CONFORMATION, PORE-FORMING ACTIVITY, AND ANTIGENICITY OF SYNTHETIC PEPTIDE ANALOGS OF A SPIRALIN PUTATIVE AMPHIPATHIC ALPHA-HELIX

Current models depict spiralin as a bitopic transmembrane protein with the transbilayer domain being an amphipathic alpha 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 (LNAVNTYATL(AVLDAIQN-NH2) and SC-R8A2 (LNAVNTYATLASAVLEAIKN-NH2), 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 alpha 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% alpha 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 mu M) 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.