Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors.
Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors.
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通过内在和外在因素调节设计的 TPR 蛋白的多态折叠。
DOI：
10.1002/pro.2018
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Phillips JJ
中科院分区：
文献类型：
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作者：
Phillips JJ
三角四肽重复序列（TPR）是一类由34个氨基酸螺旋-转角-螺旋基序组成的α-螺旋重复序列蛋白。这些堆叠在一起形成非小叶结构，这些结构通过来自一级序列中接近的残基的短程相互作用而稳定。与球状蛋白质不同，它们几乎没有（如果有的话）长程非局部稳定相互作用。对设计的TPR蛋白的几项研究表明，这种模块化结构反映在它们的折叠中，即观察到模块化的多态折叠，而不是两态折叠。在这里，我们表明，TPR多状态折叠可以通过调制的内在稳定性或外部环境变量被抑制到近似两个状态的折叠。通过比较具有不同内在稳定性的两个不同系列的共有设计的TPR蛋白在不同缓冲体系下的热力学解折叠来研究这种调节。共9个蛋白质的不同大小和不同的共识TPR基序分别进行热变性和化学变性，并分别使用差示扫描量热法（DSC）和CD/荧光监测其展开。DSC和化学变性数据的分析表明，降低每种蛋白质和重复单元的总稳定性会导致可观察到的双态解折叠。这些数据突出了全局和内在重复稳定性之间的密切联系，该联系决定了折叠是否通过可观察到的双态机制进行，或者部分去折叠是否产生稳定的中间结构，这些结构保持足够的稳定性以在平衡时填充。
Tetratricopeptide repeats (TPRs) are a class of all alpha‐helical repeat proteins that are comprised of 34‐aa helix‐turn‐helix motifs. These stack together to form nonglobular structures that are stabilized by short‐range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long‐range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two‐state folding. Here we show that TPR multistate folding can be suppressed to approximate two‐state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus‐designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two‐state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two‐state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.