Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability

A set of six mutants of the levan binding single-chain Fv (scFv) fragment A48 (ABPC48), which have the identical light chain but differ gradually in the stability of the heavy chain, was generated. This was achieved by introducing one or both of the stabilizing mutations H-K66R and H-N52S into the V-H domain of the A48 wild-type protein, which is naturally missing the conserved disulfide bridge in V-H, and into the cysteine-restored variant A48cys scFv. The stabilizing effects of these two mutations in V-H, which had been selected in the context of a disulfide-free derivative of this scFv fragment [Proba, K., et al. (1998) J. Mol. Biol. 275, 245-253], were found to be additive and transferable to the cysteine-restored variant of the A48 scFv, thereby generating extremely stable V-H domains. The equilibrium denaturation of these scFv fragments was compared with the corresponding isolated V-L domain and two of the different isolated V-H domains. In the scFv fragment, the V-L domain was found to be stabilized by a more stable V-H domain, and, conversely, the V-H domain was stabilized by a more stable V-L domain. A folding intermediate with nativelike V-H and denatured V-L was found at equilibrium, if V-H was significantly more stable than V-L. In all other cases, a cooperative unfolding of the scFv was observed. We explain this observation with different contributions of intrinsic domain stability and extrinsic stabilization provided by the partner domain in the single-chain antibodies.