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  • 1. Ambort, D.
    et al.
    Johansson, M.E.V.
    Gustafsson, J. K.
    Nilsson, Harriet E.
    Ermund, A.
    Johansson, B.R.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Hansson, G.C.
    Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 15, p. 5645-5650Article in journal (Refereed)
    Abstract [en]

    MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D'D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with full-length MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca2+ it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. TheMUC2-N aggregates were dissolved by removing Ca2+ and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.

  • 2. Andersson, Marlene
    et al.
    Jia, Qiupin
    Abella, Ana
    Lee, Xiau-Yeen
    Landreh, Michael
    Purhonen, Pasi
    KTH, School of Technology and Health (STH).
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Tenje, Maria
    Robinson, Carol V.
    Meng, Qing
    Plaza, Gustavo R.
    Johansson, Jan
    Rising, Anna
    Biomimetic spinning of artificial spider silk from a chimeric minispidroin2017In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 13, no 3, p. 262-+Article in journal (Refereed)
    Abstract [en]

    Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.

  • 3.
    B. Kumar, Ramakrishnan
    et al.
    Karolinska institutet, Sverige.
    Zhu, Lin
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska institutet, Sverige.
    Jegerschöld, Caroline
    Karolinska institutet, Sverige.
    Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy2017In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 121, article id e55148Article in journal (Refereed)
    Abstract [en]

    Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

  • 4.
    Balakrishnan Kumar, Ramakrishnan
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet, Sweden.
    Zhu, Lin
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet,.
    Idborg, Helena
    Radmark, Olof
    Jakobsson, Per-Johan
    Rinaldo-Matthis, Agnes
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet,.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet,.
    Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 3, article id e0152116Article in journal (Refereed)
    Abstract [en]

    An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron micros-copy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.

  • 5. Braniste, Viorica
    et al.
    Al-Asmakh, Maha
    Kowal, Czeslawa
    Anuar, Farhana
    Abbaspour, Afrouz
    Toth, Miklos
    Korecka, Agata
    Bakocevic, Nadja
    Guan, Ng Lai
    Kundu, Parag
    Gulyas, Balazs
    Halldin, Christer
    Hultenby, Kjell
    Nilsson, Harriet
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Volpe, Bruce T.
    Diamond, Betty
    Pettersson, Sven
    The gut microbiota influences blood-brain barrier permeability in mice2014In: Science Translational Medicine, ISSN 1946-6234, E-ISSN 1946-6242, Vol. 6, no 263, p. 263ra158-Article in journal (Refereed)
    Abstract [en]

    Pivotal to brain development and function is an intact blood-brain barrier (BBB), which acts as a gatekeeper to control the passage and exchange of molecules and nutrients between the circulatory system and the brain parenchyma. The BBB also ensures homeostasis of the central nervous system (CNS). We report that germ-free mice, beginning with intrauterine life, displayed increased BBB permeability compared to pathogen-free mice with a normal gut flora. The increased BBB permeability was maintained in germ-free mice after birth and during adulthood and was associated with reduced expression of the tight junction proteins occludin and claudin-5, which are known to regulate barrier function in endothelial tissues. Exposure of germ-free adult mice to a pathogen-free gut microbiota decreased BBB permeability and up-regulated the expression of tight junction proteins. Our results suggest that gut microbiota-BBB communication is initiated during gestation and propagated throughout life.

  • 6. Brismar, Torkel B.
    et al.
    Grishenkov, Dmitry
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Gustafsson, Björn
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Barrefelt, Åsa
    Kothapalli, Satya V. V. N.
    KTH, School of Technology and Health (STH), Medical Engineering, Neuronic Engineering.
    Margheritelli, Silvia
    Oddo, Letizia
    Caidahl, Kenneth
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Paradossi, Gaio
    Magnetite Nanoparticles Can Be Coupled to Microbubbles to Support Multimodal Imaging2012In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 13, no 5, p. 1390-1399Article in journal (Refereed)
    Abstract [en]

    Microbubbles (MBs) are commonly used as injectable ultrasound contrast agent (UCA) in modern ultrasonography. Polymer-shelled UCAs present additional potentialities with respect to marketed lipid-shelled UCAs. They are more robust; that is, they have longer shelf and circulation life, and surface modifications are quite easily accomplished to obtain enhanced targeting and local drug delivery. The next generation of UCAs will be required to support not only ultrasound-based imaging methods but also other complementary diagnostic approaches such as magnetic resonance imaging or computer tomography. This work addresses the features of MBs that could function as contrast agents for both ultrasound and magnetic resonance imaging. The results indicate that the introduction of iron oxide nanoparticles (SPIONs) in the poly(vinyl alcohol) shell or on the external surface of the MBs does not greatly decrease the echogenicity of the host MBs compared with the unmodified one. The presence of SPIONs provides enough magnetic susceptibility to the MBs to accomplish good detectability both in vitro and in vivo. The distribution of SPIONs on the shell and their aggregation state seem to be key factors for the optimization of the transverse relaxation rate.

  • 7. Busenlehner, Laura S.
    et al.
    Alander, Johan
    Jegerscohld, Caroline
    Holm, Peter J.
    Bhakat, Priyaranjan
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Morgenstern, Ralf
    Armstrong, Richard N.
    Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-12007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 10, p. 2812-2822Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione transferase-1 (MGST1) is a trimeric, membrane-bound enzyme with both glutathione (GSH) transferase and hydroperoxidase activities. As a member of the MAPEG superfamily, MGST1 aids in the detoxication of numerous xenobiotic substrates and in cellular protection from oxidative stress through the GSH-dependent reduction of phospholipid hydroperoxides. However, little is known about the location of the different substrate binding sites, including whether the transferase and peroxidase activities overlap structurally. Although molecular density attributed to GSH has been observed in the 3.2 A resolution electron crystallographic structure of MGST1, the electrophilic and phospholipid hydroperoxide substrate binding sites remain elusive. Amide H-D exchange kinetics and H-D ligand footprinting experiments indicate that GSH and hydrophobic substrates bind within similar, but distinct, regions of MGST1. Site-directed mutagenesis, guided by the H-D exchange results, demonstrates that specific residues within the GSH footprint effect transferase activity toward 1-chloro-2,4-dinitrobenzene. In addition, cytosolic residues surrounding the chemical stress sensor C49 but not modeled in the crystal structure appear to play an important role in the formation of the binding site for hydrophobic substrates. Although the fatty acid/phospholipid binding site structurally overlaps that for GSH, it does not appear to be localized to the same region as other hydrophobic substrates. Finally, H-D exchange mass spectrometry reveals a specific conformational transition that may mediate substrate binding and/or product release. Such structural changes in MGST1 are essential for activation of the enzyme and are important for its biological function.

  • 8. Busenlehner, L.S.
    et al.
    Codreanu, S.G.
    Holm, P.J.
    Bhakat, P.
    Hebert, Hans
    Karolinska Institutet.
    Morgenstern, R.
    Armstrong, R.N.
    Stress sensor triggers conformational response of the integral membrane protein microsomal glutathione transferase 12004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 35, p. 11145-11152Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione (GSH) transferase 1 (MGST1) is a trimeric, integral membrane protein involved in cellular response to chemical or oxidative stress. The cytosolic domain of MGST1 harbors the GSH binding site and a cysteine residue (C49) that acts as a sensor of oxidative and chemical stress. Spatially resolved changes in the kinetics of backbone amide H/D exchange reveal that the binding of a single molecule of GSH/trimer induces a cooperative conformational transition involving movements of the transmembrane helices and a reordering of the cytosolic domain. Alkylation of the stress sensor preorganizes the helices and facilitates the cooperative transition resulting in catalytic activation.

  • 9. Cheng, K.
    et al.
    Koeck, P.J.B.
    Idakieva, K.
    Ternström, T.
    Parvanova, K.
    Hebert, Hans
    Stainless Models of Rapana Thomasiana Hemocyanin2004In: Proc. of the 13th European Congress on Electron Microscopy, 2004, p. 22-27Conference paper (Refereed)
  • 10.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ivanova, Natalia
    Biomedicinskt centrum, Uppsala.
    Scheres, Sjores
    CSIC, Natl Biotechnol Ctr, Biocomp Unit, E-28049 Madrid, Spain .
    Pavlov, Michael Y
    Biomedicinskt centrum, Uppsala.
    Maria Carazo, Jose
    Lund Univ, Mol Biophys KILU.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ehrenberg, Måns
    Biomedicinskt centrum, Uppsala.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    tmRNA-SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes2010In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 169, no 3, p. 342-348Article in journal (Refereed)
    Abstract [en]

    Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15 A resolution structure of the tmRNA-SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA-SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation. (C) 2009 Elsevier Inc. All rights reserved.

  • 11.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Elmlund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Idakieva, Krassimira
    Parvanova, Katja
    Schwarz, Heinz
    Ternström, Tomas
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Comparison of the two Rapana thomasiana Hemocyanin isoforms: RtH1 and RtH22006In: Proc 16. International Microscopy Conference, 2006Conference paper (Refereed)
  • 12.
    Cheng, Kimberley
    et al.
    Department of Biosciences at NOVUM, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, S-141 57 Huddinge, Sweden.
    Koeck, Philip J. B.
    Department of Biosciences at NOVUM, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, S-141 57 Huddinge, Sweden.
    Elmund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ternström, Tomas
    Schwarz, Heinz
    Idakieva, Krassimira
    Parvanova, Katja
    Rapana thomasiana hemocyanin (RtH): Comparison of the two isoforms, RtH1 and RtH2, at 19 Å and 16 Å resolution2006In: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 37, no 6, p. 566-576Article in journal (Refereed)
    Abstract [en]

    Three-dimensional (3D) reconstructions of the two 8.4 MDa Rapana thomasiana hemocyanin isoforms, RtH1 and RtH2, have been obtained by cryoelectron microscopy of molecules embedded in vitreous ice and single particle image processing. The final 3D structures of the RtH1 and RtH2 didecamers at 19 angstrom and 16 angstrom resolution, respectively, are very similar to earlier reconstructions of gastropodan hemocyanins, revealing structural features such as the obliquely oriented subunits, the five- and two-fold symmetrical axes. Three new interactions are defined; two of them connecting the arch and the wall while the third is formed between the collar and the wall. The collar-wall connection and one of the arch-wall connections are positioned between two individual subunit dimers, while the second arch-wall connection is located between two subunits within the subunit dimer. All three interactions establish connections to the first tier of the wall. Furthermore, for each interaction we have allocated two first tier functional units most likely involved in forming the connections.

  • 13.
    Elmlund, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Baraznenok, Vera
    Division of Metabolic Diseases, Karolinska Institutet.
    Lindahl, Martin
    Department of Molecular Biophysics, Lund University.
    Samuelsen, Camilla O.
    Department of Genetics, Institute of Molecular Biology, Copenhagen.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH).
    Holmberg, Steen
    Department of Genetics, Institute of Molecular Biology, Copenhagen.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gustafsson, Claes M.
    Division of Metabolic Diseases, Karolinska Institutet.
    The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II2006In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 43, p. 15788-15793Article in journal (Refereed)
    Abstract [en]

    CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.

  • 14.
    Elmlund, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Baraznenok, Vera
    Division of Metabolic Diseases, Karolinska Institutet.
    Linder, Tomas
    Division of Metabolic Diseases, Karolinska Institutet.
    Rofougaran, Reza
    Dept. of Medical Biochemistry and Biophysics, Umeå University.
    Hofer, Anders
    Dept. of Medical Biochemistry and Biophysics, Umeå University.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gustafsson, Claes M.
    Dept. of Medical Biochemistry and Cell Biology, Göteborg University.
    Visualization of a massive TBP-binding coupled histone-fold domain rearrangement within the general transcription factor IIDArticle in journal (Other academic)
  • 15. Elmlund, Hans
    et al.
    Baraznenok, Vera
    Linder, Tomas
    Szilagyi, Zsolt
    Rofougaran, Reza
    Hofer, Anders
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lindahl, Martin Joakim
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gustafsson, Claes M.
    Cryo-EM Reveals Promoter DNA Binding and Conformational Flexibility of the General Transcription Factor TFIID2009In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 17, no 11, p. 1442-1452Article in journal (Refereed)
    Abstract [en]

    The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1(N-terminus) atomic structure to the THID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions.

  • 16.
    Elmlund, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lundqvist, Joakirn
    Lund Univ, Dept Mol Biophys.
    Al-Karadaghi, Salam
    Lund Univ, Dept Mol Biophys.
    Hansson, Mats
    Lund Univ, Dept Biochem.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    A new cryo-EM single-particle ab initio reconstruction method visualizes secondary structure elements in an ATP-fueled AAA+ motor2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 375, no 4, p. 934-947Article in journal (Refereed)
    Abstract [en]

    The generation of ab initio three-dimensional (3D) models is a bottleneck in the studies of large macromolecular assemblies by single-particle cryo-electron microscopy. We describe here a novel method, in which established methods for two-dimensional image processing are combined with newly developed programs for joint rotational 3D alignment of a large number of class averages (RAD) and calculation of 3D volumes from aligned projections (VolRec). We demonstrate the power of the method by reconstructing an similar to 660-kDa ATP-fueled AAA+ motor to 7.5 angstrom resolution, with secondary structure elements identified throughout the structure. We propose the method as a generally applicable automated strategy to obtain 3D reconstructions from unstained single particles imaged in vitreous ice.

  • 17. Ermund, Anna
    et al.
    Meiss, Lauren N.
    Rodriguez-Pineiro, Ana M.
    Baehr, Andrea
    Nilsson, Harriet E.
    KTH, School of Technology and Health (STH), Medical Engineering. Department of Medical Biochemistry, University of Gothenburg, SE-405 30 Gothenburg, Sweden; Department of Biosciences and Nutrition, Karolinska Institutet, and School of Technology and Health, KTH Royal Institute of Technology, Novum, SE-141 57 Huddinge, Sweden.
    Trillo-Muyo, Sergio
    Ridley, Caroline
    Thornton, David J.
    Wine, Jeffrey J.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Medical Engineering, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet, and School of Technology and Health, KTH Royal Institute of Technology, Novum, SE-141 57 Huddinge, Sweden.
    Klymiuk, Nikolai
    Hansson, Gunnar C.
    The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin2017In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 492, no 3, p. 331-337Article in journal (Refereed)
    Abstract [en]

    To understand the mucociliary clearance system, mucins were visualized by light, confocal and electron microscopy, and mucus was stained by Alcian blue and tracked by video microscopy on tracheal explants of newborn piglets. We observed long linear mucus bundles that appeared at the submucosal gland openings and were transported cephalically. The mucus bundles were shown by mass spectrometry and immunostaining to have a core made of MUC5B mucin and were coated with MUC5AC mucin produced by surface goblet cells. The transport speed of the bundles was slower than the airway surface liquid flow. We suggest that the goblet cell MUC5AC mucin anchors the mucus bundles and thus controls their transport. Normal clearance of the respiratory tree of pigs and humans, both rich in submucosal glands, is performed by thick and long mucus bundles. 

  • 18. Gustafsson, JK
    et al.
    Ermund, Anna
    Ambort, Daniel
    Johansson, MEV
    Nilsson, HE
    Thorell, K
    Hebert, Hans
    Karolinska Institutet, Sweden .
    Sjövall, H
    Hansson, Gunnar
    Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype2012In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 209, no 7, p. 1263-1272Article in journal (Refereed)
    Abstract [en]

    Cystic fibrosis (CF) is caused by a nonfunctional chloride and bicarbonate ion channel (CF transmembrane regulator [CFTR]), but the link to the phenomenon of stagnant mucus is not well understood. Mice lacking functional CFTR (CftrΔ508) have no lung phenotype but show similar ileal problems to humans. We show that the ileal mucosa in CF have a mucus that adhered to the epithelium, was denser, and was less penetrable than that of wild-type mice. The properties of the ileal mucus of CF mice were normalized by secretion into a high concentration sodium bicarbonate buffer (~100 mM). In addition, bicarbonate added to already formed CF mucus almost completely restored the mucus properties. This knowledge may provide novel therapeutic options for CF.

  • 19.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,.
    Two-dimensional crystallization of biological macromolecules2009In: Molecules: Nucleation, Aggregation and Crystallization: Beyond Medical and Other Implications, World Scientific Publishing Co. , 2009, p. 95-111Chapter in book (Other academic)
    Abstract [en]

    Biological macromolecules can be arranged periodically in single layers as twodimensional (2D) crystals. This enables crystallographic structure analysis using transmission electron microscopy. Periodic repeat of a large number of unit cells contributes to significant information in images or diffraction patterns from unstained specimens. Structural details at a resolution of a few Ångströms in all directions can be obtained. Such three-dimensional maps are used for building atomic models. The techniques have been used to determine structure and function relationships for membrane proteins. The dense packing obtained in 2D crystals can also be used for constructing devices at the molecular level.

  • 20.
    Hebert, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Jegerschold, Caroline
    The structure of membrane associated proteins in eicosanoid and glutathione metabolism as determined by electron crystallography2007In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 17, no 4, p. 396-404Article, review/survey (Refereed)
    Abstract [en]

    Membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) are involved in biosynthesis of arachidonic-derived mediators of pain, fever, and inflammation as well as in biotransformation and detoxification of electrophilic substances. Structure determination of microsomal glutathione transferase 1 using electron crystallography has provided the first atomic model of an MAPEG member. The homotrimer consists of three repeats of a four-helix transmembrane bundle with the largest extramembranous domain connecting the first and second helix and with a short proline rich loop on the same side between helices three and four. Residues of importance for intramolecular or intermolecular contacts as well as for stabilizing the active site have been identified and the results can be applied for interpreting structure-function relationship for similar MAPEG members.

  • 21. Holm, Peter J.
    et al.
    Bhakat, Priyaranjan
    Jegerschold, Caroline
    Gyobu, Nobuhiko
    Mitsuoka, Kaoru
    Fujiyoshi, Yoshinori
    Morgenstern, Ralf
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Structural basis for detoxification and oxidative stress protection in membranes2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 360, no 5, p. 934-945Article in journal (Refereed)
    Abstract [en]

    Synthesis of mediators of fever, pain and inflammation as well as protection against reactive molecules and oxidative stress is a hallmark of the MAPEG superfamily (membrane associated proteins in eicosanoid and glutathione metabolism). The structure of a MAPEG member, rat mictosomal glutathione transferase 1, at 3.2 angstrom resolution, solved here in complex with glutathione by electron crystallography, defines the active site location and a cytosolic domain involved in enzyme activation. The glutathione binding site is found to be different from that of the canonical soluble glutathione transferases. The architecture of the homotrimer supports a catalytic mechanism involving subunit interactions and reveals both cytosolic and membraneous substrate entry sites, providing a rationale for the membrane location of the enzyme.

  • 22.
    Härmark, Johan
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Koeck, Philip J B
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy2016In: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 85, p. 39-43Article in journal (Refereed)
    Abstract [en]

    Intravenously injected microbubbles (MBs) can be utilized as ultrasound contrast agent (CA) resulting in enhanced image quality. A novel CA, consisting of air filled MBs stabilized with a shell of polyvinyl alcohol (PVA) has been developed. These spherical MBs have been decorated with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as both ultrasound and magnetic resonance imaging (MRI) CA. In this study, a mathematical model was introduced that determined the shell thickness of two types of SPIONs decorated MBs (Type A and Type B). The shell thickness of MBs is important to determine, as it affects the acoustical properties. In order to investigate the shell thickness, thin sections of plastic embedded MBs were prepared and imaged using transmission electron microscopy (TEM). However, the sections were cut at random distances from the MB center, which affected the observed shell thickness. Hence, the model determined the average shell thickness of the MBs from corrected mean values of the outer and inner radii observed in the TEM sections. The model was validated using simulated slices of MBs with known shell thickness and radius. The average shell thickness of Type A and Type B MBs were 651nm and 637nm, respectively.

  • 23.
    Härmark, Johan
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Larsson, Malin K.
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Razuvajev, Anton
    Koeck, Philip JB
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Paradossi, Gaio
    Brodin, Lars-Åke
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Caidahl, Kenneth
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Bjällmark, Anna
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Investigation of the elimination process of a multimodal polymer-shelled contrast agent in rats using ultrasound and transmission electron microscopy2015In: Biomedical Spectroscopy and Imaging, ISSN 2212-8794, Vol. 4, no 1, p. 81-93Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: A novel polymer-shelled contrast agent (CA) with multimodal imaging and target specific potential was developed recently and tested for its acoustical properties using different in-vitro setups.

    OBJECTIVE: The aim of this study was to investigate the elimination of three types of the novel polymer-shelled CA, one unmodified and two shell modified versions, in rats.

    METHODS: The blood elimination time was estimated by measuring the image intensity, from ultrasound images of the common carotid artery, over time after a bolus injection of the three types of the novel CA. The commercially available CA SonoVue was used as a reference. The subcellular localization of the three CAs was investigated using transmission electron microscopy.

    RESULTS: The ultrasound measurements indicated a blood half-life of 17–85 s for the different types of the novel CA, which was significant longer than the blood half-life time for SonoVue. Additionally, CAs were exclusively found in the circulatory system, either taken up by, or found in the vicinity of macrophages.

    CONCLUSIONS: Compared to the commercially available CA SonoVue, the blood circulation times for the three types of the novel polymer-shelled CA were prolonged. Moreover, macrophages were suggested to be responsible for the elimination of the CA.

  • 24.
    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)
  • 25.
    Jegerschöld, Caroline
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Paweizik, Sven-Christian
    Purhonen, Pasi
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Bhakat, Priyaranian
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Gheorghe, Karina Roxana
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Gyobu, Nobuhiko
    Mitsuoka, Kaoru
    Morgenstern, Ralf
    Jakobsson, Per-Johan
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Structural basis for induced formation of the inflammatory mediator prostaglandin E-22008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 32, p. 11110-11115Article in journal (Refereed)
    Abstract [en]

    Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH(2) into PGE(2). MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH(2) to PGE(2) after displacement of the cytoplasmic half of the IN-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.

  • 26.
    Koeck, Philip J. B.
    et al.
    Karolinska Institutet.
    Purhonen, P.
    Alvang, R.
    Grundberg, B.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    3D-correlation-averaging for membrane-protein-crystals2008In: EMC 2008 14th European Microscopy Congress, 2008, p. 55-56Conference paper (Refereed)
    Abstract [en]

    Few 2-dimensional protein crystals can be used to determine high-resolution structures, whereas most electron crystallography projects remain at a resolution around 10 Ångström. This might be partly due to lack of flatness of many two-dimensional crystals [1]. We have investigated this problem and suggest single particle projection matching (3D-correlation averaging) of locally averaged unit cells to improve the quality of three-dimensional maps. Theoretical considerations and tests on simulated data demonstrate the feasibility of this refinement method [2].

  • 27. Koeck, Philip J. B.
    et al.
    Purhonen, P
    Alvang, R
    Grundberg, B
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Single-particle refinement in electron crystallography of membrane-proteins2007Conference paper (Refereed)
  • 28.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH).
    Purhonen, Pasi
    Alander, Johan
    Svensson, Richard
    Hoogland, Veronika
    Winerdal, Jens
    Spahiu, Linda
    Ottosson-Wadlund, Astrid
    Jegerschold, Caroline
    KTH, School of Technology and Health (STH).
    Morgenstern, Ralf
    Hebert, Hans
    KTH, School of Technology and Health (STH), Medical Engineering, Structural Biotechnology.
    Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 7897Article in journal (Refereed)
    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 crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer 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 supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.

  • 29.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH). Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Structure of potassium channels2015In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 17, no 19, p. 3677-3693Article, review/survey (Refereed)
    Abstract [en]

    Potassium channels ubiquitously exist in nearly all kingdoms of life and perform diverse but important functions. Since the first atomic structure of a prokaryotic potassium channel (KcsA, a channel from Streptomyces lividans) was determined, tremendous progress has been made in understanding the mechanism of potassium channels and channels conducting other ions. In this review, we discuss the structure of various kinds of potassium channels, including the potassium channel with the pore-forming domain only (KcsA), voltage-gated, inwardly rectifying, tandem pore domain, and ligand-gated ones. The general properties shared by all potassium channels are introduced first, followed by specific features in each class. Our purpose is to help readers to grasp the basic concepts, to be familiar with the property of the different domains, and to understand the structure and function of the potassium channels better.

  • 30.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Two-Dimensional Crystallization Procedure, from Protein Expression to Sample Preparation2015In: BioMed Research International, ISSN 2314-6133, E-ISSN 2314-6141, article id 693869Article, review/survey (Refereed)
    Abstract [en]

    Membrane proteins play important roles for living cells. Structural studies of membrane proteins provide deeper understanding of their mechanisms and further aid in drug design. As compared to other methods, electron microscopy is uniquely suitable for analysis of a broad range of specimens, from small proteins to large complexes. Of various electron microscopic methods, electron crystallography is particularly well-suited to study membrane proteins which are reconstituted into two-dimensional crystals in lipid environments. In this review, we discuss the steps and parameters for obtaining large and well-ordered twodimensional crystals. A general description of the principle in each step is provided since this information can also be applied to other biochemical and biophysical methods. The examples are taken from our own studies and published results with related proteins. Our purpose is to give readers a more general idea of electron crystallography and to share our experiences in obtaining suitable crystals for data collection.

  • 31. Kuang, Qie
    et al.
    Purhonen, Pasi
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    The projection structure of Kch, a putative potassium channel in Escherichia coli, by electron crystallography2014In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 1, p. 237-243Article in journal (Refereed)
    Abstract [en]

    The kch gene, the only potassium channel gene in Escherichia coil, has the property to express both full-length Kch and its cytosolic domain (RCK) due to a methionine at position 240. The RCK domains are believed to form an octameric ring structure and regulate the gating of the potassium channels after having bound certain ligands. Several different gating ring structures have been reported for the soluble RCK domains, however, these were studied isolated from their transmembrane parts. We previously reported an octameric structure of Kch in solution by electron microscopy and single particle reconstruction, composed of two tetrameric full-length proteins through RCK interaction. To exclude the effect of the detergent, we have now performed an electron crystallographic study of the full-length Kch in membrane bound form. Well-ordered two-dimensional crystals were grown in a natural phospholipid environment. A projection map merged from the fifteen best images extended to 6 angstrom resolution. The c12 two-sided plane group of the two-dimensional crystals showed that Kch crystallized as two symmetrically related overlapping layers. The arrangement suggests that the two layers of RCK domains are shifted with respect to each other and the RCK octameric gating ring of Kch does not form under the crystallization condition.

  • 32.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Jegerschöld, Caroline
    Köck, Philip
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, 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.

  • 33.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Purhonen, Pasi
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Pattipaka, Thirupathi
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Ayele, Yohannes H
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Köck, Philip J B
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    A Refined Single-Particle Reconstruction Procedure to Process Two-Dimensional Crystal Images from Transmission Electron Microscopy2015In: Microscopy and Microanalysis, ISSN 1431-9276, E-ISSN 1435-8115, Vol. 21, no 4, p. 876-85Article in journal (Refereed)
    Abstract [en]

    Single-particle reconstruction (SPR) and electron crystallography (EC), two major applications in electron microscopy, can be used to determine the structure of membrane proteins. The three-dimensional (3D) map is obtained from separated particles in conventional SPR, but from periodic unit cells in EC. Here, we report a refined SPR procedure for processing 2D crystal images. The method is applied to 2D crystals of melibiose permease, a secondary transporter in Escherichia coli. The current procedure is improved from our previously published one in several aspects. The "gold standard Fourier shell correlation" resolution of our final reconstruction reaches 13 A, which is significantly better than the previously obtained 17 A resolution. The choices of different refinement parameters for reconstruction are discussed. Our refined SPR procedure could be applied to determine the structure of other membrane proteins in small or locally distorted 2D crystals, which are not ideal for EC.

  • 34.
    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.

  • 35. Kumar, Ramakrishnan B.
    et al.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Jegerschold, Caroline
    Deciphering the Interaction of FLAP and 5LO2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 540A-540AArticle in journal (Other academic)
  • 36.
    Köck, Philip J. B.
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Purhonen, Pasi
    Alvang, Ronny
    KTH, School of Technology and Health (STH).
    Grundberg, Björn
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Single particle refinement in electron crystallography: A pilot study2007In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 160, no 3, p. 344-352Article in journal (Refereed)
    Abstract [en]

    Electron crystallography can be used to determine the structures of membrane proteins at near-atomic resolution in some cases. However, most electron crystallography projects remain at a resolution around 10 angstrom. This might be partly due to lack of flatness of many two-dimensional crystals. We have investigated this problem and suggest single particle processing of locally averaged unit cells to improve the quality and possibly the resolution of three-dimensional maps. Applying this method to the secondary transporter melibiose permease we have calculated a three-dimensional map that is clearer and easier to interpret than the map derived using purely electron-crystallographic methods.

  • 37. Lambert, W
    et al.
    Koeck, Philip J. B.
    Department of Bioscience, Karolinska Institutet, Novum, Sweden.
    Ahrman, E
    Purhonen, P
    Cheng, K
    Elmlund, D
    Hebert, Hans
    Department of Bioscience, Karolinska Institutet, Novum, Sweden.
    Emanuelsson, C
    Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp212011In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, no 2, p. 291-301Article in journal (Refereed)
    Abstract [en]

    Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25 degrees in relation to each other, suggesting a role for global dynamics in dodecamer function.

  • 38. Loureiro, A.
    et al.
    Nogueira, E.
    Azoia, N.G.
    Sárria, M.P.
    Abreu, A.S.
    Shimanovich, U.
    Rollett, A.
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Guebitz, G.
    Bernardes, G.J.L.
    Preto, A.
    Gomes, A.C.
    Cavaco-Paulo, A.
    Size controlled protein nanoemulsions for active targeting of folate receptor positive cells2015In: Colloids and Surfaces B: Biointerfaces, ISSN 0927-7765, E-ISSN 1873-4367, Vol. 135, p. 90-98Article in journal (Refereed)
    Abstract [en]

    Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5. mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100. nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy.

  • 39. Lundbäck, A.K.
    et al.
    Purhonen, P.
    Leblanc, G.
    Hebert, Hans
    3D structure of melibiose permease, a sugar cotransporter from Escherichia coli2004In: : Proc of the 13th European Microscopy Congress, 2004Conference paper (Refereed)
  • 40.
    Lundbäck, Anna-Karin
    et al.
    KTH, School of Technology and Health (STH).
    Mueller, Shirley A.
    Engel, Andreas
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Assembly of Kch, a putative potassium channel from Escherichia coli2009In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 168, no 2, p. 288-293Article in journal (Refereed)
    Abstract [en]

    Attempts to explore the structure and function of Kch, a putative potassium channel of Escherichia coli have yielded varying results; potassium-associated functions have been found in vivo but not in vitro. Here the kch gene is shown to produce two proteins, full-length Kch and the large C-terminal cytosolic domain (the RCK domain). Further, these two proteins are associated at the initial stages of purification. Previous structural studies of full-length Kch claim that the isolated protein forms large aggregates that are not suitable for analysis. The results presented here show that the purified protein sample, although heterogeneous, has one major population with a mass of about 400 kDa, implying the presence of two Kch tetramers in a complex form. A three dimensional reconstruction at 25 angstrom based on electron microscopy data from negatively stained particles, revealed a 210 angstrom long and 95 angstrom wide complex in which the two tetrameric Kch units are linked by their RCK domains, giving rise to a large central ring of density. The formation of this dimer of tetramers on expression or during purification, may explain why attempts to reconstitute Kch into liposomes for activity measurements have failed.

  • 41. Lundbäck, Anna-Karin
    et al.
    van den Berg, Susanne
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Berglund, Helena
    Eshaghi, Said
    Exploring the activity of tobacco etch virus protease in detergent solutions2008In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 382, no 1, p. 69-71Article in journal (Refereed)
    Abstract [en]

    Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.

  • 42.
    Lundqvist, Joakim
    et al.
    Dept. of Molecular Biophysics, Lund University.
    Elmlund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Kasperska, Dominika
    Division of Metabolic Diseases, Karolinska Institutet.
    Axelsson, Eva
    Dept. of Biochemistry, Lund University.
    Sirijovski, Nick
    Dept. of Biochemistry, Lund University.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Willows, Robert
    Department of Chemistry and Biomolecular Sciences, Macquarie University.
    Hansson, Mats
    Dept. of Biochemistry, Lund University.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Al-Karadaghi, Salam
    Dept. of Molecular Biophysics, Lund University.
    Cryo-electron microscopy reveals an ATP-fueled and Integrin-I mediated conformational transition of the AAA+ activation complex in R. capsulatus Mg-chelataseManuscript (Other academic)
  • 43. Lundqvist, Joakim
    et al.
    Elmlund, Hans
    KTH, School of Technology and Health (STH).
    Wulff, Ragna Peterson
    Berglund, Lisa
    Elmlund, Dominika
    KTH, School of Technology and Health (STH).
    Emanuelsson, Cecilia
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Willows, Robert D.
    Hansson, Mats
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Al-Karadaghi, Salam
    ATP-Induced Conformational Dynamics in the AAA plus Motor Unit of Magnesium Chelatase2010In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 18, no 3, p. 354-365Article in journal (Refereed)
    Abstract [en]

    Mg-chelatase catalyzes the first committed step of the chlorophyll biosynthetic pathway, the ATP-dependent insertion of Mg2+ into protoporphyrin IX (PPIX). Here we report the reconstruction using single-particle cryo-electron microscopy of the complex between subunits BchD and BchI of Rhodobacter capsulatus Mg-chelatase in the presence of ADP, the nonhydrolyzable ATP analog AMPPNP, and ATP at 7.5 angstrom, 14 angstrom, and 13 angstrom resolution, respectively. We show that the two AAA+ modules of the subunits form a unique complex of 3 dimers related by a three-fold axis. The reconstructions demonstrate substantial differences between the conformations of the complex in the presence of ATIP and ADP, and suggest that the C-terminal integrin-I domains of the BchD subunits play a central role in transmitting conformational changes of BchI to BchD. Based on these data a model for the function of magnesium chelatase is proposed.

  • 44. Nilsson, Harriet E.
    et al.
    Ambort, Daniel
    Bäckström, Malin
    Thomsson, Elisabeth
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Hansson, Gunnar C.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. epartment of Biosciences and Nutrition, Karolinska Institutet.
    Intestinal MUC2 Mucin Supramolecular Topology by Packing and Release Resting on D3 Domain Assembly2014In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, no 14, p. 2567-2579Article in journal (Refereed)
    Abstract [en]

    MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D'D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D'D3 domain by producing three recombinant variants, with or without Myc tag and GFP (green fluorescent protein), and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions but eluted as hexanners upon gel filtration under native conditions. Studies by electron microscopy and three-dimensional maps revealed cage-like structures with 2- and 3-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180 against each other, implicating that every second unfolded MUC2 net in mature mucus is turned upside down.

  • 45. Nogueira, Eugenia
    et al.
    Loureiro, Ana
    Nogueira, Patricia
    Freitas, Jaime
    Almeida, Catarina R.
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Moreira, Alexandra
    Carmo, Alexandre M.
    Preto, Ana
    Gomes, Andreia C.
    Cavaco-Paulo, Artur
    Liposome and protein based stealth nanoparticles2013In: Faraday discussions (Online), ISSN 1359-6640, E-ISSN 1364-5498, Vol. 166, p. 417-429Article in journal (Refereed)
    Abstract [en]

    Liposomes and protein based nanoparticles were tuned with different polymers and glycolipids to improve stealth and thus decrease their clearance by macrophages. Liposomes were coated with polyethylene glycol (PEG) and brain-tissue-derived monosialoganglioside (GM1). Bovine serum albumin (BSA) nanoparticles were produced incorporating a PEGylated surfactant (PEG-surfactant). All obtained nanoparticles were monodisperse, with sizes ranging from 80 to 120 nm, with a zeta-potential close to zero. The presented stealth strategies lead to a decrease of internalization levels by macrophages. These surface modified nanoparticles could be used for production of new drug delivery nanosystems for systemic administration (e.g. intravenous application).

  • 46. Park, Kwang-Hyun
    et al.
    An, Yan
    Jung, Taeyang
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Baek, In-Young
    Noh, Haemin
    Ahn, Woo-Chan
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Song, Ji-Joon
    Kim, Jeong-Hoon
    Oh, Byung-Ha
    Woo, Eui-Jeon
    RNA activation-independent DNA targeting of the Type III CRISPR-Cas system by a Csm complex2017In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 18, no 5, p. 826-840Article in journal (Refereed)
    Abstract [en]

    The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a transcription-coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from Thermococcus onnurineus is reconstituted with a minimalistic combination of Csm1(1)2(1)3(3)4(1)5(1), and shows RNA targeting and RNA-activated single-stranded DNA (ssDNA) targeting activities. Unexpectedly, in the absence of an RNA transcript, it cleaves ssDNA containing a sequence complementary to the bound crRNA guide region in a manner dependent on the HD domain of the Csm1 subunit. This nuclease activity is blocked by a repeat tag found in the host CRISPR loci. The specific cleavage of ssDNA without a target RNA suggests a novel ssDNA targeting mechanism of the Type III system, which could facilitate the efficient and complete degradation of foreign nucleic acids.

  • 47. Pawelzik, Sven-Christian
    et al.
    Uda, Narasimha Rao
    Spahiu, Linda
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Stenberg, Patric
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Morgenstern, Ralf
    Jakobsson, Per-Johan
    Identification of Key Residues Determining Species Differences in Inhibitor Binding of Microsomal Prostaglandin E Synthase-12010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 38, p. 29254-29261Article in journal (Refereed)
    Abstract [en]

    Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E-2. Nonsteroidal anti-inflammatory drugs prevent prostaglandin E-2 production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substratePGH(2), or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.

  • 48. Petrlova, Jitka
    et al.
    Zhu, Lin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Morgelin, Matthias
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Jegerschold, Caroline
    Voss, John C.
    Lagerstedt, Jens O.
    Structural properties of functional HDL and variants of apoA-I2012In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26Article in journal (Other academic)
  • 49. Petrova, J
    et al.
    Zhu, L
    Axelsson, A
    Mörgelin, M
    Cochran, M
    Monterrubio, A
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Voss, CV
    Roberts, LM
    Lagerstedt, JO
    Structural properties of functional HDL and amyloidogenic L178H variant of apo A-12011In: Proceedings of the IX European Symposium of the Protein Society, 2011Conference paper (Refereed)
  • 50.
    Poehlman, Melanie
    et al.
    University Baureight.
    Kothapalli, Veera Venkata Satya Naray
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Grishenkov, Dmitry
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging. Karolinska Institutet (KI), CLINTEC – Division of Medical Imaging and Technology.
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Philipp, A.
    Hoeller, Roland
    Seuss, M.
    Magerithelli, S.
    Paradossi, Gaio
    Diapartimento di Chimica, Università di Roma Tor Vergata.
    Fery, Andreas
    Magnetic microbubbles for multimodality imaging: the importance of the shell structure for low and high frequency mechanics2013Conference paper (Refereed)
    Abstract [en]

    There is a growing interest in magnetic microbubbles (MBs) for simultaneous enhanced ultrasound (US) and enhanced magnetic resonance imaging (MRI) to support well-established imaging procedures as well as new emerging diagnostic and therapeutic applications. However, the development of hybrid contrast agents is challenging, because their design needs to satisfy a variety of requirements such as a sufficient stability of the probe for the circulation within the cardiovascular system, the production of an adequate US echo signal and a reasonable reduced relaxation time of nearby located protons. The studied magnetic MBs consist of an air-filled core, which is encapsulated by a soft hydrogel-like shell composed of poly(vinyl alcohol) and superparamagnetic iron oxide nanoparticles (SPIONs)[1]. Two strategies were used to combine magnetic nanoparticles with the polymeric shell: SPIONs were either covalently attached to the shell surface via a post-chemical treatment or embedded physically inside the shell during the MBs’ synthesis. In particular, we were interested on the impact of the used SPIONs integration strategy on low and high frequency mechanics of the magnetic MBs. Therefore, we used a straightforward characterization of the MBs on the single particle level to correlate the synthesis with the MBs’ morphological properties and low frequency mechanics that were studied in quasi-static force measurements with atomic force microscopy. High frequency mechanics were investigated by exposure of an ensemble of MBs to an acoustic field. By further correlation of low and high frequency mechanics, we were able to bridge the gap between synthesis and the MBs macroscopic properties relevant for their application. The shown approach offers the possibility to sustainable design and optimize complex probes based on an improved understanding of structure/property relations.

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