Han Jiahui, Jiang Shijie, Zhou Zhengfu, Lin Min, Wang Jin
Key Laboratory of Agricultural Microbiome (MARA), Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China.
Antioxidants (Basel). 2023 May 24;12(6):1147. doi: 10.3390/antiox12061147.
G3LEA is a family of proteins that exhibit chaperone-like activity when under distinct stress. In previous research, DosH was identified as a G3LEA protein from model extremophile- R1 with a crucial core HD domain consisting of eight 11-mer motifs. However, the roles of motifs participating in the process of resistance to stress and their underlying mechanisms remain unclear. Here, eight different proteins with tandem repeats of the same motif were synthesized, named Motif1-8, respectively, whose function and structure were discussed. In this way, the role of each motif in the HD domain can be comprehensively analyzed, which can help in finding possibly crucial amino acid sites. Circular dichroism results showed that all proteins were intrinsically ordered in phosphate buffer, and changed into more α-helical ordered structures with the addition of trifluoroethanol and glycerol. Transformants expressing artificial proteins had significantly higher stress resistance to oxidation, desiccation, salinity and freezing compared with the control group; with Motif1 and Motif8 had more outstanding performance in particular. Moreover, enzymes and membrane protein protection viability suggested that Motif1 and Motif8 had more positive influences on various molecules, demonstrating a protective role in a chaperone-like manner. Based on these results, the artificial proteins synthesized according to the rule of 11-mer motifs have a similar function to wildtype protein. Regarding the sequence in all motifs, there are more amino acids to produce H bonds and α-helices, and more amino acids to promote interaction between proteins in Motif1 and Motif8; in addition, considering linkers, there are possibly more amino acids forming α-helix and binding substrates in these two proteins, which potentially provides some ideas for us to design potential ideal stress-response elements for synthetic biology. Therefore, the amino acid composition of the 11-mer motif and linker is likely responsible for its biological function.
G3LEA是一类蛋白质家族，在不同应激条件下表现出类似伴侣蛋白的活性。在先前的研究中，DosH被鉴定为来自模式嗜极端菌-R1的一种G3LEA蛋白，其具有由八个11聚体基序组成的关键核心HD结构域。然而，参与应激抗性过程的基序的作用及其潜在机制仍不清楚。在此，合成了八种具有相同基序串联重复的不同蛋白质，分别命名为Motif1-8，并对其功能和结构进行了讨论。通过这种方式，可以全面分析HD结构域中每个基序的作用，这有助于找到可能至关重要的氨基酸位点。圆二色性结果表明，所有蛋白质在磷酸盐缓冲液中本质上是有序的，并且随着三氟乙醇和甘油的加入转变为更多的α-螺旋有序结构。与对照组相比，表达人工蛋白的转化体对氧化、干燥、盐度和冷冻具有显著更高的应激抗性；特别是Motif1和Motif8表现更为突出。此外，酶和膜蛋白保护活力表明Motif1和Motif8对各种分子具有更积极的影响，以类似伴侣蛋白的方式发挥保护作用。基于这些结果，根据11聚体基序规则合成的人工蛋白具有与野生型蛋白相似的功能。关于所有基序中的序列，Motif1和Motif8中有更多的氨基酸产生氢键和α-螺旋，并且有更多的氨基酸促进蛋白质之间的相互作用；此外，考虑到连接子，这两种蛋白质中可能有更多的氨基酸形成α-螺旋并结合底物，这可能为我们设计合成生物学潜在的理想应激反应元件提供一些思路。因此，11聚体基序和连接子的氨基酸组成可能决定其生物学功能。
