Limits of cooperativity in a structurally modular protein: Response of the notch ankyrin domain to analogous alanine substitutions in each repeat
Limits of cooperativity in a structurally modular protein: Response of the notch ankyrin domain to analogous alanine substitutions in each repeat
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DOI：
10.1016/s0022-2836(02)00945-2
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发表时间：
2002-11-22
影响因子：
5.6
通讯作者：
Barrick, D
中科院分区：
文献类型：
--
作者：
Bradley, CM;Barrick, D
为了确定结构模块化蛋白质的协同性极限，我们表征了果蝇Notch受体锚蛋白重复结构域的甘氨酸变体的结构和稳定性。每个重复中类似的丙氨酸残基被替换为甘氨酸，并且允许在蛋白质的不同位置检查相同的扰动。锚蛋白结构域对重复序列中的取代不敏感，表明第一个重复序列未完全折叠。重复序列第二到七个中的甘氨酸取代会严重破坏稳定性，但变体保留了其整体二级和三级结构。光谱和量热数据与重复二至重复五甘氨酸变体以及野生型蛋白质的二态展开转变一致。这些数据表明，尽管具有模块化结构，Notch 锚蛋白结构域仍作为由六个 C 端重复组成的协作单元展开，并且在 N 端和中央重复中存在严重不稳定的取代的情况下，这种协作性得以维持。相比之下，重复六中的甘氨酸取代导致多态展开转变，这表明在野生型蛋白质中产生长程协同性的偶联可能在 C 端区域具有薄弱的连接。这种行为可以通过一个简单的统计热力学模型来捕捉，其中不稳定的 C 端区域通过强稳定界面耦合到稳定的 N 端区域。 (C) 2002 Elsevier Science Ltd. 保留所有权利。
To determine the limits of cooperativity in a structurally modular protein, we characterized the structure and stability of glycine variants of the ankyrin repeat domain from the Drosophila melangaster Notch receptor. The substitutions are of analogous alanine residues to glycine in each repeat, and allow the same perturbation to be examined at different positions in the protein. The ankyrin domain is insensitive to substitution in repeat one, suggesting that the first repeat is not fully-folded. Glycine substitutions in repeat two through seven are strongly destabilizing, but the variants retain their overall secondary and tertiary structures. Spectroscopic and calorimetric data are consistent with two-state unfolding transitions for the repeat-two through repeat-five glycine variants, and for the wild-type protein. These data indicate that, despite its modular structure, the Notch ankyrin domain unfolds as a cooperative unit consisting of the six C-terminal repeats, and that this cooperativity is maintained in the presence of severely destabilizing substitutions in the N-terminal and central repeats. In contrast, glycine substitution in repeat six leads to a multi-state unfolding transition, suggesting that the coupling that gives rise to long-range cooperativity in the wild-type protein may have a weak link in the C-terminal region. Such behavior is captured by a simple statistical thermodynamic model in which an unstable C-terminal region is coupled to a stable N-terminal region through a strongly stabilizing interface. (C) 2002 Elsevier Science Ltd. All rights reserved.