Abstract

Fas-activated serine/threonine kinase (FASTK) is a mitochondria-associated nuclear protein that inhibits Fas- and UV-induced apoptosis. This protein is generally activated during Fas-mediated apoptosis by phosphorylating a nuclear RNA-binding protein T-cell intracellular antigen-1 and thus considered as a modulator of apoptosis. In the present study, we have examined the equilibrium unfolding and conformational stability of the kinase domain of FASTK (FASTK_353-444). The kinase domain of FASTK_353-444 was cloned, expressed, and purified. The folding ↔ unfolding transitions of urea-induced denaturation was monitored with the help of circular dichroism, intrinsic fluorescence, and UV absorption spectroscopies. Analysis of transition curves obtained from different probes revealed a coincidence of denaturation curves, suggesting that folding/unfolding of FASTK follows a two-state process with the midpoint (C_m) value at 3.50 ± 0.1 M. Urea-induced denaturation curves were further analyzed to estimate change in the Gibbs free energy in the absence of urea (ΔG_D⁰) associated with the equilibrium of denaturation. To get atomistic insights into the urea-induced denaturation of FASTK, we performed an all-atom molecular dynamics simulation for 100 ns. A close agreement was noticed between experimental and computational studies. This study will help to understand the unfolding mechanism and structural stability of the kinase domain of FASTK.

Communicated by Ramaswamy H. Sarma

摘要

Fas 激活的丝氨酸/苏氨酸激酶 (FASTK) 是一种与线粒体相关的核蛋白，可抑制 Fas 和 UV 诱导的细胞凋亡。这种蛋白质通常在 Fas 介导的细胞凋亡过程中通过磷酸化核 RNA 结合蛋白 T 细胞细胞内抗原 1 被激活，因此被认为是细胞凋亡的调节剂。在本研究中，我们检查了 FASTK (FASTK _353-444 )激酶结构域的平衡展开和构象稳定性。FASTK _353-444的激酶结构域被克隆、表达和纯化。在圆二色性、固有荧光和紫外吸收光谱的帮助下监测尿素诱导的变性的折叠↔展开转变。对从不同探针获得的转变曲线的分析揭示了变性曲线的重合，表明 FASTK 的折叠/展开遵循中点 ( C _m ) 值为 3.50 ± 0.1 M的双态过程。进一步分析了尿素诱导的变性曲线估计在没有尿素的情况下吉布斯自由能的变化 (Δ G _D ⁰) 与变性平衡有关。为了深入了解尿素诱导的 FASTK 变性，我们进行了 100 ns 的全原子分子动力学模拟。注意到实验和计算研究之间存在密切的一致性。这项研究将有助于了解 FASTK 激酶结构域的展开机制和结构稳定性。

由 Ramaswamy H. Sarma 交流