Zhang Pan, Wang David, Yang Weitao, Marszalek Piotr E
Department of Chemistry, Duke University, Durham, North Carolina.
Department of Mechanical Engineering and Materials Science, Duke University, Durham, North Carolina.
Biophys J. 2020 Dec 1;119(11):2251-2261. doi: 10.1016/j.bpj.2020.10.023. Epub 2020 Oct 30.
Although the folding of single-domain proteins is well characterized theoretically and experimentally, the folding of large multidomain proteins is less well known. Firefly luciferase, a 550 residue three-domain protein, has been commonly used as a substrate to study chaperone reactions and as a model system for the study of folding of long polypeptide chains, including related phenomena such as cotranslational folding. Despite being characterized by various experimental techniques, the atomic-level contributions of various secondary structures of luciferase to its fold's mechanical stability remain unknown. Here, we developed a piecewise approach for all-atom steered molecular dynamics simulations to examine specific secondary structures that resist mechanical unfolding while minimizing the amount of computational resources required by the large water box of standard all-atom steered molecular dynamics simulations. We validated the robustness of this approach with a small NI3C protein and used our approach to elucidate the specific secondary structures that provide the largest contributions to luciferase mechanostability. In doing so, we show that piecewise all-atom steered molecular dynamics simulations can provide novel atomic resolution details regarding mechanostability and can serve as a platform for novel mutagenesis studies as well as a point for comparison with high-resolution force spectroscopy experiments.
尽管单结构域蛋白质的折叠在理论和实验上已得到充分表征，但大型多结构域蛋白质的折叠却鲜为人知。萤火虫荧光素酶是一种由550个残基组成的三结构域蛋白质，常被用作研究伴侣反应的底物以及研究长多肽链折叠的模型系统，包括共翻译折叠等相关现象。尽管通过各种实验技术对其进行了表征，但荧光素酶各种二级结构对其折叠机械稳定性的原子水平贡献仍不清楚。在此，我们开发了一种用于全原子引导分子动力学模拟的分段方法，以研究抵抗机械展开的特定二级结构，同时将标准全原子引导分子动力学模拟的大水盒所需的计算资源量降至最低。我们用一种小的NI3C蛋白质验证了该方法的稳健性，并使用我们的方法阐明了对荧光素酶机械稳定性贡献最大的特定二级结构。在此过程中，我们表明分段全原子引导分子动力学模拟可以提供有关机械稳定性的新的原子分辨率细节，并可作为新的诱变研究平台以及与高分辨率力谱实验进行比较的基准。
