Wörn A, Plückthun A
Biochemisches Institut, Universität Zürich, Switzerland.
Biochemistry. 1998 Sep 22;37(38):13120-7. doi: 10.1021/bi980712q.
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.
构建了一组六个莱文结合单链Fv（scFv）片段A48（ABPC48）的突变体，它们具有相同的轻链，但重链稳定性逐渐不同。这是通过将一个或两个稳定突变H-K66R和H-N52S引入A48野生型蛋白的VH结构域来实现的，该野生型蛋白在VH中天然缺失保守的二硫键，同时也引入到半胱氨酸恢复变体A48cys scFv中。这两个在VH中的稳定突变，是在该scFv片段的无二硫衍生物背景下筛选出来的[Proba, K., 等人 (1998) J. Mol. Biol. 275, 245 - 253]，发现它们具有加和性，并且可以转移到A48 scFv的半胱氨酸恢复变体中，从而产生极其稳定的VH结构域。将这些scFv片段的平衡变性与相应的分离VL结构域以及两个不同的分离VH结构域进行了比较。在scFv片段中，发现VL结构域通过更稳定的VH结构域得到稳定，反之，VH结构域通过更稳定的VL结构域得到稳定。如果VH比VL明显更稳定，则在平衡时会发现具有类似天然VH和变性VL的折叠中间体。在所有其他情况下，观察到scFv的协同解折叠。我们用单链抗体中内在结构域稳定性和伙伴结构域提供的外在稳定性的不同贡献来解释这一观察结果。
