Morfill Julia, Blank Kerstin, Zahnd Christian, Luginbühl Beatrice, Kühner Ferdinand, Gottschalk Kay-E, Plückthun Andreas, Gaub Hermann E
Lehrstuhl für Angewandte Physik and Center for Nanoscience, Ludwig-Maximilians-Universität München, Munich, Germany.
Biophys J. 2007 Nov 15;93(10):3583-90. doi: 10.1529/biophysj.107.112532. Epub 2007 Aug 3.
For many applications, antibodies need to be engineered toward maximum affinity. Strategies are in demand to especially optimize this process toward slower dissociation rates, which correlate with the (un)binding forces. Using single-molecule force spectroscopy, we have characterized three variants of a recombinant antibody single-chain Fv fragment. These variants were taken from different steps of an affinity maturation process. Therefore, they are closely related and differ from each other by a few mutations only. The dissociation rates determined with the atomic force microscope differ by one order of magnitude and agree well with the values obtained from surface plasmon resonance measurements. However, the effective potential width of the binding complexes, which was derived from the dynamic force spectroscopy measurements, was found to be the same for the different mutants. The large potential width of 0.9 nm indicates that both the binding pocket and the peptide deform significantly during the unbinding process.
对于许多应用而言，需要对抗体进行工程改造以实现最大亲和力。尤其需要一些策略来针对较慢的解离速率优化这一过程，解离速率与（非）结合力相关。利用单分子力谱技术，我们对一种重组抗体单链Fv片段的三种变体进行了表征。这些变体取自亲和力成熟过程的不同步骤。因此，它们密切相关，彼此之间仅相差少数几个突变。用原子力显微镜测定的解离速率相差一个数量级，并且与表面等离子体共振测量获得的值非常吻合。然而，通过动态力谱测量得出的结合复合物的有效势阱宽度对于不同的突变体而言是相同的。0.9纳米的大的势阱宽度表明，在解离过程中，结合口袋和肽段都会发生显著变形。
