VAV2蛋白DH结构域与CDC42蛋白相互作用的亲和力和热力学参数研究

Affinity and thermodynamic parameters of interaction between DH domain of VAV2 protein and CDC42 protein

目的:利用等温滴定量热技术研究VAV2蛋白的DH结构域(VAV2-DH)与CDC42的相互作用。方法 :首先分别将VAV2-DH和CDC42蛋白的编码基因克隆至pGEX-6p-1和pET28a载体上,构建VAV2-DH和CDC42蛋白的重组表达载体。再将重组表达载体转入大肠杆菌中诱导表达蛋白,并亲和层析、凝胶过滤层析纯化得到VAV2-DH和CDC42蛋白,最后利用鸟嘌呤核苷酸交换试验来验证VAV2-DH的鸟苷酸交换活性,并采用等温滴定量热技术检测这2种蛋白质相互作用的亲和力和热力学参数。结果:VAV2-DH和CDC42蛋白的平衡解离常数K_(D)=(11.44±2.12)μmol/L,其中摩尔结合焓DH=(-224.33±95.48) kJ/mol、-TDS=(195.97±95.95) kJ/mol、吉布斯自由能DG=(-28.30±0.49) kJ/mol。结论:VAV2-DH和CDC42蛋白属于中等强度的相互作用,是一个焓驱动的结合过程。

Objective:To study the interaction between the DH domain of VAV2 protein(VAV2-DH) and CDC42 by isothermal titration calorimetry. Methods:The coding sequences of VAV2-DH and CDC42 were cloned into pGEX-6 p-1 or pET28 a vectors to construct recombinant prokaryotic expression plasmids. Purified proteins were obtained by prokaryotic expression,affinity chromatography and gel filtration chromatography. Then GEF assay and isothermal titration calorimetry were employed to detect the affinity and thermodynamic parameters of the interaction between VAV2-DH and CDC42. Results:Moderate binding affinity was recorded between VAV2-DH and CDC42 with a K_(D) value of(11.44±2.12) μmol/L,in which the molar binding enthalpy DH=(-224.33±95.48) kJ/mol,-TDS =(195.97 ±95.95) kJ/mol,and Gibbs free energy DG =(-28.30 ±0.49) kJ/mol. Conclusion:VAV2-DH and CDC42 protein interact with each other with moderate intensity,which is an enthalpy-driven binding process.