Karolinska Institutet, Center of Bioscience, 141 57 Huddinge, Sweden.
Extremophiles. 2010 Mar;14(2):213-23. doi: 10.1007/s00792-009-0300-0.
The multi-domain enzyme isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix was studied by denaturant-induced unfolding. At pH 7.5, changes in circular dichroism ellipticity and intrinsic fluorescence showed a complex unfolding transition, whereas at pH 3.0, an apparently two-state and highly reversible unfolding occurred. Analytical ultracentrifugation revealed the dissociation from dimer to monomer at pH 3.0. The thermodynamic and kinetic stability were studied at pH 3.0 to explore the role of inter-domain interactions independently of inter-subunit interplay on the wild type and R211M, a mutant where a seven-membered inter-domain ionic network has been disrupted. The unfolding and folding transitions occurred at slightly different denaturant concentrations even after prolonged equilibration time. The difference between the folding and the unfolding profiles was decreased in the mutant R211M. The apparent Gibbs free energy decreased approximately 2 kcal/mol and the unfolding rate increased 4.3-fold in the mutant protein, corresponding to a decrease in activation free energy of unfolding of 0.86 kcal/mol. These results suggest that the inter-domain ionic network might be responsible for additional stabilization through a significant kinetic barrier in the unfolding pathway that could also explain the larger difference observed between the folding and unfolding transitions of the wild type.
来自嗜热菌 Aeropyrum pernix 的多结构域酶异柠檬酸脱氢酶通过变性剂诱导的展开进行了研究。在 pH7.5 时，圆二色性椭圆率和固有荧光的变化显示出复杂的展开转变，而在 pH3.0 时，发生了明显的两态和高度可逆的展开。分析超速离心显示在 pH3.0 时从二聚体解离为单体。在 pH3.0 下研究热力学和动力学稳定性，以探索在不考虑亚基相互作用的情况下，域间相互作用对野生型和 R211M（其中破坏了一个七元域间离子网络）的影响。即使经过长时间的平衡，展开和折叠转变仍在稍微不同的变性剂浓度下发生。突变体 R211M 中折叠和展开曲线之间的差异减小。在突变体蛋白中，表观吉布斯自由能降低约 2kcal/mol，展开速率增加 4.3 倍，这对应于展开的活化自由能降低了 0.86kcal/mol。这些结果表明，域间离子网络可能通过在展开途径中增加显著的动力学障碍来提供额外的稳定性，这也可以解释在野生型折叠和展开转变之间观察到的更大差异。
