Functional conformer of c-Myb DNA-binding domain revealed by variable temperature studies.
Functional conformer of c-Myb DNA-binding domain revealed by variable temperature studies.
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变温研究揭示了 c-Myb DNA 结合结构域的功能构象异构体。
DOI：
10.1111/febs.13508
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Oda M.
中科院分区：
文献类型：
--
作者：
Inaba S;Maeno A;Sakurai K;Narayanan SP;Ikegami T;Akasaka K;Oda M.
在接近生理条件下研究了c-Myb的最小DNA结合结构域重复序列2和3（R2 R3）的构象波动。发现涉及主链和侧链的全局解折叠转变发生在约30-70 °C的温度范围内，转变温度约为50 °C。此外，在1H一维和15 N/1H二维NMR谱中同时观察到NMR信号的位移变化和加宽，表明在生理温度下存在局部涨落状态。在R2中含有空腔的野生型蛋白质中，R2的局部波动比R3的局部波动更显著，而在空腔填充突变体V103 L中受到抑制。这表明R2中的空腔对构象不稳定性和向局部波动状态的转变有显著贡献。对于野生型R2 R3蛋白，估计在37 °C下存在一定程度的动态构象异构体，并且可能对其生物学功能有益：DNA结合。这一结果与激发态构象体的概念一致，激发态构象体与占主导地位的基态构象体平衡存在，并充当蛋白质的功能构象体。从本研究的结果来看，似乎没有二硫键和具有不稳定空腔的两个小结构域的串联重复序列作为宽型c-Myb DNA结合结构域的进化策略，以在37 °C下产生局部波动状态的适当部分，数据库化学位移和峰列表已保存在生物磁共振库中，11584和11585。
The conformational fluctuation in the minimum DNA‐binding domain of c‐Myb, repeats 2 and 3 (R2R3), was studied under closely physiological conditions. A global unfolding transition, involving both the main chain and the side chains, was found to take place at the approximate temperature range 30–70 °C, with a transition temperature of approximately 50 °C. In addition, the observation of simultaneous shift change and broadening of NMR signals in both1H one‐dimensional and15N/1H two‐dimensional NMR spectra indicated the occurrence of locally fluctuating state at physiological temperature. In the wild‐type protein containing a cavity in R2, the local fluctuation of R2 is more prominent than that of R3, whereas it is suppressed in the cavity‐filled mutant, V103L. This indicates that the cavity in R2 contributes significantly to the conformational instability and the transition into the locally fluctuating state. For the wild‐type R2R3 protein, the more dynamic conformer is estimated to be present to some extent at 37 °C and is likely beneficial for its biological function: DNA‐binding. This result is in agreement with the concept of an excited‐state conformer that exists in equilibrium with the dominant ground‐state conformer and acts as the functional conformer of the protein. From the findings of the present study, it appears that the tandem repeats of two small domains with no disulfide bonds and with a destabilizing cavity function as the evolutionary strategy of the wide‐type c‐Myb DNA‐binding domain to produce an appropriate fraction of the locally fluctuating state at 37 °C, which is more amenable to DNA‐binding.DatabaseChemical shifts and peak lists have been deposited in the Biological Magnetic Resonance Bank under entries 11584 and 11585.