Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc.
Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc.
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DOI：
10.3390/ijms22010055
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发表时间：
2020-12-23
影响因子：
5.6
通讯作者：
Marszalek P
中科院分区：
文献类型：
--
作者：
Ding Y;Apostolidou D;Marszalek P
NanoLuc是一种生物发光蛋白，最近被设计用于分子成像和细胞报告分析。与用于这些应用的其他生物发光蛋白相比，如萤火虫荧光素酶和Renilla荧光素酶，它的亮度约为150倍，热稳定性更高，体积更小。然而，目前还没有关于其机械特性的信息，这可能会为这种独特的蛋白质引入一套新的应用，例如一种新的生物材料或作为蛋白质活性/再折叠测定的底物。在这里，我们合成了一种合成的NanoLuc衍生蛋白，它由三个相连的NanoLuc蛋白组成，两侧各有两个人类titin I91结构域，并通过单分子力谱（SMFS）测量在单分子水平上进行了我们的力学研究。我们的研究结果表明，衍生物中的每个NanoLuc重复序列表现为单个结构域蛋白，当大约72 pN施加于蛋白质时，平均发生单个展开事件。此外，我们进行了循环测量，其中施加在单个蛋白质上的力循环升高然后降低，以使蛋白质有机会重新折叠：我们观察到蛋白质在机械变性后只有16.9%的时间能够重新折叠到正确的结构，而另外26.9%的时间有证据表明蛋白质错误折叠成潜在的非功能性构象。这些结果表明，NanoLuc是一种机械上中等弱的蛋白质，当拉伸变性时，它不能正确地重新折叠自己，这使它成为未来蛋白质折叠和错误折叠研究的一个有吸引力的模型。
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.
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DOI：
10.1126/science.1261909
发表时间：
2015-04-24
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Goldman DH;Kaiser CM;Milin A;Righini M;Tinoco I Jr;Bustamante C
通讯作者：
Bustamante C
影响因子：
16.6
作者：
Giganti D;Yan K;Badilla CL;Fernandez JM;Alegre-Cebollada J
通讯作者：
Alegre-Cebollada J
DOI：
10.1073/pnas.96.20.11288
发表时间：
1999-09-28
影响因子：
11.1
作者：
Carrion-Vazquez, M;Marszalek, PE;Fernandez, JM
通讯作者：
Fernandez, JM
影响因子：
56.9
作者：
Fernandez, JM;Li, HB
通讯作者：
Li, HB
DOI：
10.1073/pnas.0509217103
发表时间：
2006-01-31
影响因子：
11.1
作者：
Dietz, H;Rief, M
通讯作者：
Rief, M