Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan.
High Pressure Protein Research Center, Institute of Advanced Technology, Kinki University, Wakayama, Japan.
FEBS J. 2015 Dec;282(23):4497-514. doi: 10.1111/febs.13508. Epub 2015 Sep 29.
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 both (1)H one-dimensional and (15)N/(1)H 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. Database: Chemical shifts and peak lists have been deposited in the Biological Magnetic Resonance Bank under entries 11584 and 11585.
在接近生理条件下研究了 c-Myb 的最小 DNA 结合域重复 2 和 3（R2R3）的构象波动。发现一个涉及主链和侧链的全局展开转变发生在大约 30-70°C 的温度范围内，转变温度约为 50°C。此外，在（1）H 一维和（15）N/（1）H 二维 NMR 光谱中观察到 NMR 信号的同时位移变化和展宽，表明在生理温度下存在局部波动状态。在含有 R2 腔的野生型蛋白中，R2 的局部波动比 R3 更明显，而在填充腔的突变体 V103L 中则受到抑制。这表明 R2 中的腔对构象不稳定性和向局部波动状态的转变有重要贡献。对于野生型 R2R3 蛋白，估计在 37°C 下以某种程度存在更动态的构象，这可能有利于其生物学功能：DNA 结合。这一结果与存在于平衡态与主导基态构象之间的激发态构象的概念一致，作为蛋白质的功能构象。从本研究的结果来看，似乎两个没有二硫键的小域的串联重复和一个不稳定的腔作为宽型 c-Myb DNA 结合域的进化策略，在 37°C 下产生适当比例的局部波动状态，更适合 DNA 结合。数据库：化学位移和峰列表已在生物磁共振库中以条目 11584 和 11585 进行了存储。
