| Title: | Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism |
| Author(s): | Patching SG; Edara S; Ma P; Nakayama J; Hussain R; Siligardi G; Phillips-Jones MK; |
| Address: | "Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds. L32 9JT, UK. S.G.Patching@leeds.ac.uk" |
| DOI: | 10.1016/j.bbamem.2012.02.015 |
| ISSN/ISBN: | 0006-3002 (Print) 0006-3002 (Linking) |
| Abstract: | "FsrC is the membrane-bound histidine kinase component of the Fsr two-component signal transduction system involved in quorum sensing in the hospital-acquired infection agent Enterococcus faecalis. Synchrotron radiation circular dichroism spectroscopy was used here to study the intact purified protein solubilised in detergent micelles. Conditions required for FsrC stability in detergent were firstly determined and tested by prolonged exposure of stabilised protein to far-ultraviolet radiation. Using stabilised purified protein, far-ultraviolet synchrotron radiation circular dichroism revealed that FsrC is 61% alpha-helical and that it is relatively thermostable, retaining at least 57% secondary structural integrity at 90 degrees C in the presence or absence of gelatinase biosynthesis-activating pheromone (GBAP). Whilst binding of the quorum pheromone ligand GBAP did not significantly affect FsrC secondary structure, near-ultraviolet spectra revealed that the tertiary structure in the regions of the Tyr and Trp residues was significantly affected. Titration experiments revealed a calculated kd value of 2 microM indicative of relatively loose binding ofgelatinase biosynthesis-activating pheromone to FsrC. Although use of synchrotron radiation circular dichroism has been applied to membrane proteins previously, to our knowledge this is the first report of its use to determine a kd value for an intact membrane protein. Based on our findings, we suggest that synchrotron radiation circular dichroism will be a valuable technique for characterising ligand binding by other membrane sensor kinases and indeed other membrane proteins in general. It further provides a valuable screening tool for membrane protein stability under a range of detergent conditions prior to downstream structural methods such as crystallisation and NMR experiments particularly when lower detergent concentrations are used" |
| Keywords: | "Amino Acid Sequence Bacterial Proteins/chemistry/genetics/*metabolism Binding Sites/genetics Blotting, Western Circular Dichroism/*methods Enzyme Stability Hot Temperature Lactones/chemistry/*metabolism Membrane Proteins/chemistry/genetics/*metabolism Mol;" |
| Notes: | "MedlinePatching, Simon G Edara, Shalini Ma, Pikyee Nakayama, Jiro Hussain, Rohanah Siligardi, Giuliano Phillips-Jones, Mary K eng BB/D001641/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom /BB/D001641/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom Research Support, Non-U.S. Gov't Netherlands 2012/03/01 Biochim Biophys Acta. 2012 Jul; 1818(7):1595-602. doi: 10.1016/j.bbamem.2012.02.015" |
