Fig 1.
Flowchart of computational modeling and analysis protocol.
The flowchart outlines the protocol used to elucidate the structure of ghrelin based on ssNMR CS data.
Fig 2.
Outline of model ensemble selection algorithm.
The flowchart outlines the process by which the agreement with experimental data is determined for an ensemble of models selected from a large pool.
Fig 3.
Binding isotherms of ghrelin and desacyl ghrelin to DMPC/DMPG membranes.
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. (1).
Fig 4.
2H NMR spectra and order parameters of DMPC-d54/DMPS membranes in the absence and presence of ghrelin and deacylghrelin.
2H NMR spectra in DMPC-d54/DMPS membranes (5/1, mol/mol) in the presence of ghrelin (A) and ghrelin-d15 in DMPC/DMPS membranes (B). C) 2H NMR order parameters of DMPC-d54/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 2H NMR order parameters are smaller than the symbol size.
Fig 5.
Ghrelin sequence showing the isotopically labeled amino acids of the different ghrelin molecules and ssNMR spectra of membrane-embedded ghrelin.
Labeled amino acids are shown in bold italics (see S2 Table). A) 13C CP MAS NMR spectrum of ghrelin (with Gly1, Leu5, and Ser6 13C/15N labeled) in DMPC-d67/DMPS-d54 (5:1, mol/mol) membranes at a ghrelin concentration of 3.3 mol%. B) 1H-13C MAS HetCor NMR spectrum of the same preparation, all at 30°C and a MAS frequency of 7 kHz.
Fig 6.
Chemical shift analysis of ghrelin based on MAS ssNMR data.
The 13Cα–13Cβ 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.
Table 1.
Chemical shifts measured for acylated ghrelin bound to DMPC/DMPC membranes (5/1, mol/mol) using MAS solid-state NMR.
Fig 7.
1H-13C order parameters of ghrelin bound to DMPC/DMPS membranes.
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%.
Fig 8.
1H spin diffusion buildup curves of membrane-associated ghrelin.
The plot shows the integral of the respective Cα position as a function of the square root of the mixing time, corrected for T1 relaxation and normalized to 1 for the longest spin diffusion time of 900 ms [53]. Spin diffusion spectra were determined for 3.3 mol% membrane-associated ghrelin in DMPC-d67/DMPS-d54 (5:1, mol/mol) at a D2O content of 35 wt%. Spin diffusion originates from the membrane’s glycerol and the PS headgroups. Solid lines represent best-fit simulations using a lattice model with a spin diffusion coefficient of D = 0.001 nm2/s and a distance between protons of 2 Å.
Fig 9.
Structure of ghrelin based on MAS ssNMR chemical shift data.
A) Final ensemble of ghrelin selected from the ensemble selection algorithm discussed in the main text. The Ser3 Cα of each model was superimposed on the others. The manually placed octanoyl side chain is shown as sticks. The model in Panel B is shown as solid cartoon. Residues predicted to be PPII helix (21–23 and 26–27) are colored in orange. Residues predicted to be helical according to the chemical shift analysis displayed in Fig. 6 (4, 8, 10, and 12) are colored in green. The rest of the ensemble is shown as ribbon (gray). The ensemble was manually positioned on the surface of a DMPC lipid bilayer (blue) to illustrate how the highly flexible ghrelin peptide might interact with the membrane. B) Model from the final ensemble. The color scheme is the same as the peptide (cartoon) in Panel A. Positively charged residues (Arg and Lys), Ser3, Phe4, Val12, and Ala23 are displayed as lines.
Table 2.
Statistics for restraints, structural calculations, and structural quality for final ensemble of ghrelin models.
Fig 10.
Secondary structure analysis of ghrelin.
A) Ramachandran plots of various subsets of residues as labeled at the top of the plots. The torsion angles of all models generated in Rosetta (gray) and the final ensemble of models (black) are plotted. B) Weblogo (http://weblogo.threeplusone.com) of PPII-DSSP analysis of final ensemble of ghrelin models. Color key: black = random coil (C), blue = bend (S) or turn (T), and green = α-, 310-, or PPII helix (H, G, or P, respectively).