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Lookup NU author(s): Professor Paul RaceORCiD, Dr Alexandra Solovyova, Dr Mark Banfield
The translocated intimin receptor (Tir) is a key virulence factor of enteropathogenic Escherichia coli and related bacteria. During infection, Tir is translocated via a type III secretion system into host intestinal epithelial cells, where it inserts into the target cell membrane and acts as a receptor for the bacterial adhesin intimin. The effects of phosphorylation by cAMP-dependent kinase at two serine residues (Ser-434 and Ser-463) within the C-terminal domain of Tir, which may be involved in mediating structural/electrostatic changes in the protein to promote membrane insertion or intermolecular interactions, have previously been investigated. This study has focused on defining the conformation of Tir in solution and assessing any conformational changes associated with serine phosphorylation at positions 434/463. In addition to phosphorylated protein, combinations of Ala (unphosphorylatable) and Asp (phosphate-mimic) mutations of Ser-434 and Ser-463 have been generated, and a range of techniques (sodium dodecyl sulfate polyacrylamide gel electrophoresis, circular dichroism spectroscopy, analytical ultracentrifugation) used to further dissect the structural role and functional implications of changes in residue size/charge at these positions. The results have shown that under physiological NaCl concentrations, Tir is a monomer and adopts a highly elongated state in solution, consistent with a natively unfolded conformation. Despite this, perturbations in the structure in response to buffer conditions and the nature of the residues at positions 434 and 463 are apparent, and may be functionally relevant. © 2007 by the Biophysical Society.
Author(s): Race PR, Solovyova AS, Banfield MJ
Publication type: Article
Publication status: Published
Journal: Biophysical Journal
Year: 2007
Volume: 93
Issue: 2
Pages: 586-596
Print publication date: 01/07/2007
ISSN (print): 0006-3495
ISSN (electronic): 1542-0086
Publisher: Biophysical Society
URL: http://dx.doi.org/10.1529/biophysj.106.101766
DOI: 10.1529/biophysj.106.101766
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