Alamethicin: Secondary Structure in Solution and Interactions with Phospholipid Membranes

• Main Author: Banerjee, Utpal
• Format: Thesis
• Language: English
• Published: California Institute of Technology 1984
• Subjects: Chemistry; Gauche; Alvarez; DML
• Online Access: https://dx.doi.org/10.7907/9y1x-zh89; https://resolver.caltech.edu/CaltechTHESIS:10292018-120530235

The icosapeptide alamethicin [sequence included in scanned thesis' abstract, p. v] isolated from the fungus Trichoderma viride induces voltage-gated ionic conductance in black lipid film membranes (Latorre, R. & Alvarez, D. (1981) Physiol. Rev. 61, 77). Single-channel measurements have indicated that passive ion transport across membranes is mediated by alamethicin channels that fluctuate between several conduction states. While these studies provide phenomenological description of the nature of alamethicin-assisted ionic conduction, very few studies probed the molecular structure of this peptide and the nature of its interaction with lipid membranes. These issues are addressed in the present investigation. An analysis of the proton magnetic resonance spectrum is undertaken. Two-dimensional NMR is employed to achieve a complete assignment of the protons in the molecule to NMR resonances. The spectral assignment is a necessary first step towards molecular interpretations. Measurement of coupling constants and two-dimensional NOE's suggest a half-helical, half-extended dimeric structure for the molecule in methanol. This proposed model for the secondary structure, consistent with the NMR data as well as a line of other experimental observations erstwhile published, predicts that (a) the amide protons of residues 15 through 20 are intermolecularly hydrogen-bonded with the corresponding residues of the opposing molecule to create a rigid, extended parallel β-pleated structure for the C-terminal end of the molecule; (b) the proline at position 14 breaks the continuity of this structure, and amino acids 10 through 14 are forced into an open, non-hydrogen-bonded conformation, and (c) amino acids 3 through 9 are folded into an α-helix, with Gln-7 side chains from the two strands in the right juxtaposition to facilitate a hydrogen bond between them. The resultant structure is highly amphipathic: one face is completely hydrophobic with the aliphatic side chains exposed, whereas the other face is primarily hydrophilic with polar side chains and peptide groups lining the extended β-sheet region. The dimeric structure is further supported by relaxation measurements that indicate that the N-acetyl methyl groups at the N-termini of the two helices in the dimer have distinct proton spin-spin relaxation times. This difference is eliminated once the dimers are dissociated with urea. Spectral assignments in water are complicated by broadened NMR signals due to aggregation. Standard two-dimensional and decoupling techniques for assignments are inadequate for this case. A successful assignment is achieved by solvent titration from methanol. No changes in coupling constants are noted during the titration, and it is expected that the conformation in water is similar if not identical, to that in methanol. Relaxation measurements in water are consistent with a tightly bound dimeric unit that micellises to larger aggregates. The interaction of alamethicin with multilayers is inferred from a spectroscopic investigation of the phospholipid bilayer prepared from dimyristoyllecithin (DML) in the presence and absence of alamethicin, and, for contrast, in the presence of other membrane active molecules. The dynamics and conformation of phospholipid head group and chains are examined by P31 and H2 NMR. A P31 line shape calculation has helped identify the dependence of the spectrum on various motional, relaxation and conformational parameters. As part of the investigation of lipid packing and dynamics in membranes, small bilayer vesicles are also studied. Proton NMR indicates that the outside-facing and inside-facing leaflets of the bilayer in small vesicles have lipids packed in different densities. This is due to the differences in the extent and sign of curvature of the two leaflets. At the high field at which this NMR study is undertaken, the differences in packing show up as distinct proton peaks from the inside and outside chain methylene and methyl groups nthat differ in width and in chemical shift. Finally, the interaction of alamethicin with DML multilayers is characterized by P31, H2 and H1 NMR and Raman spectroscopy. The reduction in chemical shift anisotropy (Δσ) of the P31 signal is interpreted in terms of an interaction of the peptide at the water-membrane interface that causes a change in the average head group orientation. Deuterium NMR shows no changes in quadrupolar splittings (and hence C-D order parameters) of the chain deuterons, and Raman spectroscopy shows no change in the gauche-trans ratio of methylene segments in the chain. These results are contrasted with P31 and H2 NMR of the gramicidin S/DML system that shows polymorphism due to partial disruption of the multilayer structure and the chlorophyll A/DML system that exhibits a 7° C change in phase transition temperature as a clear indication of incorporation of the phytol chain into the bilayer. Taken together, these experiments unequivocally indicate that the peptide interacts with lipid bilayers at the lipid-water interface. The proposed amphiphilic aggregated solution structure for the peptide is ideally suited for such an interaction. Inasmuch as the conductance characteristics of alamethicin are only explained in terms of transmembrane pore formation, it is proposed that the large dipole moment of this aggregate facilitates the transfer of the peptide into the bilayer once a gradient of field is applied.