An antiparallel coiled-coil has been designed and characterized as a model for studying protein folding and assembly. This heterostranded antiparallel coiled-coil was formed by an interchain disulfide bond between cysteine residues at position 2 of one chain and at position 33 of the other chain. Each peptide chain has 35 residues which are composed of five heptad repeats of the sequence K-L-E-A-L-E-G with a single Leu-->Ala substitution at position 16. Two homostranded parallel coiled-coils were also formed as co-products of the oxidation reaction to form the interchain disulfide bond. The CD spectra of the parallel and antiparallel peptides were very similar and their high molar ellipticities at 220 nm did not increase in the presence of 50% trifluoroethanol. These data suggest that, like the parallel peptides, the antiparallel peptide also exists in a coiled-coil structure. Urea and guanidine hydrochloride denaturation studies, in conjunction with molecular modeling studies, suggest that there are no physical restrictions to the packing of hydrophobic residues in an antiparallel coiled-coil. However, interchain electrostatic interactions can have positive or negative contributions to the overall stability of the disulfide-bridged coiled-coil. In addition, interchain electrostatic interactions appear to play a major role in protein folding by controlling the parallel or antiparallel alignment of the alpha-helical polypeptide chains. This study is also for the first time providing us with a new understanding of the information that can be obtained from urea and guanidine hydrochloride denaturation studies of proteins concerning the contributions of hydrophobic and electrostatic interactions on stability.
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