Ge Meng, Mao Yong-Jin, Pan Xian-Ming
The Key Laboratory of Bioinformatics, Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, 100084 Beijing, China.
Extremophiles. 2009 Jan;13(1):131-7. doi: 10.1007/s00792-008-0204-4. Epub 2008 Nov 12.
The alpha/beta-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other alpha/beta-mixed or pure beta-sheet proteins are compared.
来自嗜热菌嗜热栖热放线菌的α/β混合二聚体蛋白Ssh10b是Sac10b家族的成员，该家族被认为参与染色体组织或DNA修复/重组过程。由变性剂和热诱导的Ssh10b的平衡去折叠/再折叠是完全可逆的，这表明Ssh10b可以作为蛋白质二聚体折叠/去折叠研究的良好模型。在这里，我们通过停流圆二色性（SF-CD）并使用盐酸胍（GdnHCl）作为变性剂，详细研究了Ssh10b的折叠/去折叠动力学。在去折叠反应中，天然的Ssh10b在停流死时间内迅速转变为完全去折叠的单体，没有可检测到的动力学中间体，这与平衡去折叠实验的结果非常吻合。在再折叠反应中，两个去折叠的单体在爆发阶段缔合形成二聚体中间体，该中间体经历进一步的、较慢的一级折叠过程以形成天然二聚体。我们的结果表明，二聚化对于维持蛋白质Ssh10b的天然三级相互作用至关重要。此外，还比较了Ssh10b和其他几种α/β混合或纯β-折叠蛋白的折叠机制。
