DESIGN AND CHARACTERIZATION OF AN INTRAMOLECULAR ANTIPARALLEL COILED-COIL PEPTIDE

A 56-residue polypeptide was designed to fold into a stable intramolecular antiparallel coiled coil, referred to as a coiled coil stem loop. The antiparallel orientation of the alpha-helices was dictated by the alignment of hydrophobic and ionic residues in the heptad repeat sequence (a, b, c, d, e, f, g)(n). The hydrophobic core at the coiled coil interface was occupied by leucine and valine residues in heptad positions d and a' and positions a and d', respectively. The interface border positions e and g were occupied by glutamic acid in the amino-terminal helix and lysine residues in the carboxy-terminal helix. A loop segment connecting the alpha-helices began and ended with the helix-breaking residues glycine and proline. Alanine and serine residues were placed on the exposed b, c, and f positions of both helices to increase the helical propensity and solubility of the peptide, respectively. Several lines of evidence argued that the synthetic peptide made with this design folded into a stable monomeric coiled coil stem loop conformation: (1) the peptide was highly soluble in 150 mM sodium chloride and 50 mM sodium phosphate, pH 7.4; (2) the circular dichroism spectrum was ct-helical but with relative ellipticity minima at 222 and 208 nm characteristic of a coiled coil structure; (3) the peptide exhibited an alpha-helical content near 80%, which was independent of peptide concentration and unchanged in the presence of trifluoroethanol; (4) size exclusion chromatography and sedimentation equilibrium ultracentrifuge measurements confirmed that the peptide was monomeric in aqueous solution; (5) the peptide exhibited high helical content over a wide pH range; (6) the apparent T-M for unfolding the alpha-helical structure was greater than 65 degrees C, and 3.0 M urea was required to reduce the helical structure by 50%; (7) a disulfide bond was readily formed in the monomer between the amino- and carboxy-terminal cysteine residues, confirming the antiparallel orientation of the helices; and (8) the peptide competed with fibrinogen for the GPIIbIIIa receptor indicating that the RGD residues present in the loop sequence were available for binding. This work establishes that an antiparallel alignment of alpha-helices can be achieved by designing specific hydrophobic and ionic interactions within the coiled coil. The prototype coiled coil peptide represents a sequence-simplified scaffold into which residues from alpha-helices and loops of native proteins can be inserted to form conformationally constrained mimetic recognition molecules.