Measurement of dissociation constants of high-molecular weight protein-protein complexes by transferred 15N-relaxation
Measurement of dissociation constants of high-molecular weight protein-protein complexes by transferred 15N-relaxation
复制标题
DOI：
10.1007/s10858-007-9147-9
复制
发表时间：
2007-05-01
影响因子：
2.7
通讯作者：
Otting, Gottfried
中科院分区：
文献类型：
--
作者：
Su, Xun-Cheng;Jergic, Slobodan;Otting, Gottfried
建议使用 N-15 弛豫数据来确定蛋白质-蛋白质复合物的解离常数，适用于 N-15 标记的蛋白质与高分子量的未标记蛋白质结合，并且结合蛋白质和游离蛋白质之间的化学交换在 NMR 时间尺度上很快的情况。该方法适用于使用相对较低浓度的蛋白质溶液估计微摩尔至毫摩尔范围内的解离常数。显示了大肠杆菌 DNA 聚合酶 III 复合物的两个亚基之间相互作用的示例，涉及 tau 亚基 C 端结构域的 N-15 标记片段 (15 kDa) 和未标记的 α 亚基 (130 kDa)。
The use of N-15-relaxation data for determination of the dissociation constant of a protein-protein complex is proposed for the situation where a N-15-labeled protein is bound to an unlabeled protein of high molecular weight, and the chemical exchange between bound and free protein is fast on the NMR time scale. The approach is shown to be suitable for estimating dissociation constants in the micromolar to millimolar range, using protein solutions at relatively low concentration. An example is shown for the interaction between two subunits from the Escherichia coli DNA polymerase III complex, involving a N-15-labeled fragment of the C-terminal domain of the tau subunit (15 kDa) and the unlabeled alpha subunit (130 kDa).