Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC 27708, USA.
Department of Engineering Mechanics, SVL, Xi'an Jiaotong University, Xi'an 710049, China.
Int J Mol Sci. 2020 Dec 23;22(1):55. doi: 10.3390/ijms22010055.
NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.
NanoLuc 是一种最近被设计用于分子成像和细胞报告分析的生物发光蛋白。与其他用于这些应用的生物发光蛋白（如萤火虫荧光素酶和海肾荧光素酶）相比，它的亮度高约 150 倍，热稳定性更高，体积更小。然而，关于其机械性能的信息尚不清楚，这可能为这种独特的蛋白质引入一系列新的应用，例如新型生物材料或作为蛋白质活性/重折叠分析的底物。在这里，我们生成了一种合成的 NanoLuc 衍生物蛋白，该蛋白由三个连接的 NanoLuc 蛋白组成，两侧各有两个人类肌联蛋白 I91 结构域。我们通过单分子力谱（SMFS）测量在单分子水平上进行了机械研究。我们的结果表明，衍生物中的每个 NanoLuc 重复都表现为单个结构域蛋白，当施加约 72 pN 的力时，平均会发生单个解折叠事件。此外，我们进行了循环测量，其中周期性地升高和降低施加到单个蛋白质上的力，以允许蛋白质有机会重折叠：我们观察到，在机械变性后，只有 16.9%的时间，蛋白质能够正确地折叠回其正确结构，而另外 26.9%的时间，有证据表明蛋白质错误折叠为潜在的非功能构象。这些结果表明，NanoLuc 是一种机械性能中等较弱的蛋白质，当拉伸变性时无法正确地重新折叠自身，这使其成为未来蛋白质折叠和错误折叠研究的有吸引力的模型。
