MRC Centre for Protein Engineering, Hills Road, Cambridge CB2 0QH, UK.
Nucleic Acids Res. 2011 Mar;39(6):2294-303. doi: 10.1093/nar/gkq800. Epub 2010 Nov 18.
The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37 °C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity.
肿瘤抑制因子 p53 的寡聚状态是其各种生物学功能的重要因素。它具有明确的四聚化结构域，蛋白质以单体、二聚体和四聚体平衡存在。寡聚形式之间的离解常数非常低，以至于在体外常规方法的测量极限内。在这里，我们使用单分子方法的高灵敏度，使用荧光相关光谱法（FCS）在生理温度、pH 值和离子强度下测量全长 p53 和其分离的四聚化结构域 p53tet 的寡聚化平衡和动力学，体外。全长 p53 从四聚体解离为二聚体的解离常数在 37°C 时为 50 ± 7 nM，二聚体解离为单体的相应值为 0.55 ± 0.08 nM。这两个过程的半衰期分别为 20 和 50 分钟。p53tet 的相应值分别为 150 ± 10 nM、1.0 ± 0.14 nM、2.5 ± 0.4 分钟和 13 ± 2 分钟。数据表明，未受应激的细胞中的未连接的 p53 应该主要是二聚体。单分子 FCS 是一种非常灵敏的测量离解平衡、动力学和聚集的程序。
