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Switchable reinforced streptavidin
Nanoscale ( IF 6.7 ) Pub Date : 2020/03/09 , DOI: 10.1039/d0nr00265h
Leonard C. Schendel 1, 2, 3, 4 , Steffen M. Sedlak 1, 2, 3, 4 , Hermann E. Gaub 1, 2, 3, 4
Affiliation  

The complex of the small molecule biotin and the homotetrameric protein streptavidin is key to a broad range of biotechnological applications. Therefore, the behavior of this extraordinarily high-affinity interaction under mechanical force is intensively studied by single-molecule force spectroscopy. Recently, steered molecular dynamics simulations have identified a low force pathway for the dissociation of biotin from streptavidin, which involves partial unfolding of the N-terminal β-sheet structure of monovalent streptavidin's functional subunit. Based on these results, we now introduced two mutations (T18C,A33C) in the functional subunit of monovalent streptavidin to establish a switchable connection (disulfide bridge) between the first two β-strands to prevent this unfolding. In atomic force microscopy-based single-molecule force spectroscopy experiments, we observed unbinding forces of about 350 pN (at a force-loading rate of 10 nN s−1) for pulling a single biotin out of an N-terminally anchored monovalent streptavidin binding pocket – about 1.5-fold higher compared with what has been reported for N-terminal force loading of native monovalent streptavidin. Upon addition of a reducing agent, the unbinding forces dropped back to 200 pN, as the disulfide bridge was destroyed. Switching from reducing to oxidizing buffer conditions, the inverse effect was observed. Our work illustrates how the mechanics of a receptor–ligand system can be tuned by engineering the receptor protein far off the ligand-binding pocket.

中文翻译:

可切换强化链霉亲和素

小分子生物素和同四聚体蛋白链霉亲和素的复合物是广泛的生物技术应用的关键。因此,通过单分子力谱研究了在机械力下这种异常高亲和力相互作用的行为。最近,转向分子动力学模拟已经确定了生物素从链霉亲和素解离的低力途径,这涉及单价链霉亲和素的功能亚基的N-末端β-折叠结构的部分展开。基于这些结果,我们现在在单价链霉亲和素的功能亚基中引入了两个突变(T18C,A33C),以在前两个β链之间建立可切换的连接(二硫键),以防止这种折叠。-1)用于将单个生物素从N端锚定的单价链霉亲和素结合袋中拉出-与已报道的天然一价链霉亲和素的N端力负载相比,高约1.5倍。加入还原剂后,由于二硫键被破坏,解键力降至200 pN。从还原缓冲液条件转变为氧化缓冲液条件,观察到相反的作用。我们的工作说明了如何通过远距离配体结合口袋的方式改造受体蛋白来调节受体-配体系统的力学。
更新日期:2020-03-27
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