Novel F13,F15 Gramicidin Subunits Predicted to Cross Bilayer Membranes and form Ion Channels

Abstract

Gramicidin A (formyl-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp9-D-Leu-L-Trp11-D-Leu-L-Trp13-D-Leu-L-Trp15-ethanolamine) functions as a membrane-spanning channel for monovalent cations. Each channel is composed of two subunits joined by six hydrogen bonds. The four Trp residues anchor each subunit to the aqueous interface and prevent subunits from crossing a lipid-bilayer membrane. Nevertheless, it has been observed that replacing Trp residues 13 and 15 with Phe results in subunits (designated [Phe13,15]gA) that are capable of crossing the membrane, presumably in a double-stranded conformation, to engage in channel formation. Our goal is to modify [Phe13,15]gA to increase the channel lifetime in biological membranes, while retaining its ability to cross membranes. To this end, the length of [Phe13,15]gA was increased to allow for better matching in thicker biological membranes, and Ala was introduced near the N-terminus to increase channel stability. Comparative biophysical experiments with three novel peptides, endo-Gly0B-Gly0A-[Phe13,15]gA, endo-Gly0B-D-Ala0A-[Phe13,15]gA and endo-D-Ala0A-[Phe13,15]gA, were performed in DMPC and DOPC bilayers. Deuterated alanines were included to facilitate analysis by solid-state deuterium NMR spectroscopy. The 2H NMR spectra in mechanically aligned DMPC bilayers suggest a single major membrane orientation for each peptide, with slightly different backbone torsion angles for Ala3 and Ala5. By contrast, the spectra in DOPC are poorly resolved. Circular dichroism spectra in DMPC and DOPC vesicles suggest mixtures of single- and double-stranded conformations. The Trp fluorescence emission λ-maxima for endo-Gly0B-D-Ala0A-[Phe13,15]gA (335 nm) and endo-D-Ala0A-[Phe13,15]gA (336 nm) in DOPC vesicles are shifted to higher wavelengths compared to [Phe13,15]gA (330 nm). Experiments to assess channel lifetimes and membrane-crossing ability of each peptide are underway.