PMID:12940980
| Citation |
Hubbard, JA, MacLachlan, LK, King, GW, Jones, JJ and Fosberry, AP (2003) Nuclear magnetic resonance spectroscopy reveals the functional state of the signalling protein CheY in vivo in Escherichia coli. Mol. Microbiol. 49:1191-200 |
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| Abstract |
Two-component signal transduction (TCST) pathways are regulatory systems that are highly homologous throughout the bacterial kingdom. Their established role in virulence and absence in vertebrates has made TCST an attractive target for therapeutic intervention. However, such systems have yet to yield success in the development of novel antibiotics. CheY serves as a prototype for the analysis of response regulator function. The protein structure exhibits several conformations by both X-ray and nuclear magnetic resonance (NMR) analyses. Knowledge of which structures are relevant in vivo would be valuable in a rational drug design project. Our aim was to probe the in vivo conformation and ligand binding of CheY in Escherichia coli under resting conditions by in-cell NMR methods. CheY was selectively labelled with 15N by the control of growth and expression conditions. NMR spectra obtained in vivo demonstrated that the Mg2+ complex was the predominant form even though cells were resuspended in metal-free buffers and the intracellular free Mg2+ was low. In-cell NMR also confirmed the uptake and in vivo binding mode to CheY of small-molecular-weight compounds identified in vitro. This paper reports the first observation of the structure and interactions with a potential drug of a regulator protein in its native host in vivo using NMR spectroscopy. |
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| Keywords |
Bacterial Proteins; Cations, Divalent/metabolism; Chemotaxis; Escherichia coli/chemistry; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Ligands; Magnesium/metabolism; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Models, Molecular; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Conformation; Signal Transduction |
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