Folding studies on a knotted protein
Folding studies on a knotted protein
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DOI：
10.1016/j.jmb.2004.12.055
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发表时间：
2005-03-11
影响因子：
5.6
通讯作者：
Jackson, SE
中科院分区：
文献类型：
--
作者：
Mallam, AL;Jackson, SE
YibK 是一种 160 个残基的同二聚体蛋白，属于 SPOUT 类甲基转移酶。该组中的蛋白质都表现出独特的拓扑特征；主链多肽链折叠形成深三叶结。这种打结的结构是完全不可预测的，人们认为蛋白质不可能以这种方式有效折叠。然而，它们正变得越来越常见，并且蛋白质数据库中的示例数量也越来越多。这些有趣的打结结构代表了蛋白质折叠领域的一个新的重大挑战。在这里，我们提出了 YibK 折叠的初步表征，YibK 是已识别的最小的打结蛋白之一。这是第一个关于打结蛋白的详细折叠研究的报道。我们已经建立了可以使用尿素在体外使蛋白质可逆变性的条件，从而表明该蛋白质的有效折叠不需要分子伴侣。在 400 倍的蛋白质浓度范围内进行了一系列平衡解折叠实验。二级和三级结构探针都显示出单一的、蛋白质浓度依赖性的去折叠转变，并且数据与涉及单体中间体的三态平衡变性模型最一致。从数据与该模型的拟合获得的热力学参数表明中间体是具有明显二级和三级结构的稳定物质；拓扑结是否仍处于中间状态仍有待证明。总之，这些结果表明，尽管 YibK 具有复杂的打结结构，但它能够有效折叠，并且在平衡条件下的行为与其他二聚体蛋白非常相似。 (C) 2005 Elsevier Ltd. 保留所有权利。
YibK is a 160 residue homodimeric protein belonging to the SPOUT class of methyltransferases. Proteins in this group all display a unique topological feature; the backbone polypeptide chain folds to form a deep trefoil knot. Such knotted structures were completely unpredicted, it being thought impossible for a protein to fold efficiently in this way. However, they are becoming more common and there are now a growing number of examples in the Protein Data Bank. These intriguing knotted structures represent a new and significant challenge in the field of protein folding. Here, we present an initial characterisation of the folding of YibK, one of the smallest knotted proteins to be identified. This is the first detailed folding study on a knotted protein to be reported. We have established conditions under which the protein can be denatured reversibly in vitro using urea, thereby showing that molecular chaperones are not required for the efficient folding of this protein. A series of equilibrium unfolding experiments were performed over a 400-fold range of protein concentration. Both secondary and tertiary structural probes show a single, protein concentration-dependent unfolding transition, and data are most consistent with a three-state equilibrium denaturation model involving a monomeric intermediate. Thermodynamic parameters obtained from the fit of the data to this model indicate that the intermediate is a stable species with appreciable secondary and tertiary structure; whether the topological knot remains in the intermediate state is still to be shown. Together, these results demonstrate that, despite its complex knotted structure, YibK is able to fold efficiently and behaves remarkably similarly to other dimeric proteins under equilibrium conditions. (C) 2005 Elsevier Ltd. All rights reserved.