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. 1999 Mar;76(3):1367–1376. doi: 10.1016/S0006-3495(99)77298-X

The role of proline and glycine in determining the backbone flexibility of a channel-forming peptide.

J Jacob 1, H Duclohier 1, D S Cafiso 1
PMCID: PMC1300115  PMID: 10049319

Abstract

Alamethicin is a helical 20-amino acid voltage-gated channel-forming peptide, which is known to exhibit segmental flexibility in solution along its backbone near alpha-methylalanine (MeA)-10 and Gly-11. In an alpha-helical configuration, MeA at position 10 would normally hydrogen-bond with position 14, but the presence of proline at this position prevents the formation of this interhelical hydrogen bond. To determine whether the presence of proline at position 14 contributes to the flexibility of this helix, two analogs of alamethicin were synthesized, one with proline 14 replaced by alanine and another with both proline 14 and glycine 11 replaced by alanine. The C-termini of these peptides were derivatized with a proxyl nitroxide, and paramagnetic enhancements produced by the nitroxide on the Calpha protons were used to estimate r-6 weighted distances between the nitroxide and the backbone protons. When compared to native alamethicin, the analog lacking proline 14 exhibited similar C-terminal to Calpha proton distances, indicating that substitution of proline alone does not alter the flexibility of this helix; however, the subsequent removal of glycine 11 resulted in a significant increase in the averaged distances between the C- and N-termini. Thus, the G-X-X-P motif found in alamethicin appears to be largely responsible for mediating high-amplitude bending motions that have been observed in the central helical domain of alamethicin in methanol. To determine whether these substitutions alter the channel behavior of alamethicin, the macroscopic and single-channel currents produced by these analogs were compared. Although the substitution of the G-X-X-P motif produces channels with altered characteristics, this motif is not essential to achieve voltage-dependent gating or alamethicin-like behavior.

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Selected References

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  1. Archer S. J., Ellena J. F., Cafiso D. S. Dynamics and aggregation of the peptide ion channel alamethicin. Measurements using spin-labeled peptides. Biophys J. 1991 Aug;60(2):389–398. doi: 10.1016/S0006-3495(91)82064-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barlow D. J., Thornton J. M. Helix geometry in proteins. J Mol Biol. 1988 Jun 5;201(3):601–619. doi: 10.1016/0022-2836(88)90641-9. [DOI] [PubMed] [Google Scholar]
  3. Barranger-Mathys M., Cafiso D. S. Membrane structure of voltage-gated channel forming peptides by site-directed spin-labeling. Biochemistry. 1996 Jan 16;35(2):498–505. doi: 10.1021/bi951985d. [DOI] [PubMed] [Google Scholar]
  4. Bechinger B. Structure and functions of channel-forming peptides: magainins, cecropins, melittin and alamethicin. J Membr Biol. 1997 Apr 1;156(3):197–211. doi: 10.1007/s002329900201. [DOI] [PubMed] [Google Scholar]
  5. Brandl C. J., Deber C. M. Hypothesis about the function of membrane-buried proline residues in transport proteins. Proc Natl Acad Sci U S A. 1986 Feb;83(4):917–921. doi: 10.1073/pnas.83.4.917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coronado R., Latorre R. Phospholipid bilayers made from monolayers on patch-clamp pipettes. Biophys J. 1983 Aug;43(2):231–236. doi: 10.1016/S0006-3495(83)84343-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. D'Aquino J. A., Gómez J., Hilser V. J., Lee K. H., Amzel L. M., Freire E. The magnitude of the backbone conformational entropy change in protein folding. Proteins. 1996 Jun;25(2):143–156. doi: 10.1002/(SICI)1097-0134(199606)25:2<143::AID-PROT1>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  8. Dathe M., Kaduk C., Tachikawa E., Melzig M. F., Wenschuh H., Bienert M. Proline at position 14 of alamethicin is essential for hemolytic activity, catecholamine secretion from chromaffin cells and enhanced metabolic activity in endothelial cells. Biochim Biophys Acta. 1998 Mar 6;1370(1):175–183. doi: 10.1016/s0005-2736(97)00260-5. [DOI] [PubMed] [Google Scholar]
  9. Duclohier H., Molle G., Dugast J. Y., Spach G. Prolines are not essential residues in the "barrel-stave" model for ion channels induced by alamethicin analogues. Biophys J. 1992 Sep;63(3):868–873. doi: 10.1016/S0006-3495(92)81637-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Esposito G., Carver J. A., Boyd J., Campbell I. D. High-resolution 1H NMR study of the solution structure of alamethicin. Biochemistry. 1987 Feb 24;26(4):1043–1050. doi: 10.1021/bi00378a010. [DOI] [PubMed] [Google Scholar]
  11. Fox R. O., Jr, Richards F. M. A voltage-gated ion channel model inferred from the crystal structure of alamethicin at 1.5-A resolution. Nature. 1982 Nov 25;300(5890):325–330. doi: 10.1038/300325a0. [DOI] [PubMed] [Google Scholar]
  12. Franklin J. C., Ellena J. F., Jayasinghe S., Kelsh L. P., Cafiso D. S. Structure of micelle-associated alamethicin from 1H NMR. Evidence for conformational heterogeneity in a voltage-gated peptide. Biochemistry. 1994 Apr 5;33(13):4036–4045. doi: 10.1021/bi00179a032. [DOI] [PubMed] [Google Scholar]
  13. Gibbs N., Sessions R. B., Williams P. B., Dempsey C. E. Helix bending in alamethicin: molecular dynamics simulations and amide hydrogen exchange in methanol. Biophys J. 1997 Jun;72(6):2490–2495. doi: 10.1016/S0006-3495(97)78893-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hall J. E., Vodyanoy I., Balasubramanian T. M., Marshall G. R. Alamethicin. A rich model for channel behavior. Biophys J. 1984 Jan;45(1):233–247. doi: 10.1016/S0006-3495(84)84151-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Helluin O., Bendahhou S., Duclohier H. Voltage sensitivity and conformational change of isolated S4L45 fragments from sodium channels are tuned to proline. Eur Biophys J. 1998;27(6):595–604. doi: 10.1007/s002490050171. [DOI] [PubMed] [Google Scholar]
  16. Kaduk C., Duclohier H., Dathe M., Wenschuh H., Beyermann M., Molle G., Bienert M. Influence of proline position upon the ion channel activity of alamethicin. Biophys J. 1997 May;72(5):2151–2159. doi: 10.1016/S0006-3495(97)78858-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Keith A., Bulfield G., Snipes W. Spin-labeled Neurospora mitochondria. Biophys J. 1970 Jul;10(7):618–629. doi: 10.1016/S0006-3495(70)86324-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kelsh L. P., Ellena J. F., Cafiso D. S. Determination of the molecular dynamics of alamethicin using 13C NMR: implications for the mechanism of gating of a voltage-dependent channel. Biochemistry. 1992 Jun 9;31(22):5136–5144. doi: 10.1021/bi00137a007. [DOI] [PubMed] [Google Scholar]
  19. Li S. C., Goto N. K., Williams K. A., Deber C. M. Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6676–6681. doi: 10.1073/pnas.93.13.6676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Montal M., Mueller P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3561–3566. doi: 10.1073/pnas.69.12.3561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. North C. L., Franklin J. C., Bryant R. G., Cafiso D. S. Molecular flexibility demonstrated by paramagnetic enhancements of nuclear relaxation. Application to alamethicin: a voltage-gated peptide channel. Biophys J. 1994 Nov;67(5):1861–1866. doi: 10.1016/S0006-3495(94)80667-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reimer U., el Mokdad N., Schutkowski M., Fischer G. Intramolecular assistance of cis/trans isomerization of the histidine-proline moiety. Biochemistry. 1997 Nov 11;36(45):13802–13808. doi: 10.1021/bi9713916. [DOI] [PubMed] [Google Scholar]
  23. Richardson J. S., Richardson D. C. Amino acid preferences for specific locations at the ends of alpha helices. Science. 1988 Jun 17;240(4859):1648–1652. doi: 10.1126/science.3381086. [DOI] [PubMed] [Google Scholar]
  24. Sankararamakrishnan R., Vishveshwara S. Conformational studies on peptides with proline in the right-handed alpha-helical region. Biopolymers. 1990;30(3-4):287–298. doi: 10.1002/bip.360300307. [DOI] [PubMed] [Google Scholar]
  25. Sansom M. S. Proline residues in transmembrane helices of channel and transport proteins: a molecular modelling study. Protein Eng. 1992 Jan;5(1):53–60. doi: 10.1093/protein/5.1.53. [DOI] [PubMed] [Google Scholar]
  26. Sessions R. B., Gibbs N., Dempsey C. E. Hydrogen bonding in helical polypeptides from molecular dynamics simulations and amide hydrogen exchange analysis: alamethicin and melittin in methanol. Biophys J. 1998 Jan;74(1):138–152. doi: 10.1016/S0006-3495(98)77775-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vogel H., Nilsson L., Rigler R., Meder S., Boheim G., Beck W., Kurth H. H., Jung G. Structural fluctuations between two conformational states of a transmembrane helical peptide are related to its channel-forming properties in planar lipid membranes. Eur J Biochem. 1993 Mar 1;212(2):305–313. doi: 10.1111/j.1432-1033.1993.tb17663.x. [DOI] [PubMed] [Google Scholar]
  28. Williams K. A., Deber C. M. Proline residues in transmembrane helices: structural or dynamic role? Biochemistry. 1991 Sep 17;30(37):8919–8923. doi: 10.1021/bi00101a001. [DOI] [PubMed] [Google Scholar]
  29. Woolfson D. N., Williams D. H. The influence of proline residues on alpha-helical structure. FEBS Lett. 1990 Dec 17;277(1-2):185–188. doi: 10.1016/0014-5793(90)80839-b. [DOI] [PubMed] [Google Scholar]

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