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  • 1. Barbieri, Shayla Fernanda
    et al.
    Ruthes, Andrea C.
    KTH, School of Biotechnology (BIO), Glycoscience.
    de Oliveira Petkowicz, Carmen Lucia
    Bueno de Godoy, Rossana Catie
    Sassaki, Guilherme Lanzi
    Santana Filho, Arquimedes Paixao
    Meira Silveira, Joana Lea
    Extraction, purification and structural characterization of a galactoglucomannan from the gabiroba fruit (Campomanesia xanthocarpa Berg), Myrtaceae family2017In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 174, p. 887-895Article in journal (Refereed)
    Abstract [en]

    In this study, we isolated and structurally characterized, for the first time, a galactoglucomannan (GGM) from the pulp of gabiroba, a Myrtaceae family species. The HPSEC-MALLS-RI analysis showed a homogeneous polysaccharide with molar mass of 25,340 g mol(-1). The monosaccharide composition showed that the GGM consisted of Man:Glc:Gal in a molar ratio of 1:1:0.6. Methylation and 1D and 2D NMR analyses suggested that the main chain of the GGM consisted of beta-D-Glcp and beta-D-Manp units (1 -> 4)-linked. The alpha-D-Galp substitutions occur mainly at O-6 position of beta-D-Manp units. The glycosidic linkages of the GGM were evident by the presence of the characteristic signals of 4-O-substituted residues at delta 78.6/3.69 for both beta-D-Glcp and beta-D-Manp. Furthermore, the 0-6 substitutions for both beta-D-Glcp and beta-D-Manp units were confirmed by signals at delta 67.1/4.00 and 3.93. The interglycosidic correlations, obtained through the analysis of the HMBC spectrum, further confirm the structure. (C) 2017 Elsevier Ltd. All rights reserved.

  • 2.
    Chang, Shu-Chieh
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031, Taiwan..
    Kao, Mu-Rong
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031, Taiwan.
    Saldivar, Rebecka Karmakar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031, Taiwan..
    Diaz-Moreno, Sara M
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Xing, Xiaohui
    Agr & Agrifood Canada, Lethbridge Res & Dev Ctr, Lethbridge, AB T1J 4B1, Canada.
    Furlanetto, Valentina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Yayo, Johannes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Divne, Christina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Vilaplana, Francisco
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Abbott, D. Wade
    Agr & Agrifood Canada, Lethbridge Res & Dev Ctr, Lethbridge, AB T1J 4B1, Canada.
    Hsieh, Yves S. Y.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031, Taiwan..
    The Gram-positive bacterium Romboutsia ilealis harbors a polysaccharide synthase that can produce (1,3;1,4)-β-D-glucans2023In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1Article in journal (Refereed)
    Abstract [en]

    (1,3;1,4)-β-D-Glucans are widely distributed in the cell walls of grasses (family Poaceae) and closely related families, as well as some other vascular plants. Additionally, they have been found in other organisms, including fungi, lichens, brown algae, charophycean green algae, and the bacterium Sinorhizobium meliloti. Only three members of the Cellulose Synthase-Like (CSL) genes in the families CSLF, CSLH, and CSLJ are implicated in (1,3;1,4)-β-D-glucan biosynthesis in grasses. Little is known about the enzymes responsible for synthesizing (1,3;1,4)-β-D-glucans outside the grasses. In the present study, we report the presence of (1,3;1,4)-β-D-glucans in the exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis CRIBT. We also report that RiGT2 is the candidate gene of R. ilealis that encodes (1,3;1,4)-β-D-glucan synthase. RiGT2 has conserved glycosyltransferase family 2 (GT2) motifs, including D, D, D, QXXRW, and a C-terminal PilZ domain that resembles the C-terminal domain of bacteria cellulose synthase, BcsA. Using a direct gain-of-function approach, we insert RiGT2 into Saccharomyces cerevisiae, and (1,3;1,4)-β-D-glucans are produced with structures similar to those of the (1,3;1,4)-β-D-glucans of the lichen Cetraria islandica. Phylogenetic analysis reveals that putative (1,3;1,4)-β-D-glucan synthase candidate genes in several other bacterial species support the finding of (1,3;1,4)-β-D-glucans in these species.

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  • 3.
    De Oliveira, Danilo Hirabae
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Integrative structural biology of protein fibers: Spider silk and beta-lactoglobulin nanofibrils2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins found in nature offer a vast range of exceptional materials, including high-performancebiopolymers such as spider silks and whey protein nanofibrils. Fibrous proteins possess immensepotential for developing novel materials suited for various applications, such as medicalbiomaterials or industrial products. This thesis uses an interdisciplinary approach based onexperimental and computational methods to present insights into the fundamental aspects ofprotein fibers, exploring details on the molecular level and their self-assembly.Spider silk threads exhibit strength, elasticity, and the ability to withstand high-energy loads.Additionally, silk is naturally degradable, and compatible with cell growth, and non-immunogenic.This thesis examines the molecular assembly of spider silk, particularly the secondary structurelevel, which contributes greatly to its properties. We unveil the structural details of therecombinant spidroins 4RepCT and the CT domain over hydrophobic/hydrophilic surfaces,describing their periodic oriented macrostructure and stability. Furthermore, it is reported that theCT domain form β-nanocrystalline components, revealing a specific segment (helix No4) that canself-assemble into nanofibrils in a pH-sensitive manner. In addition, we describe the method ofsortase-mediated transpeptidation reaction used to catalyze the covalent coupling of the spidroins4Rep and CT, resulting in partially isotopically labeled fibers suitable for solid-state NMRspectroscopy analyses.β-lactoglobulin is an emerging protein source used to create advanced biomaterials because of itshigh availability and ability to assemble to protein nanofibrils (PNFs). Recombinant and syntheticβ-lactoglobulin PNFs with isotopic labelling are generated and analyzed using solid-state NMRspectroscopy and atomic force microscopy. The fibrils of both species present congruenciesregarding morphology with unbranched conformation and a height of approximately 6 nm. At thesame time, their NMR spectra demonstrate accordance with their hydrophobic residues (i.e., Ala,Val, Ile, and Leu) as β-sheets. In addition, distinct inter-residue cross-peaks of Ser-Thr and LeuIle provide insights into the molecular structure of β-lactoglobulin PNF.This thesis presents new knowledge about the hierarchy of protein fibrils and the structure ofprotein-based materials at the molecular level. This knowledge can unlock the design anddevelopment of innovative protein-based materials for various applications.

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  • 4.
    De Oliveira, Danilo Hirabae
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Gowda, Vasantha
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry. Umeå University, Sweden.
    Sparrman, Tobias
    Umeå University, Sweden.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Lendel, Christofer
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Exploring the molecular structure of β-lactoglobulin fibrils using solid-state NMR spectroscopyManuscript (preprint) (Other academic)
  • 5.
    De Oliveira, Danilo Hirabae
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Gowda, Vasantha
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Sparrman, Tobias
    Umeå University, Sweden.
    Pires, Rodrigo Sanches
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Riekel, Christian
    European Synchrotron Radiation Facility, Grenoble Cedex France.
    Barth, Andreas
    Stockholm University, Sweden.
    Lendel, Christofer
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Untangling spider silk secrets: The structural basis of alpha-helix tobeta-sheet conversion of the spidroin C-terminal domain during fiber assemblyManuscript (preprint) (Other academic)
  • 6.
    De Oliveira, Danilo Hirabae
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Zillen, Sara
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Sparrman, Tobias
    Umeå University, Sweden.
    Jansson, Ronnie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Lendel, Christofer
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Sortase-mediated coupling of a labelled C-terminal domain and a non-labelled repetitivesegment of a spidroin for solid-state NMR structural characterization of silk fibersManuscript (preprint) (Other academic)
  • 7.
    Furlanetto, Valentina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Symbiotic and pathogenic factors in plant-microbe interaction: Structural basis of C-glycoside metabolism and lipoprotein transport in bacteria2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The communication between plants and bacteria involves a complex chemical signaling network that mediates responses to various biotic and abiotic stresses, as well as establishing symbiotic relationships between different organisms.

    The first part of the thesis focuses on a mechanism for symbiotic communication between plants and bacteria and more specifically on how C-glycosylated aromatic polyketide compounds produced by plants can be used as a mechanism for plants to communicate with beneficial bacteria. In their glycosylated form, these compounds are substrates for symbiotic bacteria, which in return deglycosylate them and release the sugar-free part, the active aglycone. The aglycone can then mediate several functions beneficial to the plant, for example facilitating nitrogen fixation or acting as an antibacterial agent against plant pathogens.

    Results from studies covered in the thesis show that the soil bacteria Deinococcus aerius, Streptomyces canus and Microbacterium testaceum produce enzymes that can cleave the carbon-carbon bond between the sugar and the aglycone in C-glycosyl compounds. Deglycosylation first requires oxidation of the sugar by an oxidoreductase, after which the carbon-carbon bond can be cleaved by a C-glycosyl deglycosidase (CGD). Biochemical and structural characterization as well as results from phylogenetic analyzes of the amino-acid sequences of CGD enzymes provided new knowledge about these relatively unexplored enzymatic processes, as well as increased insight into how C-glycosylated aromatic polyketides participate in the interaction between plant and bacteria.

    The second part of the thesis explores pathogenic interactions between plants and bacteria. The virulence of pathogenic bacteria is dependent on lipoproteins that are attached to the bacteria's outer membrane and that have a decisive role for the bacteria's survival and pathogenicity. The localization of lipoproteins takes place through a process abbreviated Lol. The Lol system of the notorious plant pathogen Xanthomonas campestris was studied to better understand the underlying molecular mechanisms of the localization system, which could eventually open new ways to combat the bacterium. Biochemical, structural, and phylogenetic techniques were used also in this project.

    Taken together, the results contributed several new discoveries. For the first time, a physical complex between the two proteins responsible for transporting lipoproteins could be determined and their mutual interactions studied. Furthermore, sequence analyses challenge the generally accepted model of how lipoproteins are released from the bacterial inner membrane before being transported to the outer membrane. 

    According to the standard model based on Escherichia coli, lipoproteins are extracted from the inner membrane by a membrane protein that belongs to the ABC-transporter family and whose structure forms an asymmetric heterodimer (LolCDE). However, our bioinformatic analysis show that most of these ABC transporters, including X. campestris, are likely to be homodimers and that Escherichia coli is the exception rather than the rule. The difference between an asymmetric and symmetric ABC transporter also has implications for several hypotheses about how these proteins function. Heterologous production of the X. campestris ABC transporter confirmed that the protein is a homodimer.

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  • 8.
    Gomez-Blanco, J.
    et al.
    McGill Univ, Dept Anat & Cell Biol, Montreal, PQ, Canada..
    de la Rosa-Trevin, J. M.
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Stockholm, Sweden..
    Marabini, R.
    Univ Autonoma Madrid, Escuela Politecn Super, E-28049 Madrid, Spain..
    del Cano, L.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Jimenez, A.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Martinez, M.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Melero, R.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Majtner, T.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Maluenda, D.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Mota, J.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Rancel, Y.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Ramirez-Aportela, E.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Vilas, J. L.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Carroni, M.
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Stockholm, Sweden..
    Fleischmann, S.
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Stockholm, Sweden..
    Lindahl, Erik
    KTH, Centres, SeRC - Swedish e-Science Research Centre. Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Stockholm, Sweden.;KTH Royal Inst Technol, Swedish E Sci Res Ctr, Stockholm, Sweden..
    Ashton, A. W.
    Harwell Sci & Innovat Campus, Diamond Light Source, Didcot OX11 0DE, Oxon, England..
    Basham, M.
    Harwell Sci & Innovat Campus, Diamond Light Source, Didcot OX11 0DE, Oxon, England..
    Clare, D. K.
    Harwell Sci & Innovat Campus, Diamond Light Source, Didcot OX11 0DE, Oxon, England..
    Savage, K.
    Harwell Sci & Innovat Campus, Diamond Light Source, Didcot OX11 0DE, Oxon, England..
    Siebert, C. A.
    Harwell Sci & Innovat Campus, Diamond Light Source, Didcot OX11 0DE, Oxon, England..
    Sharov, G. G.
    MRC, Lab Mol Biol, Francis Crick Ave, Cambridge CB2 OQH, England..
    Sorzano, C. O. S.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Conesa, P.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Carazo, J. M.
    CSIC, Natl Ctr Biotechnol, Biocomp Unit, C Darwin 3,Campus Univ Autonoma, Madrid 28049, Spain..
    Using Scipion for stream image processing at Cryo-EM facilities2018In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 204, no 3, p. 457-463Article in journal (Refereed)
    Abstract [en]

    Three dimensional electron microscopy is becoming a very data-intensive field in which vast amounts of experimental images are acquired at high speed. To manage such large-scale projects, we had previously developed a modular workflow system called Scipion (de la Rosa-Trevfn et al., 2016). We present here a major extension of Scipion that allows processing of EM images while the data is being acquired. This approach helps to detect problems at early stages, saves computing time and provides users with a detailed evaluation of the data quality before the acquisition is finished. At present, Scipion has been deployed and is in production mode in seven Cryo-EM facilities throughout the world.

  • 9.
    Hammarström, Björn
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics.
    Lane, Thomas J.
    Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany, Notkestrasse 85.
    Batili, Hazal
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Sierra, Raymond
    Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California 94025, United States.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Sellberg, Jonas A.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Acoustic Focusing of Protein Crystals for In-Line Monitoring and Up-Concentration during Serial Crystallography2022In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 94, no 37, p. 12645-12656Article in journal (Refereed)
    Abstract [en]

    Serial femtosecond crystallography (SFX) has become one of the standard techniques at X-ray free-electron lasers (XFELs) to obtain high-resolution structural information from microcrystals of proteins. Nevertheless, reliable sample delivery is still often limiting data collection, as microcrystals can clog both field- and flow-focusing nozzles despite in-line filters. In this study, we developed acoustic 2D focusing of protein microcrystals in capillaries that enables real-time online characterization of crystal size and shape in the sample delivery line after the in-line filter. We used a piezoelectric actuator to create a standing wave perpendicular to the crystal flow, which focused lysozyme microcrystals into a single line inside a silica capillary so that they can be imaged using a high-speed camera. We characterized the acoustic contrast factor, focus size, and the coaxial flow lines and developed a splitting union that enables up-concentration to at least a factor of five. The focus size, flow rates, and geometry may enable an upper limit of up-concentration as high as 200 fold. The novel feedback and concentration control could be implemented for serial crystallography at synchrotrons with minor modifications. It will also aid the development of improved sample delivery systems that will increase SFX data collection rates at XFELs, with potential applications to many proteins that can only be purified and crystallized in small amounts.

  • 10.
    Hassan, Noor
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Characterization and engineering of carbohydrate-active enzymes for biotechnological applications2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry.

    In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I).

    To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS.

    In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from  H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of  HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile.

    Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly.

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    Thesis
  • 11.
    Hueting, David A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    In silico protein design for the enhancement of protein stability and function2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enzymes are natures catalysts that increase the rate of a chemical reaction. The increased rate of a reaction is required to be able to sustain life. Despite the huge impact of enzymes, they are not perfect catalysts. Enzyme and protein engineering is the discipline in which proteins are characterized and engineered to have improved inherent properties. Interesting properties of an enzyme to improve include stability and activity. The aim of this work is to understand how proteins and enzymes function and use a variety of different protein engineering techniques to enhance the properties of different proteins. In this work proteins and enzymes are engineered to increase our knowledge of the target proteins for downstream biomedical applications. A mix between rational and semi-rational engineering is applied in this work. In paper I and paper II, the method used is ancestral sequence reconstruction. A method that utilizes the evolutionary relationship between homologous sequences. In paper I the method was applied to a terpene cyclase, which cyclizes a precursor terpene into potential interesting drug leads. The result was a hyperstable enzyme variant. In paper II the technique was applied to the SARS-CoV-2 Spike protein. The protein is responsible for the virus SARS-CoV-2 to enter human cells. The work yielded a stable spike protein that readily expresses and can be utilized as a vaccine lead. In paper III, the aim was to understand human oxidosqualene cyclase (hOSC). A terpene cyclase essential in cholesterol synthesis. The enzyme hOSC was rationally engineered to change the driving force of the reaction. Through targeted mutations the reaction changed from entropy driven to enthalpy driven. Finally, in paper IV, a rationally engineered PETase, which is capable of degrading PET polymers into monomers, was proven to be active in human serum and verifies the proof-of-concept of degrading plastic in human blood. To summarize, the results in this thesis show the applicability of different enzyme engineering techniques to stabilize or change the function of proteins and the potential of engineered proteins in medical applications.

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  • 12.
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Structural studies of microbubbles and molecular chaperones using transmission electron microscopy2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Ultrasound contrast agents (CAs) are typically used in clinic for perfusion studies (blood flow through a specific region) and border delineating (differentiate borders between tissue structures) during cardiac imaging. The CAs used during ultrasound imaging usually consist of gas filled microbubbles (MBs) (diameter 1-5 μm) that are injected intravenously into the circulatory system. This thesis partially involves a novel polymer-shelled ultrasound CA that consists of air filled MBs stabilized by a polyvinyl alcohol (PVA) shell. These MBs could be coupled with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as a combined CA for ultrasound and magnetic resonance imaging. The first three papers (Paper A-C) in this thesis investigate the structural characteristic and the elimination process of the CA.

    In Paper A, two types (PVA Type A and PVA Type B) of the novel CA were analyzed using transmission electron microscopy (TEM) images of thin sectioned MBs. The images demonstrated that the SPIONs were either attached to the PVA shell surface (PVA Type A) or embedded in the shell (PVA Type B). The average shell thickness of the MBs was determined in Paper B by introducing a model that calculated the shell thickness from TEM images of cross-sectioned MBs. The shell thickness of PVA Type A was determined to 651 nm, whereas the shell thickness of PVA Type B was calculated to 637 nm. In Paper C, a prolonged blood elimination time was obtained for PVA-shelled MBs compared to the lipid-shelled CA SonoVue used in clinic. In addition, TEM analyzed tissue sections showed that the PVA-shelled MBs were recognized by the macrophage system. However, structurally intact MBs were still found in the circulation 24 h post injection. These studies illustrate that the PVA-shelled MBs are stable and offer large chemical variability, which make them suitable as CA for multimodal imaging.

    This thesis also involves studies (Paper D-E) of the molecular chaperones (Hsp21 and DNAJB6). The small heat shock protein Hsp21 effectively protects other proteins from unfolding and aggregation during stress. This chaperone ability requires oligomerization of the protein. In Paper D, cryo-electron microscopy together with complementary structural methods, obtained a structure model which showed that the Hsp21 dodecamer (12-mer) is kept together by paired C-terminal interactions.The human protein DNAJB6 functions as a very efficient suppressor of polyglutamine (polyQ) and amyloid-β42 (Aβ42) aggregation. Aggregation of these peptides are associated with development of Huntington’s (polyQ) and Alzheimer’s (Aβ42) disease. In Paper E, a reconstructed map of this highly dynamic protein is presented, showing an oligomer with two-fold symmetry, indicating that the oligomers are assembled by two subunits.

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    Thesis
  • 13.
    Härmark, Johan
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Månsson, Cecilia
    Rasmussen, Morten
    Höjrup, Peter
    Al-Karadaghi, Salam
    Söderberg, Christopher G
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Emanuelsson, Cecilia
    Structural information on the oligomeric human molecular chaperone DNAJB6Manuscript (preprint) (Other academic)
  • 14.
    Jansson, Ronnie
    et al.
    Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Askarieh, Glareh
    Hedhammar, My
    Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Recombinant Spider Silk ― Yesterday, Today and Tomorrow2010Conference paper (Other academic)
  • 15.
    Jansson, Ronnie
    et al.
    Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Askarieh, Glareh
    Müller, Christian
    Rising, Anna
    Johansson, Jan
    Riekel, Christian
    Knight, Stefan
    Hedhammar, My
    Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Fibre X-Ray Diffraction of Recombinant Spider Silk Reveal β-Sheet Structure2011Conference paper (Refereed)
  • 16. Kim, Hyung Min
    et al.
    Song, Yanxue
    Hyun, Gyu Hwan
    Long, Nguyen Phuoc
    Park, Jeong Hill
    Hsieh, Yves S. Y.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Kwon, Sung Won
    Characterization and Antioxidant Activity Determination of Neutral and Acidic Polysaccharides from Panax Ginseng C. A. Meyer2020In: Molecules, ISSN 1431-5157, E-ISSN 1420-3049, Vol. 25, no 4, article id 791Article in journal (Refereed)
    Abstract [en]

    Panax ginseng (P. ginseng) is the most widely consumed herbal plant in Asia and is well-known for its various pharmacological properties. Many studies have been devoted to this natural product. However, polysaccharide’s components of ginseng and their biological effects have not been widely studied. In this study, white ginseng neutral polysaccharide (WGNP) and white ginseng acidic polysaccharide (WGAP) fractions were purified from P. ginseng roots. The chemical properties of WGNP and WGAP were investigated using various chromatography and spectroscopy techniques, including high-performance gel permeation chromatography, Fourier-transform infrared spectroscopy, and high-performance liquid chromatography with an ultra-violet detector. The antioxidant, anti-radical, and hydrogen peroxide scavenging activities were evaluated in vitro and in vivo using Caenorhabditis elegans as the model organism. Our in vitro data by ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), reducing power, ferrous ion chelating, and hydroxyl radical scavenging activity suggested that the WGAP with significantly higher uronic acid content and higher molecular weight exhibits a much stronger antioxidant effect as compared to that of WGNP. Similar antioxidant activity of WGAP was also confirmed in vivo by evaluating internal reactive oxygen species (ROS) concentration and lipid peroxidation. In conclusion, WGAP may be used as a natural antioxidant with potent scavenging and metal chelation properties.

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  • 17. Kimanius, Dari
    et al.
    Zickert, Gustav
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Mathematics (Div.).
    Nakane, Takanori
    Adler, Jonas
    Lunz, Sebastian
    Schönlieb, Carola-Bibiane
    Öktem, Ozan
    Scheres, Sjors
    Exploiting prior knowledge about biological macromolecules in cryo-EM structure determinationManuscript (preprint) (Other academic)
  • 18.
    Klinter, Stefan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Identification and characterisation of chitin and cellulose synthases in oomycetes: New tools for biochemical studies and structure determination2021Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Despite resembling ‘true’ fungi in terms of morphological features, oo­mycetes form a distinct eukaryotic lineage of filamentous microorganisms that belongs to the stramenopiles, a group of protists also comprising the closely-related brown algae and diatoms. Many oomycetes are devastating pathogens of plants and animals, globally causing significant economic los­ses in the agriculture and aquaculture industries, and posing considerable environmental damage to natural ecosystems. Although the cell wall (CW) is critical for the viability and morphogenesis of the organism it surrounds, our knowledge of oomycete CW architecture and biosynthetic enzymes is limited. Given the vast threat that pathogenic oomycetes pose, uncovering the details of CW biosynthesis and regulation in these pathogens may re­veal new opportunities for disease control.

    To this end, we aimed to elucidate the role of putative membrane-bound glycosyltransferase family 2 enzymes implicated in the biosynthesis of oo­mycete CW polysaccharides. Suitable gene candidates were identified, and their products analysed, as illustrated by the oomycete-wide discovery and phylogenetic analysis of the chitin synthase gene family (paper I), and the identification of the cellulose synthase genes in Saprolegnia parasitica (paper II) and Phytophthora capsici (paper III). Expression of promi­sing candidate genes was verified using different techniques, including gene expression analysis (papers II and III), and the effect of inhibitors on hyphal growth (papers I and II) and enzymatic activity in in vitro assays (paper II). Single enzymes representing putative chitin synthases from various organisms (unpublished data) and cellulose synthases from S. parasitica (extended data for paper II), and P. capsici cellulose syn­thase 1 (paper III) were produced, and partly enriched or even purified, in yeast strains specifically engineered to facilitate the biochemical characterisation of the recombinant proteins in in vitro enzyme assays. To advance functional investigations and structure determination of integral membrane proteins, we developed DirectMX, a method that allows the re­constitution of target proteins with their surrounding lipids directly from crude cell membranes into Salipro nanoparticles (paper IV).

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    Doctoral Thesis Stefan Klinter
  • 19.
    Kuang, Qie
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Structural studies of membrane proteins using transmission electron microscopy2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Membrane proteins play important roles for living cells. They control transportation of ions, solutes, and nutrients across the membrane and catalyze metabolic reactions. Transmission electron microscopy has its advantages in convenient sample preparation, straightforward structural determination, and wide applications for diverse specimens. In this thesis, the structure of three membrane proteins are studied by this method.

    Kch, a potassium channel in Escherichia coli, has a transmembrane part and a cytosolic domain. Large and well-ordered two dimensional crystals were obtained from both a functional mutant (KchM240L) and a modified protein possessing only the transmembrane part (KchTM). Both samples crystallize as two symmetry-related overlapping layers. Furthermore, the KchTM structure was reconstructed which showed that the transmembrane part of the two adjacent proteins are involved in forming the crystal contacts. Thus, the cytosolic domains of Kch in crystals are deduced to expose to the solvent and do not interact with each other.

    MGST1 (microsomal glutathione transferase 1) is a detoxification enzyme. It was recombinantly over-expressed in the current study, instead of purified from rat liver as before. The crystallization condition was adjusted and isomorphic crystals were obtained. The refined model was built from a combined data set consisting of previous and new diffraction patterns. More residues at the C-terminus of the transmembrane helix 1 were assigned and the residues in the transmembrane helices 3 and 4 were remodeled. Several phospholipid molecules were observed and the ligand glutathione adopts an extended conformation in the refined model.

    The structure of MelB (a sugar/sodium symporter in Escherichia coli) was determined using a refined single particle reconstruction method. This novel method is aimed for processing small or locally distorted crystals. In comparison with the previously published single particle reconstruction protocol, the current method is improved in several aspects. A more reliable reconstruction of MelB was obtained and the resolution was increased. The docking experiment indicates that MelB adopts an open conformation under the present two dimensional crystallization condition.

    Electron microscopy has developed quickly recently with the help of modern instruments, techniques, and software. This method will without doubt play a more critical role in future structural biology.

     

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    Thesis
  • 20.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Jegerschöld, Caroline
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Institutet, Sweden.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Free RCK Arrangement in Kch, a Putative Escherichia coli Potassium Channel, as Suggested by Electron Crystallography2015In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 23, no 1, p. 199-205Article in journal (Refereed)
    Abstract [en]

    The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement.

  • 21.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Ålander, Johan
    Svensson, Richard
    Hoogland, Veronika
    Winerdal, Jens
    Spahiu, Linda
    Ottosson-Wadlund, Astrid
    Armstrong, Richard
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Morgenstern, Ralf
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    A refined atomic model for microsomal glutathione transferase 1 from electron crystallographyManuscript (preprint) (Other academic)
    Abstract [en]

    Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional (2D) crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The 2D crystals were of the p6 two-sided plane group symmetry. For the refinement, information to 3.5 Å resolution from 225 electron diffraction patterns recorded from specimens at tilt angles up to 66° was used. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two hydrocarbon chains, and one glutathione (GSH) molecule. Interactions between subunits form trimers centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer. The location of GSH is supported by mutagenesis data in vitro.

  • 22. Lendel, Christofer
    et al.
    Bjerring, Morten
    Dubnovitsky, Anatoly
    Kelly, Robert T
    Filippov, Andrei
    Antzutkin, Oleg N.
    Nielsen, Niels C.
    Härd, Torleif
    A hexameric peptide barrel as building block of amyloid-β protofibrils.2014In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 53, p. 12756-12760Article in journal (Refereed)
    Abstract [en]

    Oligomeric and protofibrillar aggregates formed by the amyloid-A beta peptide (A beta) are believed to be involved in the pathology of Alzheimer's disease. Central to Alzheimer pathology is also the fact that the longer A beta(42) peptide is more prone to aggregation than the more prevalent A beta(40). Detailed structural studies of A beta oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of A beta that forms stable protofibrils and here we use solid-state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of A beta protomers into hexameric barrel-like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C-terminal hydrophobic regions of A beta, and hairpin loops extend from the core. The model accounts for why A beta(42) forms oligomers and protofibrils more easily than A beta(40).

  • 23. Liu, Chen
    et al.
    Chen, Yuting
    Zheng, Yuhan
    Bo, Jin
    Yang, Canjun
    Xu, Sun
    Zhang, Sheng
    Wear Resistance Improvement of Keeled Structure and Overlapped Distribution of Snake Scales2023In: Journal of Bionic Engineering, ISSN 1672-6529, Vol. 20, no 3, p. 1121-1131Article in journal (Refereed)
    Abstract [en]

    The movement mode of snakes is crawling, and the living environment of snakes with numerous branches and stones will cause plenty of wear for the snake scales. There are plenty of surface structures and morphology on snake scales to avoid severe wear. Among them, the research towards the keeled structure on snake scales is missing. Therefore, in this research, the wear resistance improvement of the keeled structure on the snake scales and the overlapped distribution of snake scales are investigated. The keeled and smooth snake scales were 3D printed and they were distributed on the substrate in the overlapped or paralleled ways. Besides these four samples with keeled/smooth scales and overlapped/paralleled distributed, there is also a reference sample with the same thickness. Based on the tribology test, the number of grooves of samples with the keeled structures is higher than that of samples with smooth surfaces, which indicates that the keeled structure dramatically enhances the wear resistance of snake scales, especially during the wear in the vertical direction. The experiment on surface morphology greatly compromised the result of the tribology test. In addition, the bottom portion of the keeled snake scales can be protected by the keeled structure. Besides, the overlapped distribution can protect the central region of snake scales and provide double-layer protection of the snake body. Overall, the keeled structure and the overlapped distribution play a significant part in the improvement of wear resistance of the snake skin. These findings can enhance the knowledge of the reptiles-mimic surface structure and facilitate the application of military uniforms under high-wear conditions.

  • 24. Lloris-Garcerá, Pilar
    et al.
    Klinter, Stefan
    Chen, Liuhong
    Skynner, Michael J.
    Löving, Robin
    Frauenfeld, Jens
    DirectMX – One-Step Reconstitution of Membrane Proteins From Crude Cell Membranes Into Salipro Nanoparticles2020In: Frontiers in Bioengineering and Biotechnology, E-ISSN 2296-4185, Vol. 8Article in journal (Refereed)
    Abstract [en]

    Integral membrane proteins (IMPs) are central to many physiological processes and represent ∼60% of current drug targets. An intricate interplay with the lipid molecules in the cell membrane is known to influence the stability, structure and function of IMPs. Detergents are commonly used to solubilize and extract IMPs from cell membranes. However, due to the loss of the lipid environment, IMPs usually tend to be unstable and lose function in the continuous presence of detergent. To overcome this problem, various technologies have been developed, including protein engineering by mutagenesis to improve IMP stability, as well as methods to reconstitute IMPs into detergent-free entities, such as nanodiscs based on apolipoprotein A or its membrane scaffold protein (MSP) derivatives, amphipols, and styrene-maleic acid copolymer-lipid particles (SMALPs). Although significant progress has been made in this field, working with inherently unstable human IMP targets (e.g., GPCRs, ion channels and transporters) remains a challenging task. Here, we present a novel methodology, termed DirectMX (for direct membrane extraction), taking advantage of the saposin-lipoprotein (Salipro) nanoparticle technology to reconstitute fragile IMPs directly from human crude cell membranes. We demonstrate the applicability of the DirectMX methodology by the reconstitution of a human solute carrier transporter and a wild-type GPCR belonging to the human chemokine receptor (CKR) family. We envision that DirectMX bears the potential to enable studies of IMPs that so far remained inaccessible to other solubilization, stabilization or reconstitution methods.

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  • 25.
    Natarajan Arul, Murugan
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Apostolov, Rossen Pavlov
    KTH, School of Computer Science and Communication (CSC), Centres, Centre for High Performance Computing, PDC.
    Rinkevicius, Zilvinas
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Kongsted, Jacob
    epartment of Physics, Chemistry and Pharmacy, University of Southern Denmark.
    Lindahl, Erik
    KTH, School of Engineering Sciences (SCI), Theoretical Physics, Theoretical & Computational Biophysics.
    Ågren, Hans
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Association dynamics and linear and nonlinear optical properties of an N-acetylaladanamide probe in a POPC membrane2013In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 135, no 36, p. 13590-13597Article in journal (Refereed)
    Abstract [en]

    Along with the growing evidence that relates membrane abnormalities to various diseases, biological membranes have been acknowledged as targets for therapy. Any such abnormality in the membrane structure alters the membrane potential which in principle can be captured by measuring properties of specific optical probes. There exists by now many molecular probes with absorption and fluorescence properties that are sensitive to local membrane structure and to the membrane potential. To suggest new high-performance optical probes for membrane-potential imaging it is important to understand in detail the membrane-induced structural changes in the probe, the membrane association dynamics of the probe, and its membrane-specific optical properties. To contribute to this effort, we here study an optical probe, N-acetylaladanamide (NAAA), in the presence of a POPC lipid bilayer using a multiscale integrated approach to assess the probe structure, dynamics, and optical properties in its membrane-bound status and in water solvent. We find that the probe eventually assimilates into the membrane with a specific orientation where the hydrophobic part of the probe is buried inside the lipid bilayer, while the hydrophilic part is exposed to the water solvent. The computed absorption maximum is red-shifted when compared to the gas phase. The computations of the two-photon absorption and second harmonic generation cross sections of the NAAA probe in its membrane-bound state which is of its first kind in the literature suggest that this probe can be used for imaging the membrane potential using nonlinear optical microscopy.

  • 26.
    Nilebäck, Linnea
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Arola, Suvi
    Aalto Univ, Sch Chem Engn, Dept Bioprod & Biosyst, POB 16100, FI-00076 Aalto, Finland..
    Kvick, Mathias
    AlbaNova Univ Ctr, Spiber Technol AB, S-10691 Stockholm, Sweden..
    Paananen, Arja
    VTT Tech Res Ctr Finland Ltd, Tietotie 2, FI-02150 Espoo, Finland..
    Linder, Markus B.
    Aalto Univ, Sch Chem Engn, Dept Bioprod & Biosyst, POB 16100, FI-00076 Aalto, Finland..
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism2018In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 34, no 39, p. 11795-11805Article in journal (Refereed)
    Abstract [en]

    The mechanism of silk assembly, and thus the cues for the extraordinary properties of silk, can be explored by studying the simplest protein parts needed for the formation of silk-like materials. The recombinant spider silk protein 4RepCT, consisting of four repeats of polyalanine and glycine-rich segments (4Rep) and a globular C-terminal domain (CT), has previously been shown to assemble into silk-like fibers at the liquid-air interface. Herein, we study the interfacial behavior of the two parts of 4RepCT, revealing new details on how each protein part is crucial for the silk assembly. Interfacial rheology and quartz crystal microbalance with dissipation show that 4Rep interacts readily at the interfaces. However, organized nanofibrillar structures are formed only when 4Rep is fused to CT. A strong interplay between the parts to direct the assembly is demonstrated. The presence of either a liquid-air or a liquid-solid interface had a surprisingly similar influence on the assembly.

  • 27. Orellana, Laura
    et al.
    Yoluk, Özge
    KTH, School of Engineering Sciences (SCI), Theoretical Physics, Theoretical & Computational Biophysics.
    Carrillo, Oliver
    Orozco, Modesto
    Lindahl, Erik
    KTH, School of Engineering Sciences (SCI), Theoretical Physics, Theoretical & Computational Biophysics.
    Prediction and Validation of Protein Intermediate States from Structurally Rich Ensembles and Coarse-Grained Simulations2016In: Nature Communications, E-ISSN 2041-1723, Vol. 7Article in journal (Refereed)
    Abstract [en]

    Protein conformational changes are at the heart of cell functions, from signaling to ion transport. However, the transient nature of the intermediates along transition pathways hampers their experimental detection, making the underlying mechanisms elusive. Here, we retrieve dynamic information on the actual transition routes from Principal Component Analysis (PCA) of structurally-rich ensembles and, in combination with coarse-grained simulations, explore the conformational landscapes of five well-studied proteins. Modeling them as elastic networks in a hybrid Elastic-Network Brownian Dynamics simulation (eBDIMS), we generate trajectories connecting stable end-states that spontaneously sample the crystallographic motions, predicting the structures of known intermediates along thepaths. We also show that the explored non-linear routes can delimit the lowest energy passages between end-states sampled by atomistic molecular dynamics. The integrative methodology presented here provides a powerful framework to extract and expand dynamic pathway information from the Protein Data Bank, as well as to validate sampling methods in general. 

  • 28.
    Ornithopoulou, Eirini
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Brett, Calvin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Davydok, Anton
    Roth, Stephan V.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Biocomposites. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Lundell, Fredrik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center. KTH, School of Engineering Sciences (SCI), Engineering Mechanics.
    Lendel, Christofer
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Confinement-induced self-assembly of whey protein nanofibrils into microscale fibersManuscript (preprint) (Other academic)
    Abstract [en]

    Protein conformation and self-assembly dictates function and structure on different hierarchical scales. We herein explore confinement-induced assembly of whey protein nanofibrils (PNF) into microscale fibers, using microfocused synchrotron X-ray scattering. The solvent evaporation forces the PNFs to align in an anisotropic fiber and the process is followed in situ by scattering experiments in a droplet placed between two anchor points. Controlling the temperature allows for variation of the evaporation rate and exploration of how the kinetics of the alignment process affect the final structure. Our findings show that there is an optimal temperature at which the order of the protein fiber has a maximum. This suggests that the degree of order results from a balance between the time scales of the forced alignment by the droplet surface and the rotational diffusion of the fibrils. Moreover, we observe that the assembly process depends on the nanoscale morphology of the PNFs. Stiff PNFs with a persistence length in the micrometer scale are aligned at the air-water interface and the anisotropy gradually decrease towards the center of the droplet. Flexible fibrils with a short persistence length (< 100 nm) align uniformly throughout the droplet, possibly due to stronger local entanglements.

  • 29.
    Qi, Xingmei
    et al.
    Soochow Univ, Jiangsu Key Lab Infect & Immun, Inst Biol & Med Sci, Suzhou 215123, Peoples R China..
    Wang, Yu
    Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.;Northeast Forestry Univ, Coll Wildlife & Protected Area, Harbin 150040, Peoples R China..
    Yu, Hairui
    Soochow Univ, Jiangsu Key Lab Infect & Immun, Inst Biol & Med Sci, Suzhou 215123, Peoples R China..
    Liu, Ruifang
    Soochow Univ, Jiangsu Key Lab Infect & Immun, Inst Biol & Med Sci, Suzhou 215123, Peoples R China..
    Leppert, Axel
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, S-17165 Solna, Sweden..
    Zheng, Zihan
    Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.;Xi An Jiao Tong Univ, Dept Pharmacol, Xian 710061, Shaanxi, Peoples R China..
    Zhong, Xueying
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Jin, Zhen
    Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.;Xi An Jiao Tong Univ, Dept Pharmacol, Xian 710061, Shaanxi, Peoples R China..
    Wang, Han
    Soochow Univ, Jiangsu Key Lab Infect & Immun, Inst Biol & Med Sci, Suzhou 215123, Peoples R China..
    Li, Xiaoli
    Chongqing Med Univ, Dept Pharmacol, Coll Pharm, Chongqing 400016, Peoples R China..
    Wang, Xiuzhe
    Shanghai Jiao Tong Univ, Dept Neurol, Sch Med, Shanghai Peoples Hosp 6, Shanghai 200233, Peoples R China..
    Landreh, Michael
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, S-17165 Solna, Sweden..
    Morozova-Roche, Ludmilla A.
    Umeå Univ, Dept Med Biochem & Biophys, S-90187 Umeå, Sweden..
    Johansson, Jan
    Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, 14157 Sweden.
    Xiong, Sidong
    Soochow Univ, Jiangsu Key Lab Infect & Immun, Inst Biol & Med Sci, Suzhou 215123, Peoples R China..
    Iashchishyn, Igor
    Umeå Univ, Dept Med Biochem & Biophys, S-90187 Umeå, Sweden..
    Chen, Gefei
    Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden..
    Spider Silk Protein Forms Amyloid-Like Nanofibrils through a Non-Nucleation-Dependent Polymerization Mechanism (Small 46/2023)2023In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 19, no 46, article id 2370388Article in journal (Refereed)
    Abstract [en]

    Both spider silk protein and Alzheimer's disease associated amyloid-β peptide can assemble into silk-like fibers that consist of nanofibrils. Spider silk proteins' unique primary structures have evolved to allow functional amyloid-like properties and direct their fibrillization pathways to avoid formation of cytotoxic intermediates. More details can be found in article number 2304031 by Gefei Chen and co-workers.

  • 30.
    Reichenbach, Tom
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Structural and biochemical insights into biosynthesis and degradation of N-glycans2020Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Carbohydrates are a primary energy source for all living organisms, but importantly, they also participate in a number of life-sustaining biological processes, e.g. cell signaling and cell-wall synthesis. The first part of the thesis examines glycosyltransferases that play a crucial role in the biosynthesis of N-glycans. Precursors to eukaryotic N-glycans are synthesized in the endoplasmic reticulum (ER) in the form of a lipid-bound oligosaccharide, which is then transferred to a nascent protein. The first seven sugar units are assembled on the cytoplasmic side of the ER, which is performed by glycosyltransferases that use nucleotide sugars as donors. The mannosyl transferase PcManGT is produced by the archaeon Pyrobaculum calidifontis, and the biochemical and structural results presented in the thesis suggest that the enzyme may be a counterpart to the glycosyltransferase Alg1 that participates in the biosynthesis of N-glycans in eukaryotes. Within the ER (in the lumen), activated dolichol-bound sugars are used as donor substrates instead of nucleotide sugars for glycosyltransferases that synthesize N-glycans. The glycosyltransferase dolichylphosphate mannose synthase (DPMS) catalyzes the formation of dolichylphosphate mannose, which is one of these dolichyl-bound sugars. The structure and function were studied for DPMS from Pyrococcus furiosus using protein X-ray crystallographic and biochemical methods and a new assay based on proteoliposomes was designed. The second part of the thesis focuses on glycoside hydrolases from bacteria that break down oligo- and polysaccharides. In one of the studies, a bacterial glycoside hydrolase from the acne bacterium Cutibacterium acnes was characterized. The enzyme was shown to be able to break down the host's N-glycans, which can be used as nutrients or perhaps even evade detection of the immune system. This study also suggests a cytoplasmic biosynthetic pathway for the formation of N-glycans in the acne bacterium. In another study, a glycoside hydrolase from a bacterium living in the moose rumen was characterized. The enzyme was shown to be able to break down β-1,3-glucans, which is a property that can be used industrially for biomass treatment.

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    Doctoral thesis Tom Reichenbach
  • 31.
    Reichenbach, Tom
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Gandini, Rosaria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Kalyani, Dayanand
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Divne, Christina
    KTH, Superseded Departments (pre-2005), Biotechnology. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Is Pyrococcus furiosus dolichylphosphate mannose synthase moonlighting as a biogenic flippase for dolichylphosphate mannose?Manuscript (preprint) (Other academic)
    Abstract [en]

    Dolichylphosphate mannose synthase (DPMS) performs an essential function by synthesizing the activated lipid-linked sugar intermediated required by non-Leloir mannosyltransferases involved in protein glycosylation pathways. In eukaryotes and archaea, DPMS catalyzes the transfer of a mannose unit from GDP-mannose to dolichylphosphate to generate dolichylphosphate mannose (Dol-P-Man). DPMS from the extremophilic archaeon Pyrococcus furiosus (PfDPMS) belongs to the type-III class of DPMSs with a large catalytic domain attached to a transmembrane (TM) domain with two dimers of TM helices, TMD1 and TMD2, oriented such that TMD2 forms a 45° angle with TMD1. Our earlier work showed that the TM domain is dispensable for Dol-Pmannosylation, which brought its biological relevance into question. Here, we present crystallographic and bioinformatic evidence for a role for the TM domain in membrane translocation of Dol-P-Man. We crystallized a reaction performed at a temperature where PfDPMS activity is low, and determined the crystal structure at 2.9 Å resolution. Although the experimental data is deeply convoluted by traces of different structural states, we observed a strong signal for a Dol-P-Man molecule flipped inside the protein. Dol-P-Man is found to be flipped across TMD2 to position the mannosylphosphate head group in a polar pocket between TMD1 and TMD2. A role of the TM domain in glycolipid translocation is further discussed based on its topological resemblance to the GtrA family of small membrane transporters. Additionally, we identfied a dpm2 gene candidate that may serve as the missing translocation domain for the previously characterized type-I DPMS from Saccharmomyces cerevisiae.

  • 32. Rutsdottir, Gudrun
    et al.
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Weide, Yoran
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Ib Rasmussen, Morten
    Højrup, Peter
    Söderberg, Christopher
    Emanuelsson, Cecilia
    Structure model obtained by homology modelling and cryo-EM for the Hsp21 dodecamer and evaluation of the importance of oligomerization for chaperone activityManuscript (preprint) (Other academic)
  • 33.
    Schriever, Karen
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Sequence- and structure guided engineering of proteins and enzymes for biotechnology and health applications2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins are highly diverse and sophisticated biomolecules that represent a cornerstone of biological structure and function and have been exploited in man-made applications for thousands of years. Those proteins that facilitate chemical reactions at physiologically relevant time-scales are referred to as enzymes. Understanding the connections between proteins’ functions and their structures, mechanisms and evolution allows to engineer them towards desired properties for various applications. The aim of the work presented in this thesis is to assess different protein engineering approaches and workflows in the context of health and biotechnology applications. Four proteins were studied and/or engineered towards different outcomes using either sequence‑based information, structural information or a combination thereof. In paper I a sequence-based approach was applied to optimise vaccine candidates for severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). Specifically, ancestral sequence reconstruction was used to generate highly stable and soluble antigens that could be produced in high quantities in a low-throughput and structure‑independent manner. These ancestral antigens interacted with antibodies from recovered patients and served as scaffolds to host a domain of the extant antigen to further enhance antibody engagement. Paper II and III applied enzyme engineering to terpene cyclases in a health and biocatalysis context, respectively. In paper II a structure-based approach was used to understand the fundamental principles underlying the catalytic mechanism of an enzyme in human steroid metabolism. Specifically, solvent access tunnels were identified and modified to probe the role of activation entropy in human oxidosqualene cyclase, which drastically modified the temperature dependence of catalysis. This finding may also have implications for engineering related plant enzymes for production of industrially relevant compounds in heterologous hosts. In paper III sequence- and structure based approaches were used together to engineer substrate specificity in a promiscuous bacterial terpene cyclase. Specifically, the structure of a stable reconstructed ancestor of spiroviolene synthase was determined in order to understand the molecular basis of substrate promiscuity and engineer highly selective variants that retained thermostability. The presented workflow is relevant for engineering these enzymes as biocatalysts for production of terpene-based high value compounds. In paper IV the metabolite regulation of a flux-controlling enzyme in the Calvin cycle was studied to eventually engineer it for enhanced growth of autotrophic production hosts. Specifically, interactions between a bifunctional cyanobacterial fructose‑1,6-bisphosphatase and a panel of metabolites were identified using a proteomics approach and verified by in vitro experiments. A synergistic regulation involving the enzyme’s redox state and glyceraldehyde 3‑phosphate was discovered, which has implications for integrated metabolic and enzyme engineering approaches involving this biocatalyst. In summary, the results presented herein highlight the utility of integrating several different engineering approaches for proteins used in health and biotechnology applications. 

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    Sequence- and structure guided engineering of proteins and enzymes for biotechnology and health applications - Kappa - Karen Schriever
  • 34. Schulte, T.
    et al.
    Sala, Benedetta Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Nilvebrant, Johan
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Achour, A.
    Shernyukov, A.
    Agback, T.
    Agback, P.
    Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal serine-rich repeat protein (PsrP-BR) reveal a rigid monomer in solution2020In: Biomolecular NMR Assignments, ISSN 1874-2718, E-ISSN 1874-270XArticle in journal (Refereed)
    Abstract [en]

    The pneumococcal serine rich repeat protein (PsrP) is displayed on the surface of Streptococcus pneumoniae with a suggested role in colonization in the human upper respiratory tract. Full-length PsrP is a 4000 residue-long multi-domain protein comprising a positively charged functional binding region (BR) domain for interaction with keratin and extracellular DNA during pneumococcal adhesion and biofilm formation, respectively. The previously determined crystal structure of the BR domain revealed a flat compressed barrel comprising two sides with an extended β-sheet on one side, and another β-sheet that is distorted by loops and β-turns on the other side. Crystallographic B-factors indicated a relatively high mobility of loop regions that were hypothesized to be important for binding. Furthermore, the crystal structure revealed an inter-molecular β-sheet formed between edge strands of two symmetry-related molecules, which could promote bacterial aggregation during biofilm formation. Here we report the near complete 15N/13C/1H backbone resonance assignment of the BR domain of PsrP, revealing a secondary structure profile that is almost identical to the X-ray structure. Dynamic 15N-T1, T2 and NOE data suggest a monomeric and rigid structure of BR with disordered residues only at the N- and C-termini. The presented peak assignment will allow us to identify BR residues that are crucial for ligand binding. 

  • 35.
    Svedendahl Humble, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Håkansson, Maria
    Kimbung, Yengo R
    Walse, Björn
    Abedi, Vahak
    Federsel, Hans-Jürgen
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Logan, Derek T
    Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 5, p. 779-792Article in journal (Refereed)
    Abstract [en]

    The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique 'relaxed' conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered 'roof ' over the PLP-binding site, whereas in the other apo form and the holo form the 'roof' is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the 'roof' structure may be associated with substrate binding in Cv-ωTA and some other transaminases. DATABASE: The atomic coordinates and structure factors for the Chromobacterium violaceumω-transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes 4A6U for the holoenzyme, 4A6R for the apo1 form, 4A6T for the apo2 form and 4A72 for the mixed form STRUCTURED DIGITAL ABSTRACT: •  -transaminases and -transaminases bind by dynamic light scattering (View interaction) •  -transaminase and -transaminase bind by x-ray crystallography (View interaction) •  -transaminase and -transaminase bind by x-ray crystallography (View interaction).

  • 36.
    Trillo-Muyo, Sergio
    et al.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Nilsson, Harriet
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden.;Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.
    Recktenwald, Christian V.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Ermund, Anna
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Ridley, Caroline
    Univ Manchester, Manchester Acad Hlth Sci Ctr, Wellcome Trust Ctr Cell Matrix Res, Fac Biol Med & Hlth, Manchester M13 9PT, Lancs, England..
    Meiss, Lauren N.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Baehr, Andrea
    Ludwig Maximilians Univ Munchen, Inst Mol Anim Breeding & Biotechnol, Gene Ctr, Hackerstr 27, D-85764 Oberschleissheim, Germany..
    Klymiuk, Nikolai
    Ludwig Maximilians Univ Munchen, Inst Mol Anim Breeding & Biotechnol, Gene Ctr, Hackerstr 27, D-85764 Oberschleissheim, Germany..
    Wine, Jeffrey J.
    Stanford Univ, Cyst Fibrosis Res Lab, Stanford, CA 94305 USA..
    Koeck, Philip J. B.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.
    Thornton, David J.
    Univ Manchester, Manchester Acad Hlth Sci Ctr, Wellcome Trust Ctr Cell Matrix Res, Fac Biol Med & Hlth, Manchester M13 9PT, Lancs, England..
    Hebert, Hans
    Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.;KTH Royal Inst Technol, Sch Technol & Hlth, S-14157 Huddinge, Sweden..
    Hansson, Gunnar C.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 15, p. 5746-5754Article in journal (Refereed)
    Abstract [en]

    Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.

  • 37. Vijayvargia, Ravi
    et al.
    Epand, Raquel
    Leitner, Alexander
    Jung, Taeyang
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Korea Adv Inst Sci & Technol, KAIST Inst BioCentury, Korea.
    Shin, Baehyun
    Jung, Roy
    Lloret, Alejandro
    Atwal, Randy Singh
    Lee, Hyeongseok
    Lee, Jong-Min
    Aebersold, Ruedi
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Song, Ji-Joon
    Seong, Ihn Sik
    Huntingtin's spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function2016In: eLIFE, E-ISSN 2050-084X, Vol. 5, article id e11184Article in journal (Refereed)
    Abstract [en]

    The polyglutamine expansion in huntingtin protein causes Huntington's disease. Here, we investigated structural and biochemical properties of huntingtin and the effect of the polyglutamine expansion using various biophysical experiments including circular dichroism, single particle electron microscopy and cross-linking mass spectrometry. Huntingtin is likely composed of five distinct domains and adopts a spherical alpha-helical solenoid where the amino-terminal and carboxyl-terminal regions fold to contain a circumscribed central cavity. Interestingly, we showed that the polyglutamine expansion increases alpha-helical properties of huntingtin and affects the intramolecular interactions among the domains. Our work delineates the structural characteristics of full-length huntingtin, which are affected by the polyglutamine expansion, and provides an elegant solution to the apparent conundrum of how the extreme amino-terminal polyglutamine tract confers a novel property on huntingtin, causing the disease.

  • 38.
    Zhu, Lin
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Structural Studies of High Density Lipoprotein by Electron Microscopy and Flexible FittingManuscript (preprint) (Other academic)
    Download full text (pdf)
    fulltext
  • 39.
    Zhu, Lin
    et al.
    KTH, School of Technology and Health (STH), Medical Engineering, Structural Biotechnology. Karolinska Institutet, Sweden.
    Petrlova, J.
    Gysbers, P.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Medical Engineering, Structural Biotechnology. Karolinska Institutet, Sweden.
    Wallin, S.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH). Karolinska Institutet, Sweden.
    Lagerstedt, J. O.
    Structures of apolipoprotein A-I in high density lipoprotein generated by electron microscopy and biased simulations2017In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1861, no 11, p. 2726-2738Article in journal (Refereed)
    Abstract [en]

    Background: Apolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) is a key protein for the transport of cholesterol from the vascular wall to the liver. The formation and structure of nascent HDL, composed of apoA-I and phospholipids, is critical to this process. Methods: The HDL was assembled in vitro from apoA-I, cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a 1:4:50 molar ratio. The structure of HDL was investigated in vitreous samples, frozen at cryogenic temperatures, as well as in negatively stained samples by transmission electron microscopy. Low resolution electron density maps were next used as restraints in biased Monte Carlo simulations of apolipoprotein A-I dimers, with an initial structure derived from atomic resolution X-ray structures. Results: Two final apoA-I structure models for the full-length structure of apoA-I dimer in the lipid bound conformation were generated, showing a nearly circular, flat particle with an uneven particle thickness. Conclusions: The generated structures provide evidence for the discoidal, antiparallel arrangement of apoA-I in nascent HDL, and propose two preferred conformations of the flexible N-termini.

  • 40.
    Öktem, Ozan
    Sidec, Kista, Sweden.
    Reconstruction methods in electron tomography2008In: Mathematical Methods in Biomedical Imaging and Intensity-Modulated Radiation Therapy (IMRT) / [ed] Y. Censor, Jiang M., and Louis A. K., Springer Berlin/Heidelberg, 2008, p. 289-320Chapter in book (Refereed)
    Abstract [en]

    Already in 1968 one recognized that the transmission electron micro- scope could be used in a tomographic setting as a tool for structure determination of macromolecules. However, its usage in mainstream structural biology has been limited and one reason is the devastating combination of noisy data and incomplete data problems that leads to severe ill-posedness of the inverse problem. Despite these issues, the importance of electron tomography is beginning to increase, espe- cially in drug discovery. This review begins with a brief introduction to the model for image formation, i.e. the forward operator. Next, we state the difficulties and review some of the various attempts at overcoming those in solving the inverse problem.

  • 41.
    Öktem, Ozan
    et al.
    Sidec AB, Kista, Sweden.
    Fanelli, Duccio
    University of Florens.
    Electron tomography: A short overview with an emphasis on the absorption potential model for the forward problem2008In: Inverse Problems, ISSN 0266-5611, E-ISSN 1361-6420, Vol. 24, no 1, p. 013001-Article in journal (Refereed)
    Abstract [en]

    This review of the development and current status of electron tomography deals mainly with the mathematical and algorithmic aspects. After a very brief description of the role of electron tomography in structural biology, we turn our attention to the derivation of the forward operator. Starting from the Schrodinger equation, the electron - specimen interaction is modelled as a diffraction tomography problem and the picture is completed by adding a description of the optical system of the transmission electron microscope. The first- order Born approximation enables one to explicitly express the intensity for any finite wavenumber in terms of the propagation operator acting on the specimen convolved with a point spread function, derived from the optics in the transmission electron microscope. Next, we focus on the difficulties that cause the reconstruction problem to be quite challenging. Special emphasis is put on explaining the extremely low signal- to- noise ratio in the data combined with the incomplete data problems, which lead to severe ill- posedness. The next step is to derive the standard phase contrast model used in the electron tomography community. The above- mentioned expression for the intensity generalizes the standard phase contrast model which can be obtained by replacing the propagation operator by its high- energy limit, the x- ray transform, as the wavenumber tends to infinity. The importance of more carefully including the wave nature of the electron - specimen interaction is supported by performing an asymptotic analysis of the intensity as the wavenumber tends to infinity. Next we provide an overview of the various reconstruction methods that have been employed in electron tomography and we conclude by mentioning a number of open problems. Besides providing an introduction to electron tomography written in the 'language of inverse problems', the authors hope to raise interest among experts in integral geometry and regularization theory for the mathematical and algorithmic difficulties that are encountered in electron tomography.

  • 42.
    Öktem, Ozan
    et al.
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Mathematics (Div.). KTH, School of Engineering Sciences (SCI), Centres, Center for Industrial and Applied Mathematics, CIAM.
    Quinto, Eric Todd
    Tufts University.
    Skoglund, Ulf
    Okinawa Institute of Science and Technology.
    Electron Lambda-tomography2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 51, p. 21842-21847Article in journal (Refereed)
    Abstract [en]

    Filtered back-projection and weighted back-projection have long been the methods of choice within the electron microscopy com- munity for reconstructing the structure of macromolecular assem- blies from electron tomography data. Here, we describe electron lambda-tomography, a reconstruction method that enjoys the ben- efits of the above mentioned methods, namely speed and ease of implementation, but also addresses some of their shortcomings. In particular, compared to these standard methods, electron lambda- tomography is less sensitive to artifacts that come from structures outside the region that is being reconstructed, and it can sharpen boundaries.

1 - 42 of 42
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