DE-NOVO DESIGN OF A PEPTIDE WHICH PARTITIONS BETWEEN WATER AND PHOSPHOLIPID-BILAYERS AS A MONOMERIC ALPHA-HELIX

To dissect the determinants of protein insertion into membranes, we de signed a model peptide which partitions between water and phospholipid bilayers as an alpha-helical monomer, We used a simplex method to opt imize the 'a, d hydrophobicity' and 'e, g charge' of a series of five peptides, where 'abcdefg' correspond to the positions in two turns of an alpha-helix. Circular dichroism and analytical ultracentrifugation experiments showed that the final peptide (helix5) is monomeric and ha s an alpha-helix content of approximately 89% at 0 degrees C in aqueou s solution. In the presence of large unilamellar vesicles (LUVs), heli x5 partitions between the aqueous and membranous phases with a partiti on constant well suited for measurements by electron paramagnetic reso nance (EPR) spectroscopy, EPR power saturation experiments with a cyst eine-scanning strategy showed that the alpha-helicity of helix5 is con served upon binding to LUVs and that the alpha-helix binds parallel to the membrane surface with the central axis approximately 5 Angstrom b elow the lipid phosphate groups. Helix5 should be a useful model pepti de for studies aimed at dissecting the determinants of the membrane bi nding of alpha-helices. The simplex-based strategy may be useful in th e rational design of proteins when desired structural or partitioning properties cannot be selected or screened from libraries.