Essentiality of Core hydrophobicity to the structure and function of archaeal chromatin protein Cren7.
Essentiality of Core hydrophobicity to the structure and function of archaeal chromatin protein Cren7.
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DOI：
10.1016/j.ijbiomac.2022.06.114
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发表时间：
2022-06
影响因子：
8.2
通讯作者：
L. Tian;Niannian Ding;Xuehui Liu;Yuanyuan Chen;Zhenfeng Zhang
中科院分区：
文献类型：
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作者：
L. Tian;Niannian Ding;Xuehui Liu;Yuanyuan Chen;Zhenfeng Zhang
蛋白质结构与功能关系的研究不仅有助于理解蛋白质折叠的原理，而且有助于理解蛋白质工程的基本原理。Crenarchaeal染色质蛋白Cren 7为这一问题提供了一个很好的研究模型。小蛋白采用“β-桶”折叠，由双链反平行β-折叠1与三链反平行β-折叠2紧密包装形成。Cren 7的简单结构由β-折叠之间的疏水核心稳定，该疏水核心由V8、V10、L20、V25、F41和F50的侧链组成。在本工作中，通过丙氨酸取代疏水核心中的每个残基进行突变分析。圆二色谱和核磁共振分析表明，F41突变导致Cren 7的β折叠被破坏，从而导致Cren 7的错误折叠。另一方面，突变体F41 A与野生型Cren 7（Tm > 80°C）相比，热稳定性降低（Tm为53.2°C）。生物膜干涉法和缺口闭合分析显示F41 A的DNA结合和超螺旋活性基本不变，表明Cren 7的DNA界面通常保留在F41 A中。然而，F41 A不能介导DNA桥接，可能是由于在DNA上形成寡聚体/聚合物的损伤。F41 A-DNA复合物的原子力显微镜图像也显示，F41 A几乎完全失去了将DNA压缩成高度凝聚结构的能力。我们的研究结果不仅揭示了F41在Cren 7蛋白质折叠中的关键作用，而且为热稳定蛋白质的结构-功能关系提供了新的见解。
Studies on the structure–function relationship of protein greatly help to understand not only the principles of protein folding but also the rationales of protein engineering. Crenarchaeal chromatin protein Cren7 provides an excellent research model for this issue. The small protein adopts a ‘β-barrel’ fold, formed by the double-stranded antiparallel β-sheet 1 tightly packing with the triple-stranded antiparallel β-sheet 2. The simple structure of Cren7 is stabilized by the hydrophobic core between the β-sheets, consisting of the side chains of V8, V10, L20, V25, F41 and F50. In the present work, mutation analyses by alanine substitution of each of the residues in the hydrophobic core were performed. Circular dichroism spectra and nuclear magnetic resonance analyses showed that mutation of F41 led to a significant misfolding of Cren7 through disruption of the β-sheets. Meanwhile, the mutant F41A showed a reduced thermostatility (Tm of 53.2°C), as compared with the wild-type Cren7 (Tm > 80°C). Biolayer interferometry and nick-closure assays showed the largely unchanged activities in DNA binding and supercoiling of F41A, indicating the DNA interface of Cren7 was generally retained in F41A. However, F41A was unable to mediate DNA bridging, probably due to the impairment in forming oligomers/polymers on DNA. Atomic force microscopic images of the F41A-DNA complexes also revealed that F41A nearly completely lost the ability to compact DNA into highly condensed structures. Our results not only reveal the critical role of F41 in protein folding of Cren7 but also provide new insights into the structure-function relationships of thermostable proteins.