SYNTHETIC MODEL PROTEINS - THE RELATIVE CONTRIBUTION OF LEUCINE RESIDUES AT THE NONEQUIVALENT POSITIONS OF THE 3-4 HYDROPHOBIC REPEAT TO THE STABILITY OF THE 2-STRANDED ALPHA-HELICAL COILED-COIL

Our de novo designed coiled-coil model protein consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment via interchain hydrophobic interactions and a disulfide bridge at the position 2 between two helices. To quantitate the relative contribution of leucine residues at the nonequivalent position of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil, a single alanine was systematically substituted for a leucine in each chain at position "a" (9, 16, 23, or 30) or "d" (5, 12, 19, 26, or 33). The formation and stability of the coiled-coils were determined by circular dichroism studies in the absence and presence of guanidine hydrochloride. All the proteins with an alanine substituted at position a have a similar stability ([Gdn.HCl]1/2 ranges from 2.6 to 2.9 M), while all the proteins with an alanine substituted at position d have similar stability ([Gdn.HCl]1/2 ranges from 3.6 to 4.2 M), except for the proteins with an alanine substituted in the C-terminal heptad. The greater decrease in stability observed for a Leu --> Ala mutation at position a (the average DELTA-DELTA-G(u) value is 3.3 kcal/mol) compared to those where the substitution was effected at position d (the average DELTA-DELTA-G(u) value is 2.0 kcal/mol) indicates that an Ala mutation at position a has a greater effect on the side-chain packing and hydrophobic interactions in the coiled-coil than an Ala mutation at position d. Analyses of the retention behaviors of these coiled-coils during reversed-phase chromatography and computer modeling also suggest that the isopropyl group (which is the structural difference between the side chain of Leu and Ala) of the leucine side chain is more buried at position a than at position d. The difference between the leucine residues at positions a and d in terms of their contribution to the coiled-coil stability is not dependent on the disulfide bridge location. Rather, the disulfide bridge locks the coiled-coil structure in a conformation which mimics the conformation observed in the X-ray structure of the GCN4 leucine zipper.