We have designed and synthesized a small, unique protein molecule with defined secondary, tertiary and quaternary structure. This 35-residue peptide, containing a cysteine residue at its N-terminal end, was oxidized to form a 70-residue disulfide-linked two-stranded alpha-helical coiled-coil with the two alpha-helices parallel and in-register. The major contribution to the formation and stabilization of the alpha-helical coiled-coil is hydrophobic interactions between positions 2 and 5 of the heptapeptide repeat (Lys-Leu-Glu-Ala-Leu-Glu-Gly). The protein (L-protein) contains nine leucine-leucine hydrophobic interactions between the alpha-helices of the coiled-coil. Circular dichroism studies demonstrated that this protein in its reduced ([L (r)] or oxidized (L (o)] state was essentially 100% alpha-helical ([theta]220 = -34,050 and -32,000 degrees respectively) at pH 2 (0.1% aqueous trifluoroacetic acid). Our objective was to modify systematically the structure of L to delineate the contribution that various amino acid side chains make to the formation and stabilization of its three-dimensional structure. A-protein, which contains alanine instead of leucine at positions 16 and 19 of the hydrophobic repeat in each chain of the coiled-coil, was compared to the L-protein. At pH 2, the oxidized form of the A-protein [A (o)] was essentially 100% helical. However, the protein was much less stable to temperature denaturation compared to the L-protein. The replacement of two leucine-leucine interactions by two alanine-alanine interactions has a dramatic effect on the formation and stability of the two-stranded alpha-helical coiled-coil structure. The results of this study clearly demonstrate the validity of this synthetic model protein approach to understanding the molecular aspects responsible for the folding and stabilization of protein molecules.