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  • 1. 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.

  • 2.
    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.

  • 3.
    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.

  • 4. 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.

  • 5. 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.

  • 6. Frauenfeld, Jens
    et al.
    Loving, Robin
    Armache, Jean-Paul
    Sonnen, Andreas F-P
    Guettou, Fatma
    Moberg, Per
    Zhu, Lin
    KTH, School of Technology and Health (STH), Health Systems Engineering.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Flayhan, Ali
    Briggs, John A. G.
    Garoff, Henrik
    Low, Christian
    Cheng, Yifan
    Nordlund, Par
    A saposin-lipoprotein nanoparticle system for membrane proteins2016In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 13, no 4, p. 345-351Article in journal (Refereed)
    Abstract [en]

    A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptT(S02) by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state.

  • 7.
    Grishenkov, Dmitry
    et al.
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging. Karolinska Institutet (KI), CLINTEC – Division of Medical Imaging and Technology.
    Adrian, Gonon
    Department of Clinical Physiology, Karolinska University Hospital.
    Weitzberg, Eddie
    Department of Physiology and Pharmacology, Karolinska Institutet.
    Lundberg, Jon
    Department of Physiology and Pharmacology, Karolinska Institutet, .
    Harmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Cerroni, Barbara
    Department of Chemical Sciences and Technologies, University of Rome Tor Vergata.
    Paradossi, Gaio
    Diapartimento di Chimica, Università di Roma Tor Vergata.
    Janerot Sjöberg, Birgitta
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging. CLINTEC, Department of Medical Imaging and Technology, Karolinska Institute.
    Ultrasound contrast agent loaded with nitric oxide as a theranostic microdevice: Theranostic contrast agent loaded with nitric oxide2015In: Drug Design, Development and Therapy, ISSN 1177-8881, E-ISSN 1177-8881, Vol. 9, p. 2409-2419Article in journal (Refereed)
    Abstract [en]

    The current study describes novel multifunctional polymer-shelled microbubbles (MBs) loaded with nitric oxide (NO) for integrated therapeutic and diagnostic applications, i.e. theranostics, of myocardial ischemia. We used gas filled MBs with an average diameter of 4 µm stabilized by a biocompatible shell of poly(vinyl)alcohol. In vitro acoustic tests showed a sufficient enhancement of the backscattered power (20 dB) acquired from the MBs suspension. The values of attenuation coefficient (0.8 dB/cm MHz) and phase velocities (1517 m/s) were comparable to those reported for the soft tissue. Moreover, polymer MBs demonstrate increased stability compared to clinically approved contrast agents with fracture threshold of about 900 kPa. In vitro chemiluminescence measurements demonstrated that dry powder of NO-loaded MBs releases its gas content in about 2 hours following an exponential decay profile with an exponential time constant equal 36 min. The application of high power ultrasound pulse (MI=1.2) on the MBs resuspended in saline decreases the exponential time constant from 55 to 4 min in air saturated solution and from 17 to 10 min in degased solution. Thus, ultrasound-triggered release of NO is achieved. Cytotoxicity tests indicate that phagocytosis of the MBs by macrophages starts within 6 to 8 hours. This is suitable time for initial diagnostics, treatment and monitoring of the therapeutic effect using single injection of the proposed multifunctional MBs.

  • 8.
    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.

  • 9.
    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.

  • 10.
    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.

  • 11.
    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.

  • 12.
    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)
  • 13.
    Koeck, P. J. B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Improved Zernike-type phase contrast for transmission electron microscopy2015In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 259, no 1, p. 74-78Article in journal (Refereed)
    Abstract [en]

    Zernike phase contrast has been recognized as a means of recording high-resolution images with high contrast using a transmission electron microscope. This imaging mode can be used to image typical phase objects such as unstained biological molecules or cryosections of biological tissue. According to the original proposal discussed in Danev and Nagayama (2001) and references therein, the Zernike phase plate applies a phase shift of /2 to all scattered electron beams outside a given scattering angle and an image is recorded at Gaussian focus or slight underfocus (below Scherzer defocus). Alternatively, a phase shift of -/2 is applied to the central beam using the Boersch phase plate. The resulting image will have an almost perfect contrast transfer function (close to 1) from a given lowest spatial frequency up to a maximum resolution determined by the wave length, the amount of defocus and the spherical aberration of the microscope. In this paper, I present theory and simulations showing that this maximum spatial frequency can be increased considerably without loss of contrast by using a Zernike or Boersch phase plate that leads to a phase shift between scattered and unscattered electrons of only /4, and recording images at Scherzer defocus. The maximum resolution can be improved even more by imaging at extended Scherzer defocus, though at the cost of contrast loss at lower spatial frequencies. Lay description Zernike phase contrast has been recognized as a means of recording high-resolution images with high contrast using a transmission electron microscope. This imaging mode can be used to image specimens such as unstained biological molecules or sections of biological tissue. According to the original proposal, the Zernike phase plate applies a phase shift of /2 to all scattered electron beams outside a given scattering angle and an image is recorded at or close to focus. The resulting image will be an almost perfect representation of the specimen up to a maximum resolution determined by the energy of the electrons and certain optical parameters of the microscope. In this paper, I present theory and simulations showing that this maximum resolution can be increased considerably without loss of contrast by using a Zernike phase plate that leads to a phase shift between scattered and unscattered electrons of only /4, and recording images somewhat out of focus.

  • 14.
    Koeck, P. J. B.
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Karshikoff, A.
    Limitations of the linear and the projection approximations in three-dimensional transmission electron microscopy of fully hydrated proteins2015In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 259, no 3, p. 197-209Article in journal (Refereed)
    Abstract [en]

    We establish expressions for the linear and quadratic terms in the series expansion of the phase and the phase and amplitude object description of imaging thin specimens by transmission electron microscopy. Based on these expressions we simulate the corresponding contributions to images of unstained protein complexes of varying thickness and arrive at an estimate for how much each term contributes to the contrast of the image. From this we can estimate a maximum specimen thickness for which the weak phase and the weak amplitude and phase object approximation (and therefore linear imaging) is still reasonably accurate. When discussing thick specimens it is also necessary to consider limitations due to describing the image as a filtered projection of the specimen, since the different layers of the specimen are not imaged with the same defocus value. We therefore compared simulations based on the projection approximation with the more accurate multislice model of image formation. However, we find that the errors due to nonlinear image contributions are greater than those due to the defocus gradient for the defocus values chosen for the simulations. Finally, we study how the discussed nonlinear image contributions and the defocus gradient affect the quality of three-dimensional reconstructions. We find that three-dimensional reconstructions reach high resolution when at the same time exhibiting localized systematic structural errors. Non-Technical Abstract Cryo transmission electron microscopy and three-dimensional reconstruction can be used to determine a three-dimensional model of a protein molecule. In the mathematical methods used for three-dimensional reconstruction assumptions are made about a linear relationship between the images recorded in the electron microscope and the objects being imaged. In this paper we investigate with computer simulations at what specimen thickness these assumptions start breaking down and what sort of errors can be expected in the three-dimensional reconstructions when the assumptions are not valid anymore.

  • 15.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Improved Hilbert phase contrast for transmission electron microscopy2015In: Ultramicroscopy, ISSN 0304-3991, E-ISSN 1879-2723, Vol. 154, p. 37-41Article in journal (Refereed)
    Abstract [en]

    Hilbert phase contrast has been recognized as a means of recording high resolution images with high contrast using a transmission electron microscope. This imaging mode could be used to image typical phase objects such as unstained biological molecules or cryo sections of biological tissue. According to the original proposal by (Danev et al., 2002) the Hilbert phase plate applies a phase shift of π to approximately half the focal plane (for example the right half excluding the central beam) and an image is recorded at Gaussian focus. After correction for the inbuilt asymmetry of differential phase contrast this image will have an almost perfect contrast transfer function (close to 1) from the lowest spatial frequency up to a maximum resolution determined by the wave length and spherical aberration of the microscope. In this paper I present theory and simulations showing that this maximum spatial frequency can be increased considerably almost without loss of contrast by using a Hilbert phase plate of half the thickness, leading to a phase shift of π/2, and recording images at Scherzer defocus. The maximum resolution can be improved even more by imaging at extended Scherzer defocus, though at the cost of contrast loss at lower spatial frequencies.

  • 16. Kronqvist, Nina
    et al.
    Sarr, Medoune
    Lindqvist, Anton
    Nordling, Kerstin
    Otikovs, Martins
    Venturi, Luca
    Pioselli, Barbara
    Purhonen, Pasi
    Landreh, Michael
    Biverstal, Henrik
    Toleikis, Zigmantas
    Sjöberg, Lisa
    Robinson, Carol V.
    Pelizzi, Nicola
    Jornvall, Hans
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sverige.
    Jaudzems, Kristaps
    Curstedt, Tore
    Rising, Anna
    Johansson, Jan
    Efficient protein production inspired by how spiders make silk2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 15504Article in journal (Refereed)
    Abstract [en]

    Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT star) that is pH insensitive, stabilized and hypersoluble compared to wildtype NT. NT star-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT star enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT star also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.

  • 17.
    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.

     

  • 18.
    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.

  • 19.
    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.

  • 20. 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.

  • 21.
    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.

  • 22.
    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.

  • 23.
    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.

  • 24.
    Köck, Philip
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Novum, Huddinge, Sweden.
    Annular dark field transmission electron microscopy for protein structure determination2016In: Ultramicroscopy, ISSN 0304-3991, E-ISSN 1879-2723, Vol. 161, p. 98-104Article in journal (Refereed)
    Abstract [en]

    Recently annular dark field (ADF) transmission electron microscopy (TEM) has been advocated as a means of recording images of biological specimens with better signal to noise ratio (SNR) than regular bright field images. I investigate whether and how such images could be used to determine the three-dimensional structure of proteins given that an ADF aperture with a suitable pass-band can be manufactured and used in practice. I develop an approximate theory of ADF-TEM image formation for weak amplitude and phase objects and test this theory using computer simulations. I also test whether these simulated images can be used to calculate a three-dimensional model of the protein using standard software and discuss problems and possible ways to overcome these.

  • 25. 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.

  • 26. Löw, Christian
    et al.
    Yau, Yin Hoe
    Pardon, Els
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Wåhlin, Lisa
    Quistgaard, Esben M.
    Moberg, Per
    Geifman-Shochat, Susana
    Steyaert, Jan
    Nordlund, Pär
    Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 10, p. e77984-Article in journal (Refereed)
    Abstract [en]

    Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.

  • 27. Mittal, Monica
    et al.
    Kumar, Ramakrishnan B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Balagunaseelan, Navisraj
    Hamberg, Mats
    Jegerschold, Caroline
    Rådmark, Olof
    Häggstrom, Jesper Z.
    Rinaldo-Matthis, Agnes
    Kinetic investigation of human 5-lipoxygenase with arachidonic acid2016In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 26, no 15, p. 3547-3551Article in journal (Refereed)
    Abstract [en]

    Human 5-lipoxygenase (5-LOX) is responsible for the formation of leukotriene (LT)A(4), a pivotal intermediate in the biosynthesis of the leukotrienes, a family of proinflammatory lipid mediators. 5-LOX has thus gained attention as a potential drug target. However, details of the kinetic mechanism of 5-LOX are still obscure. In this Letter, we investigated the kinetic isotope effect (KIE) of 5-LOX with its physiological substrate, arachidonic acid (AA). The observed KIE is 20 +/- 4 on k(cat) and 17 +/- 2 on k(cat)/K-M at 25 degrees C indicating a non-classical reaction mechanism. The observed rates show slight temperature dependence at ambient temperatures ranging from 4 to 35 degrees C. Also, we observed low Arrhenius prefactor ratio (A(H)/A(D) = 0.21) and a small change in activation energy (E-a(D) - E-a(H) = 3.6 J/mol) which suggests that 5-LOX catalysis involves tunneling as a mechanism of H-transfer. The measured KIE for 5-LOX involves a change in regioselectivity in response to deuteration at position C7, resulting in H-abstraction form C10 and formation of 8-HETE. The viscosity experiments influence the (H)k(cat), but not (D)k(cat). However the overall kcat/K-M is not affected for labeled or unlabeled AA, suggesting that either the product release or conformational rearrangement might be involved in dictating kinetics of 5-LOX at saturating conditions. Investigation of available crystal structures suggests the role of active site residues (F421, Q363 and L368) in regulating the donor-acceptor distances, thus affecting H-transfer as well as regiospecificity. In summary, our study shows that that the H-abstraction is the rate limiting step for 5-LOX and that the observed KIE of 5-LOX is masked by a change in regioselectivity.

  • 28. 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.

  • 29. 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).

  • 30. Nogueira, Eugenia
    et al.
    Mangialavori, Irene C.
    Loureiro, Ana
    Azoia, Nuno G.
    Sarria, Marisa P.
    Nogueira, Patricia
    Freitas, Jaime
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Inst, Sch Technol & Hlth.
    Shimanovich, Ulyana
    Rollett, Alexandra
    Lacroix, Ghislaine
    Bernardes, Goncalo J. L.
    Guebitz, Georg
    Hebert, Hans
    Moreira, Alexandra
    Carmo, Alexandre M.
    Rossi, Juan Pablo F. C.
    Gomes, Andreia C.
    Preto, Ana
    Cavaco-Paulo, Artur
    Peptide Anchor for Folate-Targeted Liposomal Delivery2015In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 16, no 9, p. 2904-2910Article in journal (Refereed)
    Abstract [en]

    Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

  • 31. 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.

  • 32.
    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.

  • 33. Poehlmann, Melanie
    et al.
    Grishenkov, Dmitry
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Kothapalli, Satya V.V.N.
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    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, Alexandra
    Hoeller, Roland
    Seuss, Maximilian
    Kuttner, Christian
    Margheritelli, Silvia
    Paradossi, Gaio
    Frey, Andreas
    On the interplay of shell structure with low- and high-frequency mechanics of multifunctional magnetic microbubbles2014In: Soft Matter, ISSN 1744-683X, E-ISSN 1744-6848, Vol. 10, no 1, p. 214-226Article in journal (Refereed)
    Abstract [en]

    Polymer-shelled magnetic microbubbles have great potential as hybrid contrast agents for ultrasound and magnetic resonance imaging. In this work, we studied US/MRI contrast agents based on air-filled poly(vinyl alcohol)-shelled microbubbles combined with superparamagnetic iron oxide nanoparticles (SPIONs). The SPIONs are integrated either physically or chemically into the polymeric shell of the microbubbles (MBs). As a result, two different designs of a hybrid contrast agent are obtained. With the physical approach, SPIONs are embedded inside the polymeric shell and with the chemical approach SPIONs are covalently linked to the shell surface. The structural design of hybrid probes is important, because it strongly determines the contrast agent's response in the considered imaging methods. In particular, we were interested how structural differences affect the shell's mechanical properties, which play a key role for the MBs' US imaging performance. Therefore, we thoroughly characterized the MBs' geometric features and investigated low-frequency mechanics by using atomic force microscopy (AFM) and high-frequency mechanics by using acoustic tests. Thus, we were able to quantify the impact of the used SPIONs integration method on the shell's elastic modulus, shear modulus and shear viscosity. In summary, the suggested approach contributes to an improved understanding of structure-property relations in US-active hybrid contrast agents and thus provides the basis for their sustainable development and optimization.

  • 34. 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)
  • 35. Rutsdottir, Gudrun
    et al.
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine. Karolinska Institutet, Sverige.
    Weide, Yoran
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sverige.
    Rasmussen, Morten I.
    Wernersson, Sven
    Respondek, Michal
    Akke, Mikael
    Højrup, Peter
    Köck, Philip J. B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sverige.
    Söderberg, Christopher A. G.
    Emanuelsson, Cecilia
    Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 19, p. 8103-8121Article in journal (Refereed)
    Abstract [en]

    Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.

  • 36. 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.

  • 37.
    Zheng, Miaomiao
    et al.
    KTH, School of Technology and Health (STH), Medical Engineering.
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Grishenkov, Dmitry
    KTH, School of Technology and Health (STH), Medical Engineering.
    Janerot Sjöberg, Birgitta
    CLINTEC, Department of Medical Imaging and Technology, Karolinska Institute.
    Polymer-Shelled Ultrasound Contrast Agents with controlled size and polydispersity.2011In: Nanomedicine: Nanotechnology, Biology & Medicine, 2011Conference paper (Refereed)
    Abstract [en]

    Ultrasound imaging techniques can be greatly improved by the use of ultrasound contrast agents (UCAs). Gas bubbles encapsulated into biocompatible polymer shell are of particular interest of this work. Shell of the bubbles produced from Poly-Vinyl-Alcohol (PVA) offers considerable chemical versatility and stability. However, questions regarding the size and polydispersity of the microbubbles must be further investigated. The ideal UCAs should not obstruct the blood flow in pulmonary capillaries which diameter is less than 10 μm. From the technical perspective UCAs should modify the acoustic properties of a region of interest, by increasing backscattered efficiency. In order to enhance the ultrasound response UCAs should be engineered with narrow size distribution. In the present work PVA-shelled UCAs with controlled size and polydispersity is manufactured under varied parameters of the manufacturing protocol. It was observed that temperature of the surrounding atmosphere has major effect on the size of the UCAs, while polydispersity is regulated by geometry and speed of the disperser. Finally, the acoustic response of these microbubbles is tested using developed ultrasound test rig. The enhancement of the backscattered power of about 25 dB from a suspension of the microbubbles is observed at 5 MHz ultrasound frequency. Keeping in mind that in clinical practice ultrasound scatter from the blood is of about 30 dB weaker than scatter from surrounding tissue, introduction of novel PVA microbubbles will potentially improve diagnosis of the cardiovascular patients.

  • 38.
    Zhu, Lin
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Structural studies of HDL and applications of EM on membrane proteins2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A large number of proteins interact with biological membranes, either integrated in the membrane (PepTSo2), embedded on a membrane surface (5-lipoxygenase) or encircling a cutout of lipid bilayer (apolipoprotein1 (apoA-I). They function as transporters, receptors or biocatalysts in cellular processes like inflammation or cholesterol transport which are touched upon here. Malfunction of specific membrane proteins are the cause for several diseases or disorders.

    Knowledge of protein structure supports understanding of its mechanism of function. Here, transmission electron microscopy (TEM) was used for structure determination. To obtain structure information to high resolution for membrane proteins, normally surrounded by lipids, demands specific methods and materials for stabilization. Stabilized in detergent the structure of the bacterial transporter PepTSo2 was shown to form a tetramer even bound to substrate. However, with a protein based stabilizer, Salipro, the structure of PepTSo2 could be determined to high resolution.

    High density lipoprotein (HDL) in blood plasma, involved in the removal of cholesterol from peripheral tissues, have a central role in cardiovascular function, metabolic syndrome and diabetes.

    The HDL-particle is composed of two copies of ApoA1 and around hundred lipid molecules. From TEM data, for the first time the clearly discoidal shape could be shown by 3-dimendional reconstructions. These were used for modelling the ApoA1 protein dimer by a "biased fitting" procedure. The results indicate how ApoA1 folds around a lipid bilayer in a disc-shaped structure.

    Modified HDL called nanodiscs were here used to show the Ca2+ dependent binding of 5-lipoxygenase on the nanodisc bilayer and thereby increased production of the inflammatory mediator leukotrieneA4. Dimerization of 5-lipoxygenase inactivates these functions.

  • 39.
    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)
1 - 39 of 39
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