ Sheffield, Med Sch, Dept Oncol & Metab, Weston Pk Canc Ctr, Sheffield, S Yorkshire, England..
    Vallin, Karl S. A.
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Sarno, Antonio
    Norwegian Univ Sci & Technol, Dept Canc Res & Mol Med, Trondheim, Norway..
    Svensson, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gokturk, Camilla
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Pham, Therese
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Jemth, Ann-Sofie
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Loseva, Olga
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Cookson, Victoria
    Univ Sheffield, Med Sch, Dept Oncol & Metab, Weston Pk Canc Ctr, Sheffield, S Yorkshire, England..
    Kiweler, Nicole
    Luxembourg Inst Hlth, Dept Oncol, Canc Metab Grp, Luxembourg, Luxembourg..
    Sandberg, Lars
    Stockholm Univ, Dept Organ Chem, Sci Life Lab, Drug Discovery & Dev Platform, Solna, Sweden..
    Rasti, Azita
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Unterlass, Judith E.
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Haraldsson, Martin
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Drug Discovery & Dev Platform, Solna, Sweden..
    Andersson, Yasmin
    Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Sci Life Lab, Drug Discovery & Dev Platform, Solna, Sweden..
    Scaletti, Emma R.
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.;Lund Univ, Dept Expt Med Sci, Lund, Sweden..
    Bengtsson, Christoffer
    Stockholm Univ, Dept Organ Chem, Sci Life Lab, Drug Discovery & Dev Platform, Solna, Sweden..
    Paulin, Cynthia B. J.
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Sanjiv, Kumar
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pudelko, Linda
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Kunz, Ben
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Desroses, Matthieu
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Iliev, Petar
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Farnegardh, Katarina
    Stockholm Univ, Dept Organ Chem, Sci Life Lab, Drug Discovery & Dev Platform, Solna, Sweden..
    Kramer, Andreas
    Goethe Univ, Inst Pharmaceut Chem, Frankfurt, Germany..
    Garg, Neeraj
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Michel, Maurice
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Haggblad, Sara
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Biochem & Cellular Screening Facil, Solna, Sweden..
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kalderen, Christina
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Jensen, Amanda Bogedahl
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Almlof, Ingrid
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Karsten, Stella
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Zhang, Si Min
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Haggblad, Maria
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Biochem & Cellular Screening Facil, Solna, Sweden..
    Eriksson, Anders
    Karolinska Inst, Karolinska High Throughput Ctr, Dept Biosci & Nutr, Huddinge, Sweden..
    Liu, Jianping
    Karolinska Inst, Karolinska High Throughput Ctr, Dept Biosci & Nutr, Huddinge, Sweden..
    Glinghammar, Bjorn
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Drug Discovery & Dev Platform, Solna, Sweden..
    Nekhotiaeva, Natalia
    Karolinska Inst, Karolinska High Throughput Ctr, Dept Biosci & Nutr, Huddinge, Sweden..
    Klingegard, Fredrik
    Stockholm Univ, Dept Organ Chem, Sci Life Lab, Drug Discovery & Dev Platform, Solna, Sweden..
    Koolmeister, Tobias
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Martens, Ulf
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Biochem & Cellular Screening Facil, Solna, Sweden..
    Llona-Minguez, Sabin
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Moulson, Ruth
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Nordström, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Parrow, Vendela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Dahllund, Leif
    Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Sci Life Lab, Drug Discovery & Dev Platform, Solna, Sweden..
    Sjoberg, Birger
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Drug Discovery & Dev Platform, Solna, Sweden..
    Vargas, Irene L.
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Vo, Duy Duc
    Uppsala Univ, Dept Med Chem, Sci Life Lab, Uppsala, Sweden..
    Wannberg, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Knapp, Stefan
    Goethe Univ, Inst Pharmaceut Chem, Frankfurt, Germany..
    Krokan, Hans E.
    Norwegian Univ Sci & Technol, Dept Canc Res & Mol Med, Trondheim, Norway..
    Arvidsson, Per, I
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Drug Discovery & Dev Platform, Solna, Sweden..
    Scobie, Martin
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Meiser, Johannes
    Luxembourg Inst Hlth, Dept Oncol, Canc Metab Grp, Luxembourg, Luxembourg..
    Stenmark, Pal
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.;Lund Univ, Dept Expt Med Sci, Lund, Sweden..
    Berglund, Ulrika Warpman
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Homan, Evert J.
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden..
    Helleday, Thomas
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Solna, Sweden.;Univ Sheffield, Med Sch, Dept Oncol & Metab, Weston Pk Canc Ctr, Sheffield, S Yorkshire, England..
    Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress2022In: NATURE CANCER, ISSN 2662-1347, Vol. 3, no 2, p. 156-Article in journal (Refereed)
    Abstract [en]

    The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors. Helleday and colleagues describe a nanomolar MTHFD2 inhibitor that causes replication stress and DNA damage accumulation in cancer cells via thymidine depletion, demonstrating a potential therapeutic strategy in AML tumors in vivo.

    Download full text (pdf)
    fulltext
  • 48.
    Brickel, Sebastian
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Demkiv, Andrey O.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Crean, Rory M.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Pinto, Gaspar P.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Georgia Inst Technol, Sch Chem & Biochem, 901 Atlantic Dr NW, Atlanta, GA 30332 USA..
    Q-RepEx: A Python pipeline to increase the sampling of empirical valence bond simulations2023In: Journal of Molecular Graphics and Modelling, ISSN 1093-3263, E-ISSN 1873-4243, Vol. 119, article id 108402Article in journal (Refereed)
    Abstract [en]

    The exploration of chemical systems occurs on complex energy landscapes. Comprehensively sampling rugged energy landscapes with many local minima is a common problem for molecular dynamics simulations. These multiple local minima trap the dynamic system, preventing efficient sampling. This is a particular challenge for large biochemical systems with many degrees of freedom. Replica exchange molecular dynamics (REMD) is an approach that accelerates the exploration of the conformational space of a system, and thus can be used to enhance the sampling of complex biomolecular processes. In parallel, the empirical valence bond (EVB) approach is a powerful approach for modeling chemical reactivity in biomolecular systems. Here, we present an open-source Python-based tool that interfaces with the Q simulation package, and increases the sampling efficiency of the EVB free energy perturbation/umbrella sampling approach by means of REMD. This approach, Q-RepEx, both decreases the computational cost of the associated REMD-EVB simulations, and opens the door to more efficient studies of biochemical reactivity in systems with significant conformational fluctuations along the chemical reaction coordinate.

    Download full text (pdf)
    fulltext
  • 49.
    Brodmerkel, Maxim
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    De Santis, Emiliano
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy.
    Konijnenberg, Albert
    Sobott, Frank
    Marklund, Erik G.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Molecular dynamics simulations reveal barrel opening during the unfolding of the outer membrane protein FhaCManuscript (preprint) (Other academic)
    Abstract [en]

    Many membrane proteins carry out gatekeeping and transport functions across the membrane, which makes them tremendously important for the control of what passes into or out from the cell. Their underlying dynamics can be very challenging to capture for structural biology techniques, for which structural heterogeneity often is problematic. Native ion mobility mass spectrometry (IM-MS) is capable of maintaining non-covalent interactions between biomolecules in vacuo, allowing for intact protein complexes from heterogeneous mixtures to be analysed with respect to their masses and structures, making it a powerful tool for structural biology. Recent collision induced unfolding (CIU) experiments, where IM-MS is used to track the unfolding of proteins after activation, were used to investigate the dynamics of the membrane protein FhaC from Bordetella pertussis. FhaC is a β-barrel transmembrane protein found in the outer membrane, where it secretes virulence factors to the outside of the bacterium, requiring notable changes to its structure. CIU cannot on its own provide detailed information about the structural changes along the unfolding pathway. Here, we use MD simulations to mimic the CIU experiments to see if the unfolding proceeds as expected, with cytoplasm-facing domains leading the unfolding, or if other parts of the structure breaks first. By separating our simulation data according to experimental CIU data from literature, we match the structures in the former to the unfolding states identified in the latter, and find that FhaC instead unfolds from a “seam” in the β-barrel. In a wider context, our investigation provides insights into the structural stability and unfolding dynamics of β-barrel membrane proteins and how they can be studied using a combination of CIU and MD.

  • 50.
    Brodmerkel, Maxim N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Theoretical and Biochemical: Advancing Protein Structure Investigations with Complementing Computations2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Life as we know it today would not exist without proteins. The functions of proteins for us and other organisms are linked to their three-dimensional structures. As such, protein structure investigations are a crucial contribution for understanding proteins and the molecular basis of life. Some methods probe the structure of proteins in the gas phase, which brings various advantages as well as complications. Amongst them is mass spectrometry, a powerful method that provides a multitude of information on gaseous protein structures. Whilst mass spectrometry shines in obtaining data of the higher-order structures, atomistic details are out of reach. Molecular dynamics simulations on the other hand allow the interrogation of proteins in high-resolution, which makes it an ideal method for their structural research, be it in or out of solution.

    This thesis aims to advance the understanding of protein structures and the methods for their study utilising classic molecular dynamics simulations. The research presented in this thesis can be divided into two themes, comprising the rehydration of vacuum-exposed structures and the interrogation of the induced unfolding process of proteins. Out of their native environment, proteins undergo structural changes when exposed to vacuum. Investigating the ability to revert those potential vacuum-induced structural changes by means of computational rehydration provided detailed information on the underlying protein dynamics and how much of the structure revert back to their solution norm. We have further shown through rehydration simulations that applying an external electric field for dipole-orientation purposes does not induce irreversible changes to the protein structures. Our investigations on the induced unfolding of protein structures allowed a detailed look into the process of unfolding, accurately pinpointing areas within the proteins that unfolded first. The details provided by our simulations enabled us to describe potential mechanisms of the unfolding processes of different proteins on an atomistic level. The obtained results thus provide a potent theoretical basis for current and future experiments, where it will be very interesting to see MD compared with or complemented to experiments.

    List of papers
    1. Stability and conformational memory of electrosprayed and rehydrated bacteriophage MS2 virus coat proteins
    Open this publication in new window or tab >>Stability and conformational memory of electrosprayed and rehydrated bacteriophage MS2 virus coat proteins
    Show others...
    2022 (English)In: Current Research in Structural Biology, E-ISSN 2665-928X, Vol. 4, p. 338-348Article in journal (Refereed) Published
    Abstract [en]

    Proteins are innately dynamic, which is important for their functions, but which also poses significant challenges when studying their structures. Gas-phase techniques can utilise separation and a range of sample manipulations to transcend some of the limitations of conventional techniques for structural biology in crystalline or solution phase, and isolate different states for separate interrogation. However, the transfer from solution to the gas phase risks affecting the structures, and it is unclear to what extent different conformations remain distinct in the gas phase, and if resolution in silico can recover the native conformations and their differences. Here, we use extensive molecular dynamics simulations to study the two distinct conformations of dimeric capsid protein of the MS2 bacteriophage. The protein undergoes notable restructuring of its peripheral parts in the gas phase, but subsequent simulation in solvent largely recovers the native structure. Our results suggest that despite some structural loss due to the experimental conditions, gas-phase structural biology techniques provide meaningful data that inform not only about the structures but also conformational dynamics of proteins.

    Place, publisher, year, edition, pages
    Elsevier, 2022
    Keywords
    Molecular dynamics simulations, Bacteriophage, Gas-phase structure, Protein structure, Solvation, Electrospray ionization
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-499621 (URN)10.1016/j.crstbi.2022.10.001 (DOI)36440379 (PubMedID)
    Funder
    Swedish Research Council, 2020-04825EU, Horizon 2020, 801406Swedish Research Council, 2021-05988
    Available from: 2023-04-03 Created: 2023-04-03 Last updated: 2023-10-19Bibliographically approved
    2. Rehydration Post-orientation: Investigating Field-Induced Structural Changes via Computational Rehydration
    Open this publication in new window or tab >>Rehydration Post-orientation: Investigating Field-Induced Structural Changes via Computational Rehydration
    2023 (English)In: The Protein Journal, ISSN 1572-3887, E-ISSN 1875-8355, Vol. 42, no 3, p. 205-218Article in journal (Refereed) Published
    Abstract [en]

    Proteins can be oriented in the gas phase using strong electric fields, which brings advantages for structure determination using X-ray free electron lasers. Both the vacuum conditions and the electric-field exposure risk damaging the protein structures. Here, we employ molecular dynamics simulations to rehydrate and relax vacuum and electric-field exposed proteins in aqueous solution, which simulates a refinement of structure models derived from oriented gas-phase proteins. We find that the impact of the strong electric fields on the protein structures is of minor importance after rehydration, compared to that of vacuum exposure and ionization in electrospraying. The structures did not fully relax back to their native structure in solution on the simulated timescales of 200 ns, but they recover several features, including native-like intra-protein contacts, which suggests that the structures remain in a state from which the fully native structure is accessible. Our fndings imply that the electric fields used in native mass spectrometry are well below a destructive level, and suggest that structures inferred from X-ray difraction from gas-phase proteins are relevant for solution and in vivo conditions, at least after in silico rehydration.

    Place, publisher, year, edition, pages
    Springer Nature, 2023
    Keywords
    Molecular dynamics simulation, Protein hydration, Electric dipole, Protein structure, Structural biology, X-rays
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-499999 (URN)10.1007/s10930-023-10110-y (DOI)000966256600001 ()37031302 (PubMedID)
    Funder
    Swedish Research Council, 2020-04825Swedish Research Council, 2018-00740Swedish Research Council, 2021-05988EU, Horizon 2020, 801406
    Available from: 2023-04-10 Created: 2023-04-10 Last updated: 2023-08-15Bibliographically approved
    3. Collision induced unfolding and molecular dynamics simulations of norovirus capsid dimers reveal strain-specific stability profiles
    Open this publication in new window or tab >>Collision induced unfolding and molecular dynamics simulations of norovirus capsid dimers reveal strain-specific stability profiles
    Show others...
    2024 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084Article in journal (Refereed) Published
    Abstract [en]

    Collision induced unfolding is method used with ion mobility mass spectrometry to examine protein structures and their stability. Such experiments yield information about higher order protein structures, yet are unable to provide details about the underlying processes. That information can however be provided using molecular dynamics simulations. Here, we investigate the collision induced unfolding of norovirus capsid dimers from the Norwalk and Kawasaki strains by employing molecular dynamics simulations over a range of temperatures, representing different levels of activation. The dimers have highly similar structures, but the activation reveals differences in the dynamics that arises in response to the activation.

    Place, publisher, year, edition, pages
    Royal Society of Chemistry, 2024
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-500271 (URN)10.1039/D3CP06344E (DOI)
    Funder
    Swedish Research Council, 2021-05988Swedish Research Council, 2020-04825Swedish Research Council, 2018-00740Swedish National Infrastructure for Computing (SNIC), 2022-22-854Swedish National Infrastructure for Computing (SNIC), 2022-22-925Swedish National Infrastructure for Computing (SNIC), 2022-22-947Swedish National Infrastructure for Computing (SNIC), 2022-5-415Swedish National Infrastructure for Computing (SNIC), 2022-23-57EU, Horizon 2020, 801406
    Available from: 2023-04-13 Created: 2023-04-13 Last updated: 2024-04-11Bibliographically approved
    4. Molecular dynamics simulations reveal barrel opening during the unfolding of the outer membrane protein FhaC
    Open this publication in new window or tab >>Molecular dynamics simulations reveal barrel opening during the unfolding of the outer membrane protein FhaC
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Many membrane proteins carry out gatekeeping and transport functions across the membrane, which makes them tremendously important for the control of what passes into or out from the cell. Their underlying dynamics can be very challenging to capture for structural biology techniques, for which structural heterogeneity often is problematic. Native ion mobility mass spectrometry (IM-MS) is capable of maintaining non-covalent interactions between biomolecules in vacuo, allowing for intact protein complexes from heterogeneous mixtures to be analysed with respect to their masses and structures, making it a powerful tool for structural biology. Recent collision induced unfolding (CIU) experiments, where IM-MS is used to track the unfolding of proteins after activation, were used to investigate the dynamics of the membrane protein FhaC from Bordetella pertussis. FhaC is a β-barrel transmembrane protein found in the outer membrane, where it secretes virulence factors to the outside of the bacterium, requiring notable changes to its structure. CIU cannot on its own provide detailed information about the structural changes along the unfolding pathway. Here, we use MD simulations to mimic the CIU experiments to see if the unfolding proceeds as expected, with cytoplasm-facing domains leading the unfolding, or if other parts of the structure breaks first. By separating our simulation data according to experimental CIU data from literature, we match the structures in the former to the unfolding states identified in the latter, and find that FhaC instead unfolds from a “seam” in the β-barrel. In a wider context, our investigation provides insights into the structural stability and unfolding dynamics of β-barrel membrane proteins and how they can be studied using a combination of CIU and MD.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-500273 (URN)
    Available from: 2023-04-13 Created: 2023-04-13 Last updated: 2023-04-25Bibliographically approved
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    UUThesis_M-Brodmerkel-2023
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