2D and 3D crystallization of a bacterial homologue of human vitamin C membrane transport proteins

J Struct Biol. 2014 Oct;188(1):87-91. doi: 10.1016/j.jsb.2014.08.004. Epub 2014 Aug 23.

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.

Keywords: Membrane protein; Projection structure; Scintillation proximity assay; Three-dimensional crystal; Transmission electron microscopy; Transport protein; Two-dimensional crystal; X-ray crystallography.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Ascorbic Acid / biosynthesis
  • Ascorbic Acid / chemistry*
  • Ascorbic Acid / genetics
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / ultrastructure
  • Crystallography, X-Ray
  • Humans
  • Ligands
  • Membrane Transport Proteins / chemistry*
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / ultrastructure
  • Microscopy, Electron, Transmission
  • Protein Binding
  • Sodium-Coupled Vitamin C Transporters / chemistry*
  • Sodium-Coupled Vitamin C Transporters / metabolism

Substances

  • Bacterial Proteins
  • Ligands
  • Membrane Transport Proteins
  • SLC23A1 protein, human
  • Sodium-Coupled Vitamin C Transporters
  • Ascorbic Acid