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  • 1.
    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.

     

  • 2.
    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.

  • 3.
    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.

  • 4.
    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.

  • 5. 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.

  • 6.
    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.

  • 7.
    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.

  • 8.
    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.

1 - 8 of 8
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