Gramicidin cation channel: an experimental determination of the right-handed helix sense and verification of beta-type hydrogen bonding.

Due to the difficulty of obtaining protein/lipid cocrystals for diffraction studies, structural research on intrinsic membrane proteins and polypeptides has been largely restricted to indirect experimental techniques. Hence, many fundamental questions associated with peptide/lipid systems remain unanswered. In particular, the handedness of the gramicidin A transmembrane ion channel incorporated into lipid bilayers has been an open question for nearly two decades. In this study, solid-state 15N NMR spectroscopy is employed to probe directly the secondary structure of the polypeptide backbone. Recent determinations of the 15N chemical shift anisotropy tensor with respect to the molecular frame enable the quantitative evaluation of the 15N chemical shift resonances obtained from oriented dimyristoylphosphatidylcholine (DMPC) bilayer samples containing specific site 15N labeled gramicidin. This direct structural approach verifies the beta-sheet hydrogen-bonding pattern proposed by Urry [Urry, D. W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676] and determines that in our DMPC bilayer preparations the gramicidin channel is right-handed. Additional structural information is provided by the 15N chemical shift data in the form of orientational constraints on the C alpha-C alpha axis orientation of individual peptides relative to the helix axis. The significance of these solid-state NMR results lies in the direct determination of the helix sense and the verification of the beta-type hydrogen bonding, in the development of the solid-state NMR methods for obtaining such information, and in emphasizing the importance of having direct structural data at atomic resolution.