Structural modeling of dual-affinity purified Pho84 phosphate transporter

Lagerstedt, Jens O.,1975Stockholms universitet,Gothenburg University,Göteborgs universitet,Wallenberglaboratoriet,Wallenberg Laboratory,Institutionen för biokemi och biofysik (författare)

The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.

NATURVETENSKAP Biologi Biokemi och molekylärbiologi hsv//swe
NATURAL SCIENCES Biological Sciences Biochemistry and Molecular Biology hsv//eng
Amino Acid Sequence
Blotting
Western
Chromatography
Affinity
Electron Spin Resonance Spectroscopy
Electrophoresis
Polyacrylamide Gel
Escherichia coli/genetics
Fungal Proteins/*chemistry/genetics/*isolation & purification/*metabolism
Models
Molecular
*Models
Structural
Molecular Sequence Data
Mutagenesis
Site-Directed
Protein Conformation
Protein Folding
Protein Structure
Secondary
Protein Structure
Tertiary
purification/*metabolism
Recombinant Fusion Proteins/metabolism
purification/*metabolism
Sequence Homology
Amino Acid
Spin Labels
Dual-affinity purification; FLAG epitope; Electron paramagnetic resonance; Phosphate transport; Pho84; Yeast
Biochemistry

Voss, J. C. (författare)
Wieslander, ÅkeStockholms universitet,Institutionen för biokemi och biofysik(Swepub:su)awies (författare)
Persson, B. L. (författare)
Göteborgs universitetWallenberglaboratoriet (creator_code:org_t)

Av författaren/redakt...: Lagerstedt, Jens ...; Voss, J. C.; Wieslander, Åke; Persson, B. L.

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