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 VH domain of the A48 wild-type protein, which is naturally missing the conserved disulfide bridge in VH, and into the cysteine-restored variant A48cys scFv. The stabilizing effects of these two mutations in VH, 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 VH domains. The equilibrium denaturation of these scFv fragments was compared with the corresponding isolated VL domain and two of the different isolated VH domains. In the scFv fragment, the VL domain was found to be stabilized by a more stable VH domain, and, conversely, the VH domain was stabilized by a more stable VL domain. A folding intermediate with nativelike VH and denatured VL was found at equilibrium, if VH was significantly more stable than VL. 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.
Wörn
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Biochemisches Institut, Universität Zürich, Switzerland.
Plückthun
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D006868:Hydrolysis; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013820:Thermolysin / Q000378:metabolism
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D013820:Thermolysin