title: 2D and 3D crystallization of a bacterial homologue of human vitamin C membrane transport proteins.
abstract: Most organisms are able to synthesize vitamin C whereas humans are not. In order to contribute to the elucidation of the molecular working mechanism of vitamin C transport through biological membranes, we cloned, overexpressed, purified, functionally characterized, and 2D- and 3D-crystallized a bacterial protein (UraDp) with 29% of amino acid sequence identity to the human sodium-dependent vitamin C transporter 1 (SVCT1). Ligand-binding experiments by scintillation proximity assay revealed that uracil is a substrate preferably bound to UraDp. For structural analysis, we report on the production of tubular 2D crystals and present a first projection structure of UraDp from negatively stained tubes. On the other hand the successful growth of UraDp 3D crystals and their crystallographic analysis is described. These 3D crystals, which diffract X-rays to 4.2Å resolution, pave the way towards the high-resolution crystal structure of a bacterial homologue with high amino acid sequence identity to human SVCT1.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland; Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012 Bern, Switzerland.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Jeckelmann
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland; Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012 Bern, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Harder
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland; Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012 Bern, Switzerland.
authors.forename: Zöhre
authors.initials: Z
authors.lastname: Ucurum
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland; Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012 Bern, Switzerland. Electronic address: dimitrios.fotiadis@ibmm.unibe.ch.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fotiadis
mesh_terms: D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D018360:Crystallography, X-Ray; D006801:Humans; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D011485:Protein Binding; D060347:Sodium-Coupled Vitamin C Transporters / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D026901:Membrane Transport Proteins; C555162:SLC23A1 protein, human; D060347:Sodium-Coupled Vitamin C Transporters; D001205:Ascorbic Acid
keywords: Membrane protein; Projection structure; Scintillation proximity assay; Three-dimensional crystal; Transmission electron microscopy; Transport protein; Two-dimensional crystal; X-ray crystallography