A Proline Kink in GWALP23

Abstract

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-ethanolamide) is a proven model membrane-spanning peptide (see JACS 130, 12584) that moves “beyond” WALP family peptides by employing, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central alpha-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thickness (see JBC 285, 31723), we utilize GWALP23 as a framework for introducing guest residues within the transmembrane sequence. For example, we have incorporated a central proline residue to give acetyl-GGALW5LALALAP12ALALALW19LAGA-ethanolamide. We have synthesized the resulting GWALP23-P12 with selected 2H and 15N labels for solid-state NMR spectroscopy, to enable analysis of the peptide orientation and segmental tilt in oriented lipid bilayer membranes using combined (2H)-GALA and (15N/1H)-PISEMA methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to proline are tilted substantially with respect to the bilayer normal, by about 34°-40° and 27°-29° (± 6°), respectively, with a proline-induced kink angle of 20°-23°. The proline places restrictions on the dynamics of both segments. As has been described previously for GWALP23, the C-terminal helix ends before Ala-21.