Nordlund Henri R, Hytönen Vesa P, Laitinen Olli H, Uotila Sanna T H, Niskanen Einari A, Savolainen Janne, Porkka Eevaleena, Kulomaa Markku S
Department of Biological and Environmental Science, P.O. Box 35, FIN-40014 University of, Jyväskylä, Finland.
FEBS Lett. 2003 Dec 18;555(3):449-54. doi: 10.1016/s0014-5793(03)01302-4.
In order to turn the subunit association and biotin binding of avidin into pH-sensitive phenomena, we have replaced individually three amino acid residues in avidin (Met96, Val115 and Ile117) with histidines in the 1-3 interface, and in combination with a histidine conversion in the 1-2 interface (Trp110). The single replacements Met96His and Val115His in the 1-3 interface were found to have a clear effect on the quaternary structure of avidin, since subunit associations of these mutants became pH-dependent. The histidine replacement in the 1-2 interface affected the biotin-binding properties of the mutants, in particular reversibility of binding and protein-ligand complex formation were pH-sensitive, as measured by IAsys biosensor and fluorescence correlation spectroscopy, respectively. The possibility of regulating the quaternary structure and function of avidin in a controlled and predictable manner, due to introduced interface histidines, will expand even further the range and versatility of the avidin-biotin technology.
为了使抗生物素蛋白的亚基缔合和生物素结合转变为对pH敏感的现象，我们在1-3界面处单独将抗生物素蛋白中的三个氨基酸残基（Met96、Val115和Ile117）替换为组氨酸，并结合在1-2界面处（Trp110）的组氨酸转换。发现在1-3界面处的单取代Met96His和Val115His对抗生物素蛋白的四级结构有明显影响，因为这些突变体的亚基缔合变得依赖于pH。在1-2界面处的组氨酸取代影响了突变体的生物素结合特性，特别是结合的可逆性和蛋白质-配体复合物的形成对pH敏感，分别通过IAsys生物传感器和荧光相关光谱法测量。由于引入了界面组氨酸，以可控且可预测的方式调节抗生物素蛋白的四级结构和功能的可能性将进一步扩大抗生物素蛋白-生物素技术的范围和多功能性。
