Biophysical Journal
Volume 53, Issue 1, January 1988, Pages 67-76
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Articles
Conformation and Orientation of Gramicidin a in Oriented Phospholipid Bilayers Measured by Solid State Carbon-13 NMR

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Three analogues of the helical ionophore gramicidin A have been synthesized with 13C-labeled carbonyls (13C=O) incorporated at either Gly2, Ala3, or Val7. A fourth compound incorporated 13C at both the carbonyl and α-carbon of Gly2 within the same molecule. These labels were studied using solid-state, proton-enhanced, 13C nuclear magnetic resonance (NMR) in hydrated dispersions of dimyristoylphosphatidylcholine (DMPC)-gramicidin A. The dispersions were aligned on glass coverslips whose orientation to the magnetic field could be varied through 180°. The orientation dependence of the NMR spectrum was used to obtain an accurate measurement of the 13C chemical shift anisotropy (CSA), and in the case of the fourth compound, the 13C—13C dipolar coupling constant. From the measured CSA and estimates of the orientation of the 13C shielding tensor, we are able to determine the direction of the 13C=O bonds and to compare these with the predictions of the various reported models for the configuration of gramicidin A in phospholipid bilayers. Our results are consistent with the left-handed ππ6.3LD single-stranded helix (Urry, D. W., J. T. Walker, and T. L. Trapane. 1982. J. Membr. Biol. 69:225–231). The right-handed ππ6.3LD single-stranded helix observed for gramicidin A in sodium dodecyl sulfate micelles (Arseniev, A. S., I. L. Barsukov, V. F. Bystrov, A. L. Loize, and Yu A. Ovchinnikov. 1985. FEBS (Fed. Eur. Biochem. Soc.) Lett. 186:168–174) yields a poorer fit to the data. However, the width of the carbonyl resonances suggests a distribution of molecular geometries possibly resulting from a spread in the helix pitch and handedness. Double-stranded helices and β sheet structures are excluded. In dispersions in which the lipid is in the Lα phase, the gramicidin A undergoes rapid reorientation about an axis which is centered on the normal to the plane of the coverslips. When the supporting lipid is in the Lβ′ phase the helices are rigid on the timescale of 13C-NMR. The configuration of gramicidin A is unaltered by Lα-Lβ′ phase transition of the bilayer lipid.

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