Nikolaeva Olga P, Orlov Victor N, Bobkov Andrey A, Levitsky Dmitrii I
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University; and A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.
Eur J Biochem. 2002 Nov;269(22):5678-88. doi: 10.1046/j.1432-1033.2002.03279.x.
The thermal unfolding of myosin subfragment 1 (S1) cleaved by trypsin was studied by differential scanning calorimetry. In the absence of nucleotides, trypsin splits the S1 heavy chain into three fragments (25, 50, and 20 kDa). This cleavage has no appreciable influence on the thermal unfolding of S1 examined in the presence of ADP, in the ternary complexes of S1 with ADP and phosphate analogs, such as orthovanadate (Vi) or beryllium fluoride (BeFx), and in the presence of F-actin. In the presence of ATP and in the complexes S1.ADP.Vi or S1.ADP.BeFx, trypsin produces two additional cleavages in the S1 heavy chain: a faster cleavage in the N-terminal region between Arg23 and Ile24, and a slower cleavage at the 50 kDa fragment. It has been shown that the N-terminal cleavage strongly decreases the thermal stability of S1 by shifting the maximum of its thermal transition by about 7 degrees C to a lower temperature, from 50 degrees C to 42.4 degrees C, whereas the cleavage at both these sites causes dramatic destabilization of the S1 molecule leading to total loss of its thermal transition. Our results show that S1 with ATP-induced N-terminal cleavage is able, like uncleaved S1, to undergo global structural changes in forming the stable ternary complexes with ADP and Pi analogs (Vi, BeFx). These changes are reflected in a pronounced increase of S1 thermal stability. However, S1 cleaved by trypsin in the N-terminal region is unable, unlike S1, to undergo structural changes induced by interaction with F-actin that are expressed in a 4-5 degrees C shift of the S1 thermal transition to higher temperature. Thus, the cleavage between Arg23 and Ile24 does not significantly affect nucleotide-induced structural changes in the S1, but it prevents structural changes that occur when S1 is bound to F-actin. The results suggest that the N-terminal region of the S1 heavy chain plays an important role in structural stabilization of the entire motor domain of the myosin head, and a long-distance communication pathway may exist between this region and the actin-binding sites.
通过差示扫描量热法研究了经胰蛋白酶切割的肌球蛋白亚片段1（S1）的热解折叠过程。在不存在核苷酸的情况下，胰蛋白酶将S1重链切割成三个片段（25 kDa、50 kDa和20 kDa）。这种切割对在ADP存在下、S1与ADP和磷酸盐类似物（如原钒酸盐（Vi）或氟化铍（BeFx））形成的三元复合物中以及F-肌动蛋白存在下所检测的S1热解折叠没有明显影响。在ATP存在下以及在S1·ADP·Vi或S1·ADP·BeFx复合物中，胰蛋白酶在S1重链上产生另外两个切割位点：一个在N端区域的Arg23和Ile24之间的较快切割，以及在50 kDa片段处的较慢切割。已经表明，N端切割通过将其热转变最大值从50℃降低约7℃至较低温度42.4℃，从而强烈降低S1的热稳定性，而这两个位点的切割会导致S1分子显著不稳定，导致其热转变完全丧失。我们的结果表明，具有ATP诱导的N端切割的S1能够像未切割的S1一样，在与ADP和Pi类似物（Vi、BeFx）形成稳定的三元复合物时经历整体结构变化。这些变化表现为S1热稳定性显著增加。然而，与S1不同，在N端区域被胰蛋白酶切割的S1无法经历与F-肌动蛋白相互作用诱导的结构变化，这种变化表现为S1热转变向更高温度移动4 - 5℃。因此，Arg23和Ile24之间的切割不会显著影响核苷酸诱导的S1结构变化，但它会阻止S1与F-肌动蛋白结合时发生的结构变化。结果表明，S1重链的N端区域在肌球蛋白头部整个运动结构域的结构稳定中起重要作用，并且该区域与肌动蛋白结合位点之间可能存在长距离通讯途径。
