Proba K, Honegger A, Plückthun A
Biochemisches Institut Universität Zürich, Switzerland.
J Mol Biol. 1997 Jan 17;265(2):161-72. doi: 10.1006/jmbi.1996.0726.
While the disulfide bridge is highly conserved within the immunoglobulin fold, a few antibody variable domains lack one of the essential cysteine residues. In the levan binding antibody ABPC48 one of the essential cysteine residues (Cys H92) of the heavy chain variable domain is replaced by tyrosine. We expressed scFv fragments with the ABPC48 sequence and a mutant in which the VH disulfide bond has been restored in Escherichia coli, purified both proteins by antigen affinity chromatography and characterized them by equilibrium denaturation. While the ABPC48 protein was found to be significantly less stable than an average scFv molecule, the restored disulfide increased its stability above that of other, unrelated scFv fragments, explaining why it tolerates the disulfide loss. Surprisingly, we observed that under some refolding conditions, the unpaired cysteine residue of functional scFv of ABPC48 is derivatized by glutathione. It is easily accessible to other reagents and thus appears to be solvent-exposed, in contrast to the deeply buried disulfide of ordinary variable domains. This implies a very unusual conformation of stand b containing the unpaired Cys H22, which might be stabilized by interactions with the tyrosine residue in position H92.
虽然二硫键在免疫球蛋白折叠中高度保守，但一些抗体可变结构域缺少一个必需的半胱氨酸残基。在果聚糖结合抗体ABPC48中，重链可变结构域的一个必需半胱氨酸残基（Cys H92）被酪氨酸取代。我们在大肠杆菌中表达了具有ABPC48序列的单链抗体片段（scFv）以及重链可变区（VH）二硫键已恢复的突变体，通过抗原亲和层析纯化了这两种蛋白，并通过平衡变性对其进行了表征。虽然发现ABPC48蛋白的稳定性明显低于平均scFv分子，但恢复的二硫键提高了其稳定性，使其高于其他不相关的scFv片段，这解释了它为何能耐受二硫键的缺失。令人惊讶的是，我们观察到在某些重折叠条件下，ABPC48功能性scFv的未配对半胱氨酸残基会被谷胱甘肽衍生化。与普通可变结构域深埋的二硫键不同，它很容易与其他试剂反应，因此似乎是暴露在溶剂中的。这意味着包含未配对Cys H22的β链具有非常不寻常的构象，可能通过与H92位酪氨酸残基的相互作用而稳定。
