Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53.
Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53.
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DOI：
10.1093/nar/gkq800
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发表时间：
2011-03
影响因子：
14.9
通讯作者：
Fersht AR
中科院分区：
文献类型：
--
作者：
Rajagopalan S;Huang F;Fersht AR
肿瘤抑制因子p53的寡聚化状态是其多种生物学功能的重要因素。它有一个明确的四聚化结构域，蛋白质以单体、二聚体和四聚体的形式平衡存在。低聚物形式之间的解离常数是如此之低，以至于它们处于通过体外常规方法测量的极限。在这里，我们已经使用了高灵敏度的单分子方法来测量的平衡和动力学的全长p53和它的孤立的四聚化结构域，p53tet，在生理温度，pH值和离子强度，使用荧光相关光谱（FCS）在体外的寡聚化。对于全长p53，在37 ° C下解离成二聚体的四聚体的解离常数为50 ± 7 nM，并且二聚体解离成单体的相应值为0.55 ± 0.08 nM。这两个过程的半衰期分别为20和50分钟。p53 tet的当量为150 ± 10 nM、1.0 ± 0.14 nM、2.5 ± 0.4 min和13 ± 2 min。这些数据表明，在未应激的细胞中未连接的p53应该主要是二聚体。单分子FCS是一个有用的程序，用于测量解离平衡，动力学和聚集在极端灵敏度。
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.
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影响因子：
14.9
作者：
Rajagopalan, Sridharan;Sade, Robert S.;Fersht, Alan R.
通讯作者：
Fersht, Alan R.
DOI：
10.1073/pnas.0501459102
发表时间：
2005-03-29
影响因子：
11.1
作者：
Fernandez-Fernandez, MR;Veprintsev, DB;Fersht, AR
通讯作者：
Fersht, AR
影响因子：
3.9
作者：
Petitjean, Audrey;Mathe, Ewy;Olivier, Magali
通讯作者：
Olivier, Magali
影响因子：
4.8
作者：
Friedler, A;Veprintsev, DB;Fersht, AR
通讯作者：
Fersht, AR
影响因子：
64.5
作者：
Kruse JP;Gu W
通讯作者：
Gu W