Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea.
Anal Chem. 2011 Aug 1;83(15):6011-7. doi: 10.1021/ac201013p. Epub 2011 Jul 7.
Understanding of the molecular relationships in carbohydrate-protein interactions provides useful information on biological processes in living organisms and is also helpful for development of potent biomedical agents. Herein, the interaction unbinding force between GM1 pentasaccharide and Vibrio cholera toxin (ctx) proteins was measured using atomic force microscopy (AFM), which enabled us to determine the interaction of ctx holotoxin (ctxAB) with GM1 and the interactive formation. First, the interaction force measured between A and B subunits (ctxA-ctxB) was 184.2 ± 4.5 pN, and the unbinding forces were evaluated to confirm the role of ctxA in ctxAB complex formation and were determined to be 443.7 ± 7.5 and 535.7 ± 25.9 pN for GM1-ctxB and GM1-ctxAB complexes, respectively. The force difference of ∼90 pN between GM1-ctxB and GM1-ctxAB might be due to the formation of the cholera toxin complex. Importantly, from the analogue analyses, we understand how structural and binding positional differences in complex carbohydrates affect the interaction with protein and surmise that the GM1-ctxAB complex makes a "two-finger grip" formation through the conformational change of a flexible carbohydrate. In conclusion, using AFM force analysis, we successfully quantified and characterized the interactive configuration of carbohydrate-protein molecules.
理解碳水化合物-蛋白质相互作用的分子关系提供了关于生物体内生物过程的有用信息，也有助于开发有效的生物医学试剂。在此，我们使用原子力显微镜（AFM）测量了 GM1 五糖与霍乱弧菌毒素（ctx）蛋白之间的相互作用解吸力，这使我们能够确定 ctx 全毒素（ctxAB）与 GM1 的相互作用以及相互作用的形成。首先，测量了 A 亚基和 B 亚基（ctxA-ctxB）之间的相互作用力为 184.2 ± 4.5 pN，并且评估了非键合力以确认 ctxA 在 ctxAB 复合物形成中的作用，分别确定 GM1-ctxB 和 GM1-ctxAB 复合物的非键合力为 443.7 ± 7.5 和 535.7 ± 25.9 pN。GM1-ctxB 和 GM1-ctxAB 之间约 90 pN 的力差可能是由于霍乱毒素复合物的形成。重要的是，通过类似物分析，我们了解了复合碳水化合物的结构和结合位置差异如何影响与蛋白质的相互作用，并推测 GM1-ctxAB 复合物通过柔性碳水化合物的构象变化形成“双指抓握”结构。总之，我们使用 AFM 力分析成功地定量和表征了碳水化合物-蛋白质分子的相互作用构型。
