Track: High Throughput Protein Science Thermal and mechanical stability of highly-luminescent protein NanoLuc in presence and absence of chaperones; Award Winners/Abstracts from the 36th Annual Symposium of The Protein Society, July 7 – 10, 2022
Track: High Throughput Protein Science Thermal and mechanical stability of highly-luminescent protein NanoLuc in presence and absence of chaperones; Award Winners/Abstracts from the 36th Annual Symposium of The Protein Society, July 7 – 10, 2022
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赛道：高通量蛋白质科学 高发光蛋白 NanoLuc 在存在和不存在分子伴侣的情况下的热稳定性和机械稳定性；
DOI：
10.1002/pro.4494
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发表时间：
2023
期刊：
影响因子：
8
通讯作者：
Marszalek, Piotr E.
中科院分区：
文献类型：
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作者：
Apostolidou, Dimitra;Ding, Yue;Marszalek, Piotr E.
尽管NanoLuc蛋白作为一种报告酶被广泛应用，但其力学行为从未被研究过。与传统使用的萤火虫荧光素酶相比，NanoLuc具有高热稳定性，这一未开发的领域引起了我们的兴趣。我们基于原子力显微镜的单分子力光谱实验对NanoLuc的各种多蛋白结构进行了研究，使我们能够探索这种蛋白质展开和可能错误折叠的各种情况。我们的研究结果有力地表明，尽管所有的结构具有相似的展开行为，但重折叠行为不同。当蛋白质与自身连接时，NanoLuc的成功重折叠记录的百分比大大降低。这与NanoLuc重复序列被其他蛋白质分离的结构或单一NanoLuc蛋白的结构相反。此外，NanoLuc的定向分子动力学模拟为蛋白质的展开途径提供了有价值的见解，其中蛋白质的Cterminus端首先与蛋白质的其余部分分离。最后，热变性实验表明，在变性温度为58℃时，聚NanoLuc蛋白的热稳定性突然下降，而在相同条件下，单体NanoLuc蛋白的热稳定性基本保持折叠状态。将大肠杆菌DnaK/DnaJ/GrpE伴侣系统添加到poly-NanoLuc蛋白中，可使初始生物发光恢复70%。在没有伴侣的情况下，自发恢复显示没有恢复，进一步支持poly-NanoLuc蛋白是强大的伴侣底物。
Despite the extensive application of NanoLuc protein as a reporter enzyme, its mechanical behavior has never been investigated. This unexplored area intrigued our interest given NanoLuc's high thermal stability in comparison to the traditionally used Firefly Luciferase. Our Atomic Force Microscopy based Single Molecule Force Spectroscopy experiments on various polyprotein constructs of NanoLuc enabled us to explore various scenarios of this protein's unfolding and possibly misfolding. Our results strongly demonstrated that despite all the constructs had similar unfolding behavior, the refolding behavior differed. The percentage of successful refolding recordings of NanoLuc was greatly decreased when the protein was linked to itself. This was a contrary result from the construct in which the NanoLuc repeats were separated by other proteins or in the construct with a single NanoLuc protein. Additionally, Steered Molecular Dynamics Simulations of NanoLuc provided valuable insight into the unfolding pathways, in which the Cterminus end of the protein was the first to break apart from the rest of the protein. Lastly, thermal denaturation experiments of poly-NanoLuc proteins showed a sudden decrease in thermal stability at the denaturation temperature of 58C, while the monomeric NanoLuc remained mostly folded in same conditions. Addition of the E. coli DnaK/DnaJ/GrpE chaperone system to the poly-NanoLuc proteins resulted to a 70% recovery of the initial bioluminescence. The spontaneous recovery in the absence of the chaperones showed no recovery, further supporting how poly-NanoLuc proteins are robust chaperone substrates.