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. 1988 Jan;53(1):67–76. doi: 10.1016/S0006-3495(88)83066-2

Conformation and Orientation of Gramicidin a in Oriented Phospholipid Bilayers Measured by Solid State Carbon-13 NMR

Bruce A Cornell, Frances Separovic, Attilio J Baldassi, Ross Smith
PMCID: PMC1330122  PMID: 19431717

Abstract

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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arseniev A. S., Barsukov I. L., Bystrov V. F., Lomize A. L., Ovchinnikov YuA 1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices. FEBS Lett. 1985 Jul 8;186(2):168–174. doi: 10.1016/0014-5793(85)80702-x. [DOI] [PubMed] [Google Scholar]
  2. Bamberg E., Apell H. J., Alpes H. Structure of the gramicidin A channel: discrimination between the piL,D and the beta helix by electrical measurements with lipid bilayer membranes. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2402–2406. doi: 10.1073/pnas.74.6.2402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cavatorta P., Spisni A., Casali E., Lindner L., Masotti L., Urry D. W. Intermolecular interactions of gramicidin A' transmembrane channels incorporated into lysophosphatidylcholine lipid systems. Biochim Biophys Acta. 1982 Jul 14;689(1):113–120. doi: 10.1016/0005-2736(82)90195-x. [DOI] [PubMed] [Google Scholar]
  4. Chapman D., Cornell B. A., Ellasz A. W., Perry A. Interactions of helical polypepetide segments which span the hydrocarbon region of lipid bilayers. Studies of the gramicidin A lipid-water system. J Mol Biol. 1977 Jul 5;113(3):517–538. doi: 10.1016/0022-2836(77)90236-4. [DOI] [PubMed] [Google Scholar]
  5. Datema K. P., Pauls K. P., Bloom M. Deuterium nuclear magnetic resonance investigation of the exchangeable sites on gramicidin A and gramicidin S in multilamellar vesicles of dipalmitoylphosphatidylcholine. Biochemistry. 1986 Jul 1;25(13):3796–3803. doi: 10.1021/bi00361a010. [DOI] [PubMed] [Google Scholar]
  6. Eisenman G., Sandblom J. P. Modeling the gramicidin channel: interpretation of experimental data using rate theory. Biophys J. 1984 Jan;45(1):88–90. doi: 10.1016/S0006-3495(84)84119-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fossel E. T., Veatch W. R., Ovchinnikov U. A., Blout E. R. A 13C nuclear magnetic resonance study of gramicidin A in monomer and dimer forms. Biochemistry. 1974 Dec 17;13(26):5264–5275. doi: 10.1021/bi00723a003. [DOI] [PubMed] [Google Scholar]
  8. Haigh E. A., Thulborn K. R., Sawyer W. H. Comparison of fluorescence energy transfer and quenching methods to establish the position and orientation of components within the transverse plane of the lipid bilayer. Application to the gramicidin A--bilayer interaction. Biochemistry. 1979 Aug 7;18(16):3525–3532. doi: 10.1021/bi00583a014. [DOI] [PubMed] [Google Scholar]
  9. Hladky S. B., Haydon D. A. Ion transfer across lipid membranes in the presence of gramicidin A. I. Studies of the unit conductance channel. Biochim Biophys Acta. 1972 Aug 9;274(2):294–312. doi: 10.1016/0005-2736(72)90178-2. [DOI] [PubMed] [Google Scholar]
  10. Katz E., Demain A. L. The peptide antibiotics of Bacillus: chemistry, biogenesis, and possible functions. Bacteriol Rev. 1977 Jun;41(2):449–474. doi: 10.1128/br.41.2.449-474.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Killian J. A., Borle F., de Kruijff B., Seelig J. Comparative 2H- and 31P-NMR study on the properties of palmitoyllysophosphatidylcholine in bilayers with gramicidin, cholesterol and dipalmitoylphosphatidylcholine. Biochim Biophys Acta. 1986 Jan 16;854(1):133–142. doi: 10.1016/0005-2736(86)90073-8. [DOI] [PubMed] [Google Scholar]
  12. Killian J. A., de Kruijff B. Importance of hydration for gramicidin-induced hexagonal HII phase formation in dioleoylphosphatidylcholine model membranes. Biochemistry. 1985 Dec 31;24(27):7890–7898. doi: 10.1021/bi00348a007. [DOI] [PubMed] [Google Scholar]
  13. Killian J. A., de Kruijff B. Thermodynamic, motional, and structural aspects of gramicidin-induced hexagonal HII phase formation in phosphatidylethanolamine. Biochemistry. 1985 Dec 31;24(27):7881–7890. doi: 10.1021/bi00348a006. [DOI] [PubMed] [Google Scholar]
  14. Koeppe R. E., 2nd, Hodgson K. O., Stryer L. Helical channels in crystals of gramicidin A and of a cesium--gramicidin A complex: an x-ray diffraction study. J Mol Biol. 1978 May 5;121(1):41–54. doi: 10.1016/0022-2836(78)90261-9. [DOI] [PubMed] [Google Scholar]
  15. Koeppe R. E., 2nd, Schoenborn B. P. 5-A Fourier map of gramicidin A phased by deuterium-hydrogen solvent difference neutron diffraction. Biophys J. 1984 Mar;45(3):503–507. doi: 10.1016/S0006-3495(84)84186-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mackay D. H., Berens P. H., Wilson K. R., Hagler A. T. Structure and dynamics of ion transport through gramicidin A. Biophys J. 1984 Aug;46(2):229–248. doi: 10.1016/S0006-3495(84)84016-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Monoi H. Ionic interactions and anion binding in the gramicidin channel. An electrostatic calculation. J Theor Biol. 1983 May 7;102(1):69–99. doi: 10.1016/0022-5193(83)90263-1. [DOI] [PubMed] [Google Scholar]
  18. Nabedryk E., Gingold M. P., Breton J. Orientation of gramicidin A transmembrane channel. Infrared dichroism study of gramicidin in vesicles. Biophys J. 1982 Jun;38(3):243–249. doi: 10.1016/S0006-3495(82)84555-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Naik V. M., Krimm S. The structure of crystalline and membrane-bound gramicidin A by vibrational analysis. Biochem Biophys Res Commun. 1984 Dec 28;125(3):919–925. doi: 10.1016/0006-291x(84)91371-8. [DOI] [PubMed] [Google Scholar]
  20. Prasad K. U., Alonso-Romanowski S., Venkatachalam C. M., Trapane T. L., Urry D. W. Synthesis, characterization, and black lipid membrane studies of [7-L-alanine] gramicidin A. Biochemistry. 1986 Jan 28;25(2):456–463. doi: 10.1021/bi00350a027. [DOI] [PubMed] [Google Scholar]
  21. Prasad K. U., Trapane T. L., Busath D., Szabo G., Urry D. W. Synthesis and characterization of 1-(13) C-D X Leu12, 14 gramicidin A. Int J Pept Protein Res. 1982 Feb;19(2):162–171. [PubMed] [Google Scholar]
  22. Rajan S., Kang S. Y., Gutowsky H. S., Oldfield E. Phosphorus nuclear magnetic resonance study of membrane structure. Interactions of lipids with protein, polypeptide, and cholesterol. J Biol Chem. 1981 Feb 10;256(3):1160–1166. [PubMed] [Google Scholar]
  23. Rice D., Oldfield E. Deuterium nuclear magnetic resonance studies of the interaction between dimyristoylphosphatidylcholine and gramicidin A'. Biochemistry. 1979 Jul 24;18(15):3272–3279. doi: 10.1021/bi00582a012. [DOI] [PubMed] [Google Scholar]
  24. SARGES R., WITKOP B. GRAMICIDIN A. V. THE STRUCTURE OF VALINE- AND ISOLEUCINE-GRAMICIDIN A. J Am Chem Soc. 1965 May 5;87:2011–2020. doi: 10.1021/ja01087a027. [DOI] [PubMed] [Google Scholar]
  25. Sarin V. K., Kent S. B., Tam J. P., Merrifield R. B. Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction. Anal Biochem. 1981 Oct;117(1):147–157. doi: 10.1016/0003-2697(81)90704-1. [DOI] [PubMed] [Google Scholar]
  26. Smith R., Cornell B. A. The dynamics of the intrinsic membrane polypeptide gramicidin a in phospholipid bilayers: a solid state carbon-13 NMR study. Biophys J. 1986 Jan;49(1):117–118. doi: 10.1016/S0006-3495(86)83617-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Urry D. W., Shaw R. G., Trapane T. L., Prasad K. U. Infrared spectra of the Gramicidin A transmembrane channel: the single-stranded-beta 6-helix. Biochem Biophys Res Commun. 1983 Jul 18;114(1):373–379. doi: 10.1016/0006-291x(83)91637-6. [DOI] [PubMed] [Google Scholar]
  28. Urry D. W., Spisni A., Khaled A. Characterization of micellar-packaged gramicidin A channels. Biochem Biophys Res Commun. 1979 Jun 13;88(3):940–949. doi: 10.1016/0006-291x(79)91499-2. [DOI] [PubMed] [Google Scholar]
  29. Urry D. W. The gramicidin A transmembrane channel: a proposed pi(L,D) helix. Proc Natl Acad Sci U S A. 1971 Mar;68(3):672–676. doi: 10.1073/pnas.68.3.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Urry D. W., Trapane T. L., Prasad K. U. Is the gramicidin a transmembrane channel single-stranded or double-stranded helix? A simple unequivocal determination. Science. 1983 Sep 9;221(4615):1064–1067. doi: 10.1126/science.221.4615.1064. [DOI] [PubMed] [Google Scholar]
  31. Urry D. W., Venkatachalam C. M., Spisni A., Bradley R. J., Trapane T. L., Prasad K. U. The malonyl gramicidin channel: NMR-derived rate constants and comparison of calculated and experimental single-channel currents. J Membr Biol. 1980 Jun 30;55(1):29–51. doi: 10.1007/BF01926368. [DOI] [PubMed] [Google Scholar]
  32. Urry D. W., Walker J. T., Trapane T. L. Ion interactions in (1-13C)D-Val8 and D-Leu14 analogs of gramicidin A, the helix sense of the channel and location of ion binding sites. J Membr Biol. 1982;69(3):225–231. doi: 10.1007/BF01870401. [DOI] [PubMed] [Google Scholar]
  33. Veatch W. R., Blout E. R. The aggregation of gramicidin A in solution. Biochemistry. 1974 Dec 17;13(26):5257–5264. doi: 10.1021/bi00723a002. [DOI] [PubMed] [Google Scholar]
  34. Veatch W. R., Fossel E. T., Blout E. R. The conformation of gramicidin A. Biochemistry. 1974 Dec 17;13(26):5249–5256. doi: 10.1021/bi00723a001. [DOI] [PubMed] [Google Scholar]
  35. Wallace B. A. Ion-bond forms of the gramicidin a transmembrane channel. Biophys J. 1984 Jan;45(1):114–116. doi: 10.1016/S0006-3495(84)84131-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wallace B. A. Structure of gramicidin A. Biophys J. 1986 Jan;49(1):295–306. doi: 10.1016/S0006-3495(86)83642-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wallace B. A., Veatch W. R., Blout E. R. Conformation of gramicidin A in phospholipid vesicles: circular dichroism studies of effects of ion binding, chemical modification, and lipid structure. Biochemistry. 1981 Sep 29;20(20):5754–5760. doi: 10.1021/bi00523a018. [DOI] [PubMed] [Google Scholar]
  38. Weinstein S., Durkin J. T., Veatch W. R., Blout E. R. Conformation of the gramicidin A channel in phospholipid vesicles: a fluorine-19 nuclear magnetic resonance study. Biochemistry. 1985 Jul 30;24(16):4374–4382. doi: 10.1021/bi00337a019. [DOI] [PubMed] [Google Scholar]
  39. Weinstein S., Wallace B. A., Blout E. R., Morrow J. S., Veatch W. Conformation of gramicidin A channel in phospholipid vesicles: a 13C and 19F nuclear magnetic resonance study. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4230–4234. doi: 10.1073/pnas.76.9.4230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Weinstein S., Wallace B. A., Morrow J. S., Veatch W. R. Conformation of the gramicidin A transmembrane channel: A 13C nuclear magnetic resonance study of 13C-enriched gramicidin in phosphatidylcholine vesicles. J Mol Biol. 1980 Oct 15;143(1):1–19. doi: 10.1016/0022-2836(80)90121-7. [DOI] [PubMed] [Google Scholar]

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