Ion Interactions of Gramicidin A using Multidimensional Proton Solid State NMR

Gramicidin A, an enzymatic product from the bacterium Bacillus brevis, is a fifteen residue peptide that induces cell lysis by forming a transmembrane channel that conducts monovalent cations and water molecules into a cell. The dimer of gramicidin A forms a right-handed β-helix, creating a pore of 4 A that supports a single column of water molecules. The cation transport properties of the channel formed by the gramicidin A dimer is very similar to physiologically important channels, and therefore makes gramicidin A an attractive model to study.A two-dimensional heteronuclear correlation (HETCOR) solid state NMR spectroscopy was used to correlate 1H and 15N chemical shifts and to obtain the 1H-15N dipolar couplings of aligned gramicidin A samples in lamellar phase lipid environments. The 1H-15N dipolar splittings obtained from HETCOR are not sensitive to the 1H carrier frequency. The additional 1H chemical shift information may give new insights and therefore offers a great basis for studies looking for new orientation restraints such as 1H chemical shifts.Ions were introduced to gramicidin A since previous studies have shown ion binding to affect 15N chemical shift tensors, and may affect 1H chemical shift tensors likewise. Initial results yielded a 2 ppm shift in 1H chemical shift upon addition of K+ ions at the Trp15 site of gramicidin A. A shift of 1.2 kHz in the 1H-15N dipolar couplings was also observed. Since proton chemical shifts are highly sensitive to the surrounding environment, the 2 ppm difference in 1H chemical shift upon the addition of K+ ions suggest a significant change in peptide plane orientation. We will fully report the effects of ions on chemical shift tensors in gramicidin A and demonstrate the ability of the HETCOR experiment to produce high resolution spectra.