Solid-State NMR Studies on the Membrane Bound Acylated Peptide Hormone Ghrelin

Abstract

Ghrelin is a 28-amino acid peptide, which is known to play a key role in the regulation of food intake and body weight. Ghrelin is the only known hormone that carries an octanoyl lipid modification at residue Ser 3. Here, we have studied the structure and dynamics of the molecule bound to DMPC/DMPS (80/20) membranes by solid-state NMR spectroscopy. We have synthesized several Ghrelin peptides with varying 13C/15N containing amino acids covering 10 out of the 28 residues in the sequence. 13C NMR spectra recorded under magic-angle spinning conditions provide backbone torsion angles for the membrane-associated peptide. Further, the molecular dynamics of Ghrelin was studied using motional averaged dipolar coupling measurements. Overall, the data indicates a rather flexible peptide conformation on the membrane surface. We have further studied the conformation and membrane insertion of the octanoyl lipid modification of Ghrelin using 2H NMR. Ghrelin binding leads to a small alteration in the order parameter profile of the host membrane. Further, we have also measured the 2H NMR spectrum of a Ghrelin molecule with a deuterated octanoyl chain. This spectrum showed clear indications of a membrane inserted lipid chain, however, the chain order parameters of the lipid modification are smaller translating into a somewhat shorter chain length compared to the host membrane. Experimentally, we determined a membrane binding energy of ∼40 kJ/mol for Ghrelin, which cannot solely be explained by the insertion of hydrophobic amino acids and the octanoyl chain. In addition, electrostatic forces play a role arising from the 7 positively charged residues in the peptide sequence. In the course of action, Ghrelin binds to the GHS receptor, so membrane binding might be an important step increasing the Ghrelin concentration at the membrane prior to binding to its receptor.