13 research outputs found
Integrating solid-state NMR and computational modeling to investigate the structure and dynamics of membrane-associated ghrelin.
The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide's secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide's positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8-17 form an α-helix, while residues 21-23 and 26-27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane
<sup>2</sup>H NMR spectra and order parameters of DMPC-<i>d</i><sub>54</sub>/DMPS membranes in the absence and presence of ghrelin and deacylghrelin.
<p><sup>2</sup>H NMR spectra in DMPC-<i>d</i><sub>54</sub>/DMPS membranes (5/1, mol/mol) in the presence of ghrelin (A) and ghrelin-<i>d</i><sub>15</sub> in DMPC/DMPS membranes (B). C) <sup>2</sup>H NMR order parameters of DMPC-<i>d</i><sub>54</sub>/DMPS (5:1, mol/mol) membranes in the presence or absence of ghrelin or desacylghreliln (1:30 protein to lipid molar ratio) at a temperature of 30°C and a buffer content of 35 wt%. Error bars of the <sup>2</sup>H NMR order parameters are smaller than the symbol size.</p
Chemical shifts measured for acylated ghrelin bound to DMPC/DMPC membranes (5/1, mol/mol) using MAS solid-state NMR.
<p>Chemical shifts measured for acylated ghrelin bound to DMPC/DMPC membranes (5/1, mol/mol) using MAS solid-state NMR.</p
Binding isotherms of ghrelin and desacyl ghrelin to DMPC/DMPG membranes.
<p>The amount of bound ghrelin (black squares) and desacyl ghrelin (red circles) as a function of lipid concentration is given. The binding data were fitted according to Eq. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122444#pone.0122444.e001" target="_blank">1</a>).</p
Statistics for restraints, structural calculations, and structural quality for final ensemble of ghrelin models.
<p>Statistics for restraints, structural calculations, and structural quality for final ensemble of ghrelin models.</p
Chemical shift analysis of ghrelin based on MAS ssNMR data.
<p>The <sup>13</sup>Cα–<sup>13</sup>Cβ CS values (chemical shift index) for reach residue are plotted. Positive values greater than 1 ppm indicate a tendency for α-helical structure, whereas values less than −1 ppm suggest β-sheet character. Amino acids with a CS index close to 0 ppm are considered to have no secondary structure. Asterisks indicate that no CSs were available for that residue.</p
<sup>1</sup>H-<sup>13</sup>C order parameters of ghrelin bound to DMPC/DMPS membranes.
<p>Order parameters were determined for 3.3 mol% ghrelin bound to DMPC/DMPS membranes (5:1, mol/mol) at a temperature of 30°C and a water content of 35 wt%.</p
Ghrelin sequence showing the isotopically labeled amino acids of the different ghrelin molecules and ssNMR spectra of membrane-embedded ghrelin.
<p>Labeled amino acids are shown in bold italics (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122444#pone.0122444.s010" target="_blank">S2 Table</a>). A) <sup>13</sup>C CP MAS NMR spectrum of ghrelin (with Gly1, Leu5, and Ser6 <sup>13</sup>C/<sup>15</sup>N labeled) in DMPC-<i>d</i><sub>67</sub>/DMPS-<i>d</i><sub>54</sub> (5:1, mol/mol) membranes at a ghrelin concentration of 3.3 mol%. B) <sup>1</sup>H-<sup>13</sup>C MAS HetCor NMR spectrum of the same preparation, all at 30°C and a MAS frequency of 7 kHz.</p