title: Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes.
abstract: The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli. refolded, and reconstituted into bilayer membranes. The molecule was characterized by15N and13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility,1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40-50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.
authors.affiliation: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Schrottke
authors.affiliation: Institute of Biochemistry, University of Leipzig, Brüderstr. 34, D-04103 Leipzig, Germany.
authors.forename: Anette
authors.initials: A
authors.lastname: Kaiser
authors.affiliation: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Vortmeier
authors.affiliation: Institute of Biochemistry, University of Leipzig, Brüderstr. 34, D-04103 Leipzig, Germany.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Els-Heindl
authors.affiliation: Institute of Biochemistry, University of Leipzig, Brüderstr. 34, D-04103 Leipzig, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Worm
authors.affiliation: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Bosse
authors.affiliation: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Schmidt
authors.affiliation: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany.
authors.forename: Holger A
authors.initials: HA
authors.lastname: Scheidt
authors.affiliation: Institute of Biochemistry, University of Leipzig, Brüderstr. 34, D-04103 Leipzig, Germany.
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
authors.affiliation: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Huster
mesh_terms: D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D054439:Ghrelin / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D010713:Phosphatidylcholines / Q000737:chemistry; D054440:Receptors, Ghrelin / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D054439:Ghrelin; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D054440:Receptors, Ghrelin; D011994:Recombinant Proteins; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; D004134:Dimyristoylphosphatidylcholine