The helix-turn-helix motif as an ultrafast independently folding domain:: The pathway of folding of Engrailed homeodomain
The helix-turn-helix motif as an ultrafast independently folding domain:: The pathway of folding of Engrailed homeodomain
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DOI：
10.1073/pnas.0703434104
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发表时间：
2007-05-29
影响因子：
11.1
通讯作者：
Fersht, Alan R.
中科院分区：
文献类型：
--
作者：
Religa, Tomasz L.;Johnson, Christopher M.;Fersht, Alan R.
Engrailed同源结构域（EnHD）的螺旋2和3形成螺旋-转角-螺旋（HTH）基序。这个共同的基序被认为不是独立折叠的，这是基序而不是结构域的特征。但我们发现EnHD HTH基序是单体的，在溶液中折叠，具有与全长蛋白质基本相同的结构。它有一个S形热变性转变。天然骨架和局部三级相互作用在25 ℃下以4 × 10（5）s（-1）同时形成，分别通过IR和荧光T-跳跃动力学监测，与EnHD折叠中的快速相相同的速率常数。因此，HTH基序是一种超快速折叠的天然蛋白质结构域。其独立的稳定性和适当的折叠动力学解释了EnHD的逐步折叠，完全满足通路上中间体的标准，并解释了整个同源结构域家族折叠机制的变化。用不同探针对亲本蛋白质的突变和工程化片段进行的实验允许将观察到的动力学阶段分配给特定事件，以表明EnHD不是单态下坡折叠的例子。
Helices 2 and 3 of Engrailed homeodomain (EnHD) form a helix-turn-helix (HTH) motif. This common motif is believed not to fold independently, which is the characteristic feature of a motif rather than a domain. But we found that the EnHD HTH motif is monomeric and folded in solution, having essentially the same structure as in full-length protein. It had a sigmoidal thermal denaturation transition. Both native backbone and local tertiary interactions were formed concurrently at 4 x 10(5) s(-1) at 25 degrees C, monitored by IR and fluorescence T-jump kinetics, respectively, the same rate constant as for the fast phase in the folding of EnHD. The HTH motif, thus, is an ultrafast-folding, natural protein domain. Its independent stability and appropriate folding kinetics account for the stepwise folding of EnHD, satisfy fully the criteria for an on-pathway intermediate, and explain the changes in mechanism of folding across the homeodomain family. Experiments on mutated and engineered fragments of the parent protein with different probes allowed the assignment of the observed kinetic phases to specific events to show that EnHD is not an example of one-state downhill folding.