Structural modeling of dual-affinity purified Pho84 phosphate transporter

• 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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

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

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)