Wang Yejing, He Huawei, Liu Lina, Gao Chunyan, Xu Shui, Zhao Ping, Xia Qingyou
State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing, China; College of Biotechnology, Southwest University, Beibei, Chongqing, China.
State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing, China.
PLoS One. 2014 Sep 25;9(9):e107932. doi: 10.1371/journal.pone.0107932. eCollection 2014.
The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase.
研究了尿素和盐酸胍（GdnHCl）对嗜热栖热菌HB27来源的一种热稳定低分子量蛋白质——蛋白质酪氨酸磷酸酶（PTPase）的活性、构象及去折叠过程的影响。酶活性测定表明，尿素和GdnHCl均能使PTPase失活，且呈浓度和时间依赖性。失活动力学分析表明，尿素和GdnHCl诱导的PTPase失活均为单相且可逆的过程，尿素和GdnHCl对PTPase的影响类似于混合型可逆抑制剂。远紫外（UV）圆二色性（CD）、色氨酸和1-苯胺基萘-8-磺酸（ANS）荧光光谱分析表明，在尿素和GdnHCl诱导的去折叠过程中，分别存在部分活性和无活性的类熔球中间体。基于序列比对和同源Tt1001蛋白结构，我们讨论了尿素和GdnHCl诱导的PTPase可能的构象转变，并详细比较了这些去折叠中间体的构象与牛PTPase的瞬态状态及其复合物结构。我们的结果或许能够为揭示PTPase的结构与酶活性之间的关系以及去折叠途径和机制提供一些有价值的线索。
