Langedijk A C, Honegger A, Maat J, Planta R J, van Schaik R C, Plückthun A
Biochemisches Institut Universität Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland.
J Mol Biol. 1998;283(1):95-110. doi: 10.1006/jmbi.1998.2064.
Monoclonal antibody mAb 03/01/01, directed against the musk odorant traseolide, carries a serine residue instead of the conserved Cys H92 in the heavy chain variable domain, and is thus lacking the highly conserved disulfide bridge. We investigated the energetic consequence of restoring the disulfide bond and the nature of residue H6 (Glu or Gln), which is poised to interact with Ser H92 in the recombinant scFv fragment obtained from this antibody. In the scFv fragment derived from this antibody, the stabilizing effect of Gln H6 over Glu was found to be as large as the effect of reintroducing the disulfide bond. We have analyzed the conformation and hydrogen bond pattern of Gln H6 and Glu H6 in antibodies carrying these residues and suggest mechanisms by which this residue could contribute to VH domain stability. We also show that the unpaired cysteine H22 is buried, and conforms to the expected VH structure. The antibody appears to have acquired two somatic mutations (Ser H52 and Arg H66), which had been previously characterized as having a positive effect on VH stability. The overall domain stability is the decisive factor for generating functional, disulfide-free antibody domains, and several key residues play dominant roles.
针对麝香气味剂吐纳麝香的单克隆抗体mAb 03/01/01，在重链可变域中携带的是丝氨酸残基而非保守的半胱氨酸H92，因此缺少高度保守的二硫键。我们研究了恢复二硫键的能量后果以及残基H6（谷氨酸或谷氨酰胺）的性质，该残基在从该抗体获得的重组单链抗体片段中与丝氨酸H92相互作用。在源自该抗体的单链抗体片段中，发现谷氨酰胺H6相对于谷氨酸的稳定作用与重新引入二硫键的作用一样大。我们分析了携带这些残基的抗体中谷氨酰胺H6和谷氨酸H6的构象和氢键模式，并提出了该残基有助于重链可变区稳定性的机制。我们还表明，未配对的半胱氨酸H22被掩埋，并且符合预期的重链可变区结构。该抗体似乎获得了两个体细胞突变（丝氨酸H52和精氨酸H66），先前已证明它们对重链可变区稳定性有积极影响。整体结构域稳定性是产生功能性、无二硫键抗体结构域的决定性因素，几个关键残基起主导作用。
