Conformation, pore-forming activity, and antigenicity of synthetic peptide analogues of a spiralin putative amphipathic α helix

Abstract

Current models depict spiralin as a bitopic transmembrane protein with the transbilayer domain being an amphipathic α helix. However, though secondary structure prediction methods suggest a helical conformation for the hypothetical transmembrane segment of spiralin, no potential transmembrane helices could be detected in this protein using the method of Von Heijne ( Von Heijne, G. (1992) J. Mol. Biol. 225, 487–494). Therefore, we have reconsidered the spiralin topological model by investigating the properties of the chemically synthesized peptides SM-BC3 (LNAVNTYATLAKAVLDAIQN-NH 2) and SC-R8A2 (LNAVNTYATLASAVLEAIKN-NH 2), corresponding to the hypothetical transmembrane segments of spiralins of two distinct spiroplasma species. The hydrophobic moment plot method suggests that these spiralin amino acid stretches are class G amphipathic α helices (i.e., helices localized on the surface of a globular protein domain). Circular dichroism spectra showed that both peptides have little ordered structure in aqueous solutions but adopt a mainly helical conformation in the presence of 25% trifluoroethanol or in detergent micelles (up to 74% α helix). Both peptides formed concentration- and voltage-dependent pores in planar lipid bilayers with a unitary conductance of 130 pS in 1 M KCl and with mean numbers of monomers per conducting aggregates of 6 for SC-R8A2 and 9 for SM-BC3. However, the two peptides displayed a haemolytic activity only at high concentrations (≫ 250 μM) and reacted with antibodies raised against membrane-bound spiralin. Together with previously published results, these data suggest that spiralin is a monotopic membrane protein anchored at the surface of the spiroplasma cell and that the 20-residue amphipathic segment is most probably a class G helix containing a B-cell epitope.