Thermodynamic identification of stable folding intermediates in the B-subunit of cholera toxin.
Thermodynamic identification of stable folding intermediates in the B-subunit of cholera toxin.
复制标题
霍乱毒素 B 亚基中稳定折叠中间体的热力学鉴定。
DOI：
10.1021/bi00234a031
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Freire,E
中科院分区：
文献类型：
--
作者：
Bhakuni,V;Xie,D;Freire,E
马里兰州巴尔的摩约翰霍普金斯大学生物学和生物计量中心系，1990年12月27日收到；修订稿收到1991年3月5日摘要：霍乱毒素五聚体B亚单位的结构稳定性和结构域结构已被测量为不同扰动的函数，以评估B亚单位内相互作用的大小。在这些研究中，以温度、盐酸胍(GuHCl)和pH为扰动，通过高灵敏度差示扫描量热法、等温反应热法、荧光光谱分析和部分酶消化法测定了它们的影响。在pH 7.5和没有任何附加扰动的情况下，B亚基五聚体的热展开具有以77℃为中心的热容函数的单峰，其特征热容函数为328千卡/摩尔，AHvJAHCII为0.3。将pH降至4或将盐酸铵加入到2M以下，均可使量热焓降低，但对范霍夫热无明显影响。相变热随pH呈S形下降，拐点位于pH 5.3附近。等温滴定量热法研究表明，B亚基五聚体在27℃时发生了以pH 5.3为中心的转变，其热变为27千卡/摩尔，在此pH以下，展开转变的热变约为B亚基五聚体的100千卡/摩尔。在这种情况下，在0.5M盐酸盐下观察到第一个转变，在3M盐酸盐下观察到第二个转变。胰酶消化研究表明，在pH为5.0时，B亚基比在pH为7.0时更易消化4倍，在pH为5.0时，有限的蛋白质降解会产生~7和~5 kDa的两个片段。这些研究提供了强有力的证据，证明霍乱毒素的B-亚基由两个折叠/展开结构域组成，同一亚单位内两个结构域之间的相互作用以及亚单位之间的相互作用能够解释整个五聚体环的合作行为。
Department of Biology and Biocalorimetry Center, The Johns Hopkins University, Baltimore, Maryland 21218 Received December 27, 1990; Revised Manuscript Received March 5, 1991 abstract: The structural stability and domain structure of the pentameric B-subunit of cholera toxin have been measured as a function of different perturbante in order to assess the magnitude of the interactions within the B-subunits. For these studies, temperature, guanidine hydrochloride (GuHCl), and pH were used as perturbante, and the effects were measured by high-sensitivity differential scanning calorimetry, isothermal reaction calorimetry, fluorescence spectroscopy, and partial protease digestion. At pH 7.5 and in the absence of any additional perturbante, the thermal unfolding of the B-subunit pentamer is characterized by a single peak in the heat capacityfunction centered at 77 C and characterized by a] of 328 kcal/mol of B-subunit pentamerand AHvJAHCii of 0.3. Lowering the pH down to 4 or adding GuHCl up to 2 M results in a decrease of the calorimetric enthalpy with no significant effect on the van’t Hoff enthalpy. The transition enthalpy decreases in a sigmoidal fashion with pH, with an inflection point centered at pH 5.3. Isothermal titration calorimetric studies as a function of pH also report a transition centered at pH 5.3 and characterized by an enthalpy change of 27 kcal/mol of B-subunit pentamer at 27 C. Below this pH, the enthalpy change for the unfolding transition is reduced to approximately 100 kcal/mol of B-subunit pentamer.Similar behavior is obtained with GuHCl. In this case, a first transition is observed at 0.5 M GuHCl and a second one at 3 M GuHCl. Trypsin digestionstudies show that at pH 5.0 the B-subunit is 4 times more susceptible to digestion than at pH 7.0 and that at pH 5.0 limited proteolysis results in two fragments of~ 7 and~ 5 kDa. These studies provide strong evidence that the B-subunits of cholera toxin are composed of two folding/unfolding domains and that the interactions between the two domains within the same subunit and between subunits are able to account for the cooperative behavior of the entire pentameric ring.