Asymmetry of the GroEL-GroES complex under physiological conditions as revealed by small-angle X-ray scattering
Asymmetry of the GroEL-GroES complex under physiological conditions as revealed by small-angle X-ray scattering
复制标题
DOI：
10.1529/biophysj.107.114710
复制
发表时间：
2008-02-15
影响因子：
3.4
通讯作者：
Kuwajima, Kunihiro
中科院分区：
文献类型：
--
作者：
Inobe, Tomonao;Takahashi, Kazunobu;Kuwajima, Kunihiro
尽管在伴侣蛋白循环过程中GroEL-GroES复合物形成的功能重要性是众所周知的，但复合物的化学计量尚未阐明。该复合物可以以不对称的1：1 GroEL-GroES复合物或对称的1：2 GroEL-GroES复合物的形式存在，尽管在生理条件下哪种类型是主要的仍然不确定。为了解决这个问题，我们通过小角X射线散射研究了生理条件下GroEL-GroES复合物的结构，这是一种直接观察溶液中蛋白质复合物结构的强大技术。我们评估了分子结构参数，回转半径和复合物的最大尺寸，在三个不同的温度下（10摄氏度，25摄氏度，和37摄氏度），在各种核苷酸条件下（3 mM ADP，3 mM ATPgS，和3 mM ATP在10 mM MgCl 2和100 mM KCl）的X-射线散射图案。然后，我们比较了实验观察到的散射图案与从已知的X-射线晶体学结构的GroEL GroES复杂的计算。结果清楚地表明，在生理条件下，不对称复合物必须是稳定存在于溶液中的主要物种。另一方面，在ATP（3 mM）和氟化铍（10 mM NaF和300 μ m BeCl 2）的存在下，我们观察到一个稳定的对称复合物的形成，表明存在一个瞬时形成的对称复合物在伴侣蛋白循环。
Despite the well-known functional importance of GroEL-GroES complex formation during the chaperonin cycle, the stoichiometry of the complex has not been clarified. The complex can occur either as an asymmetric 1:1 GroEL-GroES complex or as a symmetric 1:2 GroEL-GroES complex, although it remains uncertain which type is predominant under physiological conditions. To resolve this question, we studied the structure of the GroEL-GroES complex under physiological conditions by small-angle x-ray scattering, which is a powerful technique to directly observe the structure of the protein complex in solution. We evaluated molecular structural parameters, the radius of gyration and the maximum dimension of the complex, from the x-ray scattering patterns under various nucleotide conditions (3 mM ADP, 3 mM ATPgS, and 3 mM ATP in 10 mM MgCl2 and 100 mM KCl) at three different temperatures (10 degrees C, 25 degrees C, and 37 degrees C). We then compared the experimentally observed scattering patterns with those calculated from the known x-ray crystallographic structures of the GroEL-GroES complex. The results clearly demonstrated that the asymmetric complex must be the major species stably present in solution under physiological conditions. On the other hand, in the presence of ATP (3 mM) and beryllium fluoride (10mM NaF and 300 mu m BeCl2), we observed the formation of a stable symmetric complex, suggesting the existence of a transiently formed symmetric complex during the chaperonin cycle.