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We have determined the conformation of the channel-forming polypeptide antibiotic gramicidin A in phosphatidylcholine vesicles by using '3C and 19F NMR spectroscopy. The models previously proposed for the conformation of the dimer channel differ in the surface localization of the NH2 and COOH termini. We have incorporated specific 13C and 19F nuclei at both the NH2, and COOH termini of gramicidin and have used 13C and 19F chemical shifts and spin lattice relaxation time measurements to determine the accessibility of these labels to three paramagnetic NMR probes-two in aqueous solution and one attached to a phosphatidylcholine fatty acid chain. All of our results indicate that the COOH terminus of gramicidin in the channel is located near the surface of the membrane and the NH2 terminus is buried deep within the lipid bilayer. These findings strongly favor an NH2-terminal to NHZ-terminal helical dimer as the major conformation for the gramicidin channel in phosphatidylcholine vesicles.Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across membranes (1-4) by forming transmembrane channels (4, 5), each of which is made up of two molecules of gramicidin (6-10). The amino acid sequence of valine gramicidin A (11) is:HCOVeatch et al. (7) The indole protons of gramicidin were resolved from the lipid resonances. It was argued that these indole protons were moderately exposed to the solution, on the basis of their Tm3+-induced chemical shift change; however, due to the lack of markers on or near the NH2 terminus, specific conformational conclusions could not be drawn. It should be noted that the nominal NH2 terminus is, in fact, formylated, and the nominal COOH terminus is, in fact, an ethanolamide.Four classes of models have been proposed for the conformation of the dimer gramicidin transmembrane channel ( (15) as one of the dimer conformations found for gramicidin in nonpolar organic solvents (15, 16). Model D is a parallel-f double helix with both NH2 termini at one end of the channel. A distinctive feature of both double-helical models is the presence of both the NH2 and COOH termini at the surfaces of the membrane (Fig. 1 C and D). For the NH2-terminal to NH2-terminal dimer (Fig. 1A) only the COOH termini are on the surfaces, and for the COOH-terminal to COOH-terminal dimer (Fig. 1B) only the NH2 termini are on the surface.We have incorporated specific 13C and 19F nuclei at both the NH2 and COOH termini (Fig. 2) and have carried out NMR experiments to determine the relative accessibility of these 13C and 19F nuclei to paramagnetic NMR probes. Two of these probes are localized in the aqueous solution-manganous ion (Mn2+) and thulium ion (Tm3+)-and the third is localized in the membrane interior-a nitroxide spin label covalently att Present address:

Crystals, diffracting best to around 3 A Ê , have been grown from intact large and small ribosomal subunits. The bright synchrotron radiation necessary for the collection of the higher-resolution X-ray diffraction data introduces signi®cant decay even at cryo temperatures. Nevertheless, owing to the reasonable isomorphism of the recently improved crystals of the small ribosomal subunits, reliable phases have been extracted at medium resolution (5±6 A Ê ) and an interpretable ®ve-derivative MIR map has been constructed. For the crystals of the large subunits, however, the situation is more complicated because at higher resolution (2.7±7 A Ê ) they suffer from substantial radiation sensitivity, a low level of isomorphism, instability of the longest unit-cell axis and nonisotropic mosaicity. The 8 A Ê MIR map, constructed to gain insight into this unusual system, may provide feasible reasoning for the odd combination of the properties of these crystals as well as hints for future improvement. Parallel efforts, in which electron-microscopy-reconstructed images are being exploited for molecular-replacement studies, are also discussed.