Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064, India; Homi Bhabha National Institute, Anushaktinagar, Mumbai 400 094, India.
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064, India.
Int J Biol Macromol. 2020 Jul 1;154:904-915. doi: 10.1016/j.ijbiomac.2020.03.166. Epub 2020 Mar 21.
PIN1 proteins are a class of peptidyl prolyl cis-trans isomerases (PPIases), which have been implicated in numerous cellular functions like cell cycle progression, transcriptional control, signal transduction, promotion of oncogenesis and host-parasite interactions. In this work, the unfolding mechanism of a single domain PIN1 from Leishmania major (LmPIN1) has been characterized during thermal and denaturant-induced unfolding by differential scanning calorimetry (DSC), fluorescence and circular dichroism. Further, MD simulations have been performed to structurally probe the possible stages of its unfolding process. Both the fluorescence and CD data confirm classical two-state unfolding transitions for urea and GdnHCl. The thermal unfolding of LmPIN1, characterized by DSC, could optimally be fitted to a non two-state transition curve exhibiting two T's (53 °C and 57 °C) suggesting the possibility of an intermediate. Thermal unfolding of the modeled LmPIN1 by MD simulation shows that the unfolding process is initiated by increased fluctuations (dynamics) spanning residues 70-80, followed by perturbations in the sheet system and disjuncture of helix-sheet packing. Importantly, simulation and fluorescence quenching studies clearly suggest the possibility of the presence of residual structures of LmPIN1 even after complete denaturation.
PIN1 蛋白是一类肽基脯氨酰顺反异构酶（PPIases），它们参与了许多细胞功能，如细胞周期进程、转录控制、信号转导、促进肿瘤发生和宿主-寄生虫相互作用。在这项工作中，通过差示扫描量热法（DSC）、荧光和圆二色性研究了来自利什曼原虫（LmPIN1）的单个结构域 PIN1 的解折叠机制，在热和变性剂诱导的解折叠过程中。此外，还进行了 MD 模拟，以结构上探测其可能的解折叠过程阶段。荧光和 CD 数据均证实了脲和 GdnHCl 的经典两态解折叠转变。通过 DSC 对 LmPIN1 的热解折叠进行了表征，最优化拟合为非两态转变曲线，显示两个 T（53°C 和 57°C），表明存在中间态的可能性。MD 模拟对模型化的 LmPIN1 的热解折叠表明，解折叠过程由跨越残基 70-80 的增加的波动（动力学）引发，随后是片层系统的干扰和螺旋-片层包装的断裂。重要的是，模拟和荧光猝灭研究清楚地表明，即使在完全变性后，LmPIN1 仍可能存在残余结构。
