| Type of Submission |
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Submission Type: |
Poster Presentation |
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Subject Category: |
Structure and Function |
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| Session Information |
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Presentation Date: |
May 26, 2003 |
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Abstract ID: |
F6 |
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Session: |
Poster 1 |
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Time: |
14:00 |
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| Presenting Author |
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| Other Authors |
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H. LE MOUAL, McGill University
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| Title |
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Reconstitution as a Tool for Studying the Salmonella enterica PhoQ Sensor Kinase |
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| Abstract Text |
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Problem under investigation: The PhoP/PhoQ two-component regulatory system of Salmonella enterica plays an essential role in coordinating the expression of various virulence factors in response to environmental Mg2+ levels. Most in vitro work conducted with the transmembrane sensor kinase PhoQ has been done using membrane fractions overexpressing the protein. PhoQ activity has yet to be shown in the absence of other membrane proteins. Here, we report that PhoQ retained activity upon reconstitution into proteoliposomes. Methods: A C-terminal his-tagged variant of PhoQ was overexpressed in E. coli. Membrane fractions were prepared, solubilized and the PhoQHis protein was purified by Ni2+-NTA affinity chromatography. Several detergents were initially tested for their suitability for reconstitution of PhoQHis. Detergent was added to liposomes prepared from E. coli phospholipids and mixed with purified PhoQHis before removing the detergent by adsorption to polystyrene beads. The global catalytic activity of PhoQHis was measured by incubating either soluble PhoQHis or proteoliposomes with purified PhoP in the presence of radiolabeled ATP and MgCl2. Results: We showed that dodecyl-ß-D-maltoside solubilized PhoQHis has an essentially similar level of activity to that of non-solubilized PhoQHis. Reconstitution using liposomes solubilized with dodecyl-ß-D-maltoside and PhoQHis purified with decyl-ß-D-maltopyranoside resulted in the highest global activity. The reconstitution efficiency and the orientation of PhoQHis in proteoliposomes were also determined. Conclusions: The role of Mg2+ acting as a signaling molecule in the periplasmic domain and as a cofactor for autophosphorylation in the cytoplasmic domain can now be dissected. This system will also allow further investigations into Mg2+ regulation of PhoQ catalytic activities. |
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