Kataeva Irina A, Brewer John M, Uversky Vladimir N, Ljungdahl Lars G
Department of Biochemistry and Molecular Biology, A216 Fred Davison Life Sciences Complex, University of Georgia, Athens, GA 30602, USA.
FEBS Lett. 2005 Aug 15;579(20):4367-73. doi: 10.1016/j.febslet.2005.06.074.
Cellobiohydrolase A (CbhA) from Clostridium thermocellum is composed of an N-terminal carbohydrate-binding domain 4 (CBD4), an immunoglobulin-like domain (Ig), a glycoside hydrolase 9 (GH9), X1(1) and X1(2) domains, a CBD3, and a dockerin domain. All domains, except the Ig, bind Ca2+. The following constructs were made: X1(2), X1(1)X1(2), CBD3, X1(1)X1(2)-CBD3, Ig, GH9, Ig-GH9, Ig-GH9-X1(1)X1(2), and Ig-GH9-X1(1)X1(2)-CBD3. Interactions between domains in (1) buffer, (2) with Ca2+, or (3) ethylenediaminetetraacetic acid (EDTA) were studied by differential scanning calorimetry. Thermal unfoldings of all constructs were irreversible. Calcium increased T(d) and cooperativity of unfolding. Multi-domain constructs exhibited more cooperative unfolding in buffer and in the presence of EDTA than did individual domains. They denatured by mechanism simpler than expected from their modular architecture. The results indicate that domain coupling in thermophilic proteins constitutes a significant stabilizing factor.
来自嗜热栖热菌的纤维二糖水解酶A（CbhA）由一个N端碳水化合物结合结构域4（CBD4）、一个免疫球蛋白样结构域（Ig）、一个糖苷水解酶9（GH9）、X1(1)和X1(2)结构域、一个CBD3以及一个dockerin结构域组成。除Ig结构域外，所有结构域均结合Ca2+。构建了以下几种结构：X1(2)、X1(1)X1(2)、CBD3、X1(1)X1(2)-CBD3、Ig、GH9、Ig-GH9、Ig-GH9-X1(1)X1(2)以及Ig-GH9-X1(1)X1(2)-CBD3。通过差示扫描量热法研究了（1）缓冲液中、（2）含Ca2+时或（3）乙二胺四乙酸（EDTA）存在时各结构域之间的相互作用。所有构建体的热解折叠都是不可逆的。钙增加了解折叠的熔解温度（T(d)）和协同性。与单个结构域相比，多结构域构建体在缓冲液中和EDTA存在时表现出更多的协同解折叠。它们通过比预期的模块化结构更简单的机制变性。结果表明嗜热蛋白中的结构域偶联构成了一个重要的稳定因素。
