Wang Iren, Chen Szu-Yu, Hsu Shang-Te Danny
†Institute of Biological Chemistry, Academia Sinica, 128, Section 2, Academia Road, Taipei 11529, Taiwan.
‡Institute of Biochemical Sciences, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan.
J Phys Chem B. 2015 Mar 26;119(12):4359-70. doi: 10.1021/jp511029s. Epub 2015 Mar 17.
Understanding the mechanism by which polypeptide chains thread themselves into topologically knotted structures has emerged to be a challenging subject not least because of the additional complexity associated with the spontaneous and efficient knotting and folding events. While recent theoretical calculations have made significant progress in establishing the atomistic folding pathways for a number of knotted proteins, experimental data on the folding stabilities and kinetic pathways of knotted proteins has been sparse. Using MJ0366 from Methanocaldococcus jannaschii, the smallest knotted protein known to date, as a model system, we set out to systematically investigate its folding equilibrium, kinetics, and internal dynamics under native and chemically denatured states. NMR hydrogen-deuterium exchange analysis indicates that the knotted region is the most stable structural element within the novel fold. Additionally, (15)N spin relaxation analysis reveals the presence of residual structures in urea-denatured MJ0366. Despite the apparent two-state equilibrium unfolding behavior during chemical denaturation, the kinetic unfolding pathway of MJ0366 involves the dissociation of the homodimeric native state into a native-like monomeric intermediate followed by unfolding into a denatured state. Our results provide comprehensive structural information regarding the folding dynamics and kinetic pathways of MJ0366, whose small size is ideal for converging experimental and theoretical findings to better understand the underlying principles of the folding of knotted proteins.
理解多肽链如何自行形成拓扑打结结构的机制已成为一个具有挑战性的课题，这尤其是因为与自发且高效的打结和折叠事件相关的额外复杂性。虽然最近的理论计算在确定多种打结蛋白质的原子水平折叠途径方面取得了重大进展，但关于打结蛋白质折叠稳定性和动力学途径的实验数据却很稀少。我们以来自詹氏甲烷球菌（Methanocaldococcus jannaschii）的MJ0366（迄今为止已知的最小的打结蛋白质）作为模型系统，着手系统地研究其在天然状态和化学变性状态下的折叠平衡、动力学及内部动力学。核磁共振氢氘交换分析表明，打结区域是这种新型折叠结构中最稳定的结构元件。此外，氮-15自旋弛豫分析揭示了在尿素变性的MJ0366中存在残余结构。尽管在化学变性过程中呈现出明显的两态平衡去折叠行为，但MJ0366的动力学去折叠途径涉及同二聚体天然态解离为类似天然态的单体中间体，随后再去折叠为变性态。我们的结果提供了关于MJ0366折叠动力学和动力学途径的全面结构信息，其小尺寸非常适合融合实验和理论研究结果，以更好地理解打结蛋白质折叠的潜在原理。
