Contribution of the intramolecular disulfide bridge to the folding stability of REIv, the variable domain of a human immunoglobulin kappa light chain.

BACKGROUND

Immunoglobulin domains contain about 100 amino acid residues folded into two beta-sheets and stabilized in a sandwich by a conserved central disulfide bridge. Whether antibodies actually require disulfide bonds for stability has long been a matter of debate. The contribution made by the central disulfide bridge to the overall folding stability of the immunoglobulin REIv, the variable domain of a human kappa light chain, was investigated by introducing stabilizing amino acid replacements followed by removal of the disulfide bridge via chemical reduction or genetic substitution of the cysteine residues.

RESULTS

Nine REIv variants were constructed by methods of protein engineering that have folding stabilities elevated relative wild-type REIv by (up to) 16.0 kJ mol-1. Eight of these variants can be cooperatively refolded after unfolding and chemical reduction of the disulfide bridge-in contrast to wildtype REIv. The stabilizing effect of one of these residue replacements (T39K) was rationalized by determining the structure of the respective REIv variant at 1.7 A. The loss of folding stability caused by reduction of the intramolecular disulfide bond is on average 19 kJ mol-1. Removal of the disulfide bridge by genetic substitution of C23 for valine resulted in a stable immunoglobulin domain in the context of the stabilizing Y32H amino acid exchange; again, REIv-C23V/Y32H has 18 kJ mol-1 less folding stability than REIv-Y32H. The data are consistent with the notion that all variants studied have the same overall three-dimensional structure with the disulfide bridge opened or closed.

CONCLUSIONS

A comparison of the magnitude of the stabilizing effect exerted by the disulfide bond and the length of the mainchain loop framed by it suggests lowering of the entropy of the unfolded state as the sole source of the effect. Disulfide bonds are not necessary for proper folding of immunoglobulin variable domains and can be removed, provided the loss of folding stability is at least partly compensated by stabilizing amino acid exchanges.