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1 Departments of Molecular Biosciences and 2 Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66045, USA
(RECEIVED July 27, 2005; FINAL REVISION November 9, 2005; ACCEPTED December 9, 2005)
Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular "needle" that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37°C and MxiH having a value near 42°C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (
G°0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein–protein interactions.
Keywords: type III secretion; MxiH; Shigella; needle protein; protein stability; protein structure/folding; stability and mutagenesis; structure/function studies; other spectroscopies; circular dichroism; fluorescence
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051733506.
Reprint requests to: William D. Picking, Division of Biology, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA; e-mail: picking{at}ku.edu; fax: (785) 864-5294.
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