Kotani S, Kawai G, Aizawa H, Yokoyama S, Sakai H
Department of Biochemical Engineering and Science, Faculty of Computer Science, Kyushu Institute of Technology, Fukuoka, Japan.
J Biol Chem. 1990 Jan 25;265(3):1286-92.
The structure of microtubule assembly inhibitor protein (MIP) was studied by proton nuclear magnetic resonance (NMR) and limited proteolysis. Spin-diffusion experiments revealed that MIP has a tightly folded structure, a "rigid globule." This globule was irreversibly denatured by heat treatment at 80 degrees C, and the denatured MIP showed little ability to inhibit microtubule assembly. This indicates that the native globular structure is essential for the activity. By spin-diffusion and spin-echo experiments, the other part of the molecule was found to be highly mobile. We termed the region "highly mobile coils." Most of the acidic residues appeared to be clustered in the highly mobile coils. By limited proteolysis using subtilisin, the mobile region was digested into shorter pieces, and a single 17-kDa fragment remained. Proton NMR spectrum of this fragment was much the same as the spin-diffusion subspectra of the rigid globule in the intact MIP molecule. Furthermore, the 17-kDa fragment was found to retain the activity to inhibit the microtubule assembly. These results indicate that MIP consists of two moieties; one domain forms a rigid globule which is essential for its activity to inhibit microtubule assembly, and the other acidic one is highly mobile and tails from the globule. The tertiary structures of these two domains appear to be independent from each other. These domains may be responsible for two different functions of MIP, the interaction with the cytoskeleton and the interaction with, for example, nuclear components.
通过质子核磁共振（NMR）和有限蛋白酶解研究了微管组装抑制蛋白（MIP）的结构。自旋扩散实验表明，MIP具有紧密折叠的结构，即“刚性球体”。该球体在80℃热处理后不可逆地变性，变性的MIP抑制微管组装的能力很弱。这表明天然球状结构对其活性至关重要。通过自旋扩散和自旋回波实验，发现分子的另一部分具有高度的流动性。我们将该区域称为“高度流动的线圈”。大多数酸性残基似乎聚集在高度流动的线圈中。使用枯草杆菌蛋白酶进行有限蛋白酶解后，流动区域被消化成更短的片段，只剩下一个17 kDa的片段。该片段的质子NMR谱与完整MIP分子中刚性球体的自旋扩散子谱非常相似。此外，发现17 kDa片段保留了抑制微管组装的活性。这些结果表明，MIP由两个部分组成；一个结构域形成一个刚性球体，这对其抑制微管组装的活性至关重要，另一个酸性部分高度流动，从球体延伸出来。这两个结构域的三级结构似乎彼此独立。这些结构域可能负责MIP的两种不同功能，即与细胞骨架的相互作用以及与例如核成分的相互作用。
