title: Toxoplasma gondii: biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6.
abstract: The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6-8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8-15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed.
authors.affiliation: CNRS, UMR 5163, 38042 Grenoble, France; Université Grenoble Alpes, 38042 Grenoble, France.
authors.forename: Amina
authors.initials: A
authors.lastname: Bittame
authors.affiliation: Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble, France; CNRS, IBS, 38044 Grenoble, France; CEA, IBS, 38044 Grenoble, France; Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble, France.
authors.forename: Grégory
authors.initials: G
authors.lastname: Effantin
authors.affiliation: CNRS, UMR 5163, 38042 Grenoble, France; Université Grenoble Alpes, 38042 Grenoble, France.
authors.forename: Graciane
authors.initials: G
authors.lastname: Pètre
authors.affiliation: CNRS, UMR 5163, 38042 Grenoble, France; Université Grenoble Alpes, 38042 Grenoble, France.
authors.forename: Pauline
authors.initials: P
authors.lastname: Ruffiot
authors.affiliation: CNRS, UMR 5163, 38042 Grenoble, France; Université Grenoble Alpes, 38042 Grenoble, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Travier
authors.affiliation: Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble, France; CNRS, IBS, 38044 Grenoble, France; CEA, IBS, 38044 Grenoble, France; Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble, France.
authors.forename: Guy
authors.initials: G
authors.lastname: Schoehn
authors.affiliation: Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble, France; CNRS, IBS, 38044 Grenoble, France; CEA, IBS, 38044 Grenoble, France; Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble, France.
authors.forename: Winfried
authors.initials: W
authors.lastname: Weissenhorn
authors.affiliation: CNRS, UMR 5163, 38042 Grenoble, France; Université Grenoble Alpes, 38042 Grenoble, France.
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Cesbron-Delauw
authors.affiliation: CNRS, UMR 5163, 38042 Grenoble, France; Université Grenoble Alpes, 38042 Grenoble, France.
authors.forename: Jean
authors.initials: J
authors.lastname: Gagnon
authors.affiliation: CNRS, UMR 5163, 38042 Grenoble, France; Université Grenoble Alpes, 38042 Grenoble, France. Electronic address: corinne.mercier@ujf-grenoble.fr.
authors.forename: Corinne
authors.initials: C
authors.lastname: Mercier
mesh_terms: D000953:Antigens, Protozoan / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D008027:Light; D046529:Microscopy, Electron, Transmission; D017510:Protein Folding; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012542:Scattering, Radiation; D012995:Solubility
chemical_list: D000953:Antigens, Protozoan; C095047:GRA6 protein, Toxoplasma gondii; C106990:Gra2 protein, Toxoplasma gondii; D015800:Protozoan Proteins; D011994:Recombinant Proteins
keywords: Amphipathic alpha-helices; Circular dichroism; Dense granule proteins (GRA); Dynamic light scattering; Toxoplasma gondii; Transmission electron microscopy