萤火虫协议
荧光素酶
动力学(音乐)
分子动力学
生物物理学
化学
生物系统
计算生物学
生物
物理
进化生物学
生物化学
计算化学
基因
声学
转染
作者
Piotr E. Marszałek,Dimitra Apostolidou,Vishal Lashkari,Harshi Jasti
标识
DOI:10.1016/j.bpj.2023.11.1290
摘要
Firefly luciferase (Fluc) is a bioluminescent 62 kDa protein that finds numerous applications in cell and molecular biology research. Fluc has been a “gold standard” substrate in chaperone research because its bioluminescence is easy to measure and bioluminescence decrease and recovery is related to Fluc misfolding and chaperone-assisted refolding, respectively. Fluc is composed of a large ∼450 amino acid N-terminal domain and a smaller ∼100 amino acid C-terminal domain. Fluc is moderately stable at room temperature and at elevated temperatures above ∼38 °C quickly loses its ability to produce light, due to alterations in its structure. Even though “thermal denaturation” is critical for using Fluc in chaperone-assisted refolding reactions, very little is known about what happens to Fluc’s structure at elevated temperatures (>30 °C). In this project, we use all-atom molecular dynamics simulations to systematically explore the structure of Fluc at temperatures ranging from 5 to 80 °C. We find that simulations not longer than 100 ns (previous studies) may not reach an equilibrium. We therefore extended our calculations to 500 ns, for each temperature. Our results suggest that relatively small structural alterations are induced in Fluc at moderate temperatures, and confirming an earlier observation, we find that at these temperatures Fluc may populate an alternative stable state with domains rotated, which may not be functional. This second state is a possible substrate for chaperones. Thus, it is possible that the chaperone task may be simpler than assumed before, as it would involve only a moderate reorientation of the two domains, rather than the refolding of the totally denatured polypeptide chain of Fluc. This work was supported by the NSF grant number MCB 1817556 and MCB 2118357.
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