title: Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism.
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.
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds. L32 9JT, UK. S.G.Patching@leeds.ac.uk
authors.forename: Simon G
authors.initials: SG
authors.lastname: Patching
authors.forename: Shalini
authors.initials: S
authors.lastname: Edara
authors.forename: Pikyee
authors.initials: P
authors.lastname: Ma
authors.forename: Jiro
authors.initials: J
authors.lastname: Nakayama
authors.forename: Rohanah
authors.initials: R
authors.lastname: Hussain
authors.forename: Giuliano
authors.initials: G
authors.lastname: Siligardi
authors.forename: Mary K
authors.initials: MK
authors.lastname: Phillips-Jones
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002942:Circular Dichroism / Q000379:methods*; D004795:Enzyme Stability; D006358:Hot Temperature; D007783:Lactones / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017356:Synchrotrons; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C409033:AgrCfs protein, Enterococcus faecalis; D001426:Bacterial Proteins; D007783:Lactones; D008565:Membrane Proteins; D010456:Peptides, Cyclic; C438382:gelatinase biosynthesis-activating pheromone; D014443:Tyrosine; D014364:Tryptophan