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Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells
Published on: March 3, 2015
Putative interhelical interactions within the PheP protein revealed by second-site suppressor analysis
C Dogovski1, J Pi, A J Pittard
1Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia.
Journal of Bacteriology
|October 18, 2003
Summary
Highly conserved glycine residues in the PheP transporter are crucial for its function. Mutations reveal critical interactions between protein spans, impacting amino acid transport activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- The phenylalanine and tyrosine transporter PheP is vital for amino acid uptake.
- Conserved residues in transmembrane spans are often critical for transporter function.
Purpose of the Study:
- To investigate the functional importance of conserved glycine residues in PheP spans I and II.
- To elucidate the structural interactions within the PheP transporter.
Main Methods:
- Site-directed mutagenesis of conserved glycine residues in PheP.
- Transport activity assays to assess protein function.
- Second-site suppression studies to identify protein-protein interactions.
Main Results:
- Replacement of key glycine residues with larger amino acids progressively reduced PheP transport activity.
- Evidence for tight packing between spans I and II, and an interaction between spans I and III.
- A conserved GXXIG motif in span I is proposed as a potential dimerization motif.
Conclusions:
- Specific glycine residues in PheP spans I and II are essential for transporter function.
- Structural interactions between transmembrane spans are critical for PheP activity.
- The GXXIG motif may mediate PheP dimerization within the amino acid-polyamine-organocation transporter family.
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