Banerjee Tuhina, Kishore Nand
Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India.
J Phys Chem B. 2005 Dec 1;109(47):22655-62. doi: 10.1021/jp053757r.
The interaction of 2,2,2-trifluoroethanol (TFE) with concanavalin A has been investigated by using a combination of differential scanning calorimetry, isothermal titration calorimetry (ITC), circular dichroism (CD), and fluorescence spectroscopy at pH 2.5 and 5.2. All of the calorimetric transitions at both the pH values were found to be irreversible. In the presence of 4 mol kg(-1) TFE at pH 2.5, concanavalin A is observed to be in a partially folded state with significant loss of native tertiary structure. The loss of specific side chain interactions in the transition from native to the TFE-induced partially folded state is demonstrated by the loss of cooperative thermal transition and reduction of the CD bands in the aromatic region. Acrylamide quenching, 8-anilinonaphthalene sulfonate (ANS) binding, and energy transfer also suggest that in the presence of 4 mol kg(-1) TFE at pH 2.5 concanavalin A is in a molten globule state. ITC has been used for the first time to characterize the energetics of ANS binding to the molten globule state. ITC results indicate that the binding of ANS to the molten globule state and acid-induced state at pH 2.5 displays heterogeneity with two classes of non-interacting binding sites. The results provide insights into the role of hydrophobic and electrostatic interactions in the binding of ANS to concanavalin A. The results also demonstrate that ITC can be used to characterize the partially folded states of the protein both qualitatively and quantitatively.
通过结合差示扫描量热法、等温滴定量热法（ITC）、圆二色性（CD）和荧光光谱法，在pH 2.5和5.2条件下研究了2,2,2-三氟乙醇（TFE）与伴刀豆球蛋白A的相互作用。发现在这两个pH值下所有的量热转变都是不可逆的。在pH 2.5且存在4 mol kg⁻¹ TFE的情况下，观察到伴刀豆球蛋白A处于部分折叠状态，天然三级结构有显著损失。从天然状态转变为TFE诱导的部分折叠状态时特定侧链相互作用的丧失，通过协同热转变的丧失和芳香区CD谱带的减弱得以证明。丙烯酰胺猝灭、8-苯胺基萘磺酸（ANS）结合和能量转移也表明，在pH 2.5且存在4 mol kg⁻¹ TFE的情况下，伴刀豆球蛋白A处于熔球态。ITC首次被用于表征ANS与熔球态结合的能量学。ITC结果表明，ANS在pH 2.5时与熔球态和酸诱导态的结合表现出异质性，有两类非相互作用的结合位点。这些结果为疏水和静电相互作用在ANS与伴刀豆球蛋白A结合中的作用提供了见解。结果还表明，ITC可用于定性和定量地表征蛋白质的部分折叠状态。
