Disulfide bond contribution to protein stability: Positional effects of substitution in the hydrophobic core of the two-stranded .alpha.-helical coiled-coil

To investigate the positional effect of the disulfide bond on the structure and stability of a two-stranded alpha-helical coiled-coil, an interchain disulfide bond was systematically introduced into the hydrophobic core of a de novo designed model coiled-coil at the N-terminus (position 2), C-terminus (position 33), and nonterminal positions a (positions 9, 16, 23, and 30) and d (positions 5, 12, 19, and 26). The rate of formation of a disulfide bond is faster at position d compared to at the corresponding position a under nondenaturing conditions, suggesting that position d is more suitable for engineering a disulfide bond. The structure and stability of the reduced and oxidized coiled-coils were determined by circular dichroism studies in the absence and presence of guanidine hydrochloride. Our results demonstrate that the improvement of protein stability by introduction of a disulfide bond is very relevant to its location and the most effective disulfide bonds are those that can be introduced in the hydrophobic core without any disruption of the protein structure. The disulfide bond at position d with near-optimal geometry does not perturb the coiled-coil structure and makes the largest contribution to coiled-coil stability. In contrast, the inappropriate geometry of the disulfide bond at nonterminal position a introduces a high strain energy on the disulfide bond which disrupts the coiled-coil structure. At positions a, the closer the disulfide bridge is to the center of the coiled-coil, the larger the disruption on the coiled-coil structure and the smaller the contribution the disulfide bond makes to coiled-coil stability. The computer modeling results also suggest that an insertion of an interchain disulfide bond at position a in the GCN4 leucine zipper X-ray structure has a higher potential energy than insertion at position d. The energy-minimized coiled-coil structure with an interchain disulfide bond at position a has a larger root mean square difference from the X-ray structure of GCN4 than the coiled-coil with a disulfide bond at position d. Because interhelical interactions are common in globular proteins as well as coiled-coils, the results obtained in this study will have general utility for selecting the sites for engineering disulfide bonds between alpha-helices.