Mallam Anna L, Jackson Sophie E
Chemistry Department, Cambridge, UK.
J Mol Biol. 2006 Jun 23;359(5):1420-36. doi: 10.1016/j.jmb.2006.04.032. Epub 2006 May 2.
The homodimeric protein YibK from Haemophilus influenzae belongs to a recently discovered superfamily of knotted proteins that has brought about a new protein-folding conundrum. Members of the alpha/beta-knot clan form deep trefoil knots in their native backbone structure, a topological feature that is currently unexplained in the protein-folding field. To help solve the puzzle of how a polypeptide chain can efficiently knot itself, the folding kinetics of YibK have been studied extensively and the results are reported here. Folding was monitored using probes for changes in both secondary and tertiary structure, and the monomer-dimer equilibrium was perturbed with a variety of solution conditions to allow characterisation of otherwise inaccessible states. Multiphasic kinetics were observed in the unfolding and refolding reactions of YibK, and under conditions where the dimer is favoured, dissociation and association were rate-limiting, respectively. A folding model consistent with all kinetic data is proposed: YibK appears to fold via two parallel pathways, partitioned by proline isomerisation events, to two distinct monomeric intermediates. These form a common third intermediate that is able to fold to native dimer. Kinetic simulations suggest that all intermediates are on-pathway. These results provide the valuable groundwork required to further understand how Nature codes for knot formation.
来自流感嗜血杆菌的同二聚体蛋白YibK属于最近发现的一类具有纽结结构的蛋白超家族，这引发了一个新的蛋白质折叠难题。α/β-纽结家族的成员在其天然主链结构中形成深三叶纽结，这一拓扑特征在蛋白质折叠领域目前尚无解释。为了帮助解决多肽链如何高效地自我纽结这一谜题，我们对YibK的折叠动力学进行了广泛研究，并在此报告结果。使用探测二级和三级结构变化的探针监测折叠过程，通过多种溶液条件扰动单体-二聚体平衡，以表征其他难以接近的状态。在YibK的去折叠和重折叠反应中观察到多相动力学，在二聚体占优势的条件下，解离和缔合分别是限速步骤。我们提出了一个与所有动力学数据一致的折叠模型：YibK似乎通过两条平行途径折叠，由脯氨酸异构化事件分隔，形成两种不同的单体中间体。这些中间体形成一个共同的第三个中间体，能够折叠成天然二聚体。动力学模拟表明所有中间体都在折叠途径上。这些结果为进一步理解自然如何编码纽结形成提供了有价值的基础。
