Moradi Maryam, Hosseinkhani Saman, Arab Seyed Shahriar, Khammari Anahita
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Iran J Biotechnol. 2020 Oct 1;18(4):e2423. doi: 10.30498/IJB.2020.2423. eCollection 2020 Oct.
IP3-induced Ca2+ release, mediated by IP3R, is one of the most momentous cellular signaling mechanisms that regulate in a wide variety of essential cellular functions. Involvement of disrupted IP3 signaling pathways in numerous pathophysiology conditions is implicated to find the best methods for its measurement. Hence, several different biosensors have developed to monitor temporal changes of IP3 by using the IP3-binding domain of IP3 receptors.
Based on a previous study, we developed and characterized a series of bioluminescent biosensors using the human type-II IP3 receptor ligand binding domain (residues 1-604), named LAIRE (luminescent analyzer for IP3 receptor element) to study the effect of flexible and rigid linkers on the luminescence intensity of split luciferase. The effect of a mutation in IP3 binding residues and suppressor domain in the IP3 binding domain on luciferase complementary assay is also investigated.
In the present study, first IP3-binding domain (residues 1-604) of IP3-receptor type 2 (LAIRE) was fused between complementary non-functional fragments of firefly luciferase and then the rigid linker sequence (LLRAIEAQQHLL), selected by ProDA database, introduced between Nluc and ligand binding domain and compared with that of the flexible linker ((GGGGS)2) in LAIRE chimera. The IP3-insensitive mutant of the biosensor was constructed using the Stratagene QuikChange® procedure. In order to the analysis of the dynamical movements of selected structures in the large-scale, coarse-graining method of molecular dynamics simulation (1µs) was applied.
As expected, the flexible linker brings two inactive fragments of luciferase together relative to the rigid linker and leads to complementation of luciferase activity, which is detected using luciferin. However, this conformational flexibility in linker increases background to noise ratio and attenuates fold induction.
It seems that the ligand binding properties of IP3 binding core make it more suitable for the design of biosensor than the ligand binding domain.
由IP3受体介导的IP3诱导的Ca2+释放是调节多种重要细胞功能的最重要的细胞信号传导机制之一。IP3信号通路的破坏与多种病理生理状况有关，这意味着要找到测量它的最佳方法。因此，已经开发了几种不同的生物传感器，通过使用IP3受体的IP3结合结构域来监测IP3的时间变化。
基于先前的一项研究，我们开发并表征了一系列使用人II型IP3受体配体结合结构域（第1至604位氨基酸残基）的生物发光生物传感器，命名为LAIRE（IP3受体元件发光分析仪），以研究柔性和刚性接头对分裂荧光素酶发光强度的影响。还研究了IP3结合结构域中IP3结合残基和抑制结构域的突变对荧光素酶互补测定的影响。
在本研究中，首先将IP3受体2型（LAIRE）的IP3结合结构域（第1至604位氨基酸残基）融合在萤火虫荧光素酶的互补无功能片段之间，然后将通过ProDA数据库选择的刚性接头序列（LLRAIEAQQHLL）引入Nluc和配体结合结构域之间，并与LAIRE嵌合体中的柔性接头（(GGGGS)2）进行比较。使用Stratagene QuikChange®程序构建生物传感器的IP3不敏感突变体。为了大规模分析选定结构的动态运动，应用了分子动力学模拟的粗粒度方法（1微秒）。
正如预期的那样，相对于刚性接头，柔性接头使荧光素酶的两个无活性片段聚集在一起，并导致荧光素酶活性互补，这可以使用荧光素检测到。然而，接头中的这种构象灵活性增加了背景噪声比并减弱了倍数诱导。
似乎IP3结合核心的配体结合特性使其比配体结合结构域更适合用于生物传感器的设计。
