Urea-induced denaturation of human calcium/calmodulin-dependent protein kinase IV: a combined spectroscopic and MD simulation studies

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a multifunctional enzyme which belongs to the Ser/Thr kinase family. CaMKIV plays important role in varieties of biological processes such as gene expression regulation, memory consolidation, bone growth, T-cell maturation, sperm motility, regulation of microtubule dynamics, cell-cycle progression, and apoptosis. To measure stability parameters, urea-induced denaturation of CaMKIV was carried out at pH 7.4 and 25°C, using three different probes, namely far-UV CD, near-UV absorption, and tryptophan fluorescence. A coincidence of normalized denaturation curves of these optical properties suggests that urea-induced denaturation is a two-state process. Analysis of these denaturation curves gave values of 4.20 ± 0.12 kcal mol⁻¹, 2.95 ± 0.15 M, and 1.42 ± 0.06 kcal mol⁻¹ M⁻¹ for (Gibbs free energy change (Δ<i>G</i>_D) in the absence of urea), <i>C</i>_m (molar urea concentration ([urea]) at the midpoint of the denaturation curve), and <i>m</i> (=∂Δ<i>G</i>_D/∂[urea]), respectively. All these experimental observations have been fully supported by 30 ns molecular dynamics simulation studies.

钙/钙调蛋白依赖性蛋白激酶IV（CaMKIV）是一种多功能酶，隶属于丝氨酸/苏氨酸激酶家族（Ser/Thr kinase family）。CaMKIV在诸多生物学过程中发挥关键调控作用，涵盖基因表达调控、记忆巩固、骨骼生长、T细胞成熟、精子活力维持、微管动力学调控、细胞周期进程以及细胞凋亡。为测定该酶的稳定性参数，研究人员在pH 7.4、25℃的实验条件下，采用远紫外圆二色谱（far-UV CD）、近紫外吸收光谱及色氨酸荧光光谱三种检测手段，开展了尿素诱导的CaMKIV变性实验。上述三种光学特性的归一化变性曲线高度重合，证实尿素诱导的变性过程属于两态过程。对这些变性曲线的分析结果表明，无尿素条件下的吉布斯自由能变化（Δ<i>G</i>_D）、变性曲线中点处的尿素摩尔浓度（<i>C</i>_m）以及斜率参数<i>m</i>（<i>m</i>=∂Δ<i>G</i>_D/∂[urea]）的数值分别为4.20±0.12 kcal·mol⁻¹、2.95±0.15 M和1.42±0.06 kcal·mol⁻¹·M⁻¹。所有上述实验观测结果均得到了30纳秒分子动力学模拟研究的充分支持。