Bioluminescence resonance energy transfer detection system for the monitoring of protein-protein interactions, post-translational modifications and cell signalling.

该研究工具和仪器赠款计划的申请是为了获得适用于高灵敏度和中等通量生物发光共振能量转移（BRET）测量的多模式酶标仪。对天然存在的BRET的操纵使得能够设计真实的检测各种细胞事件的传感器。主要申请人的实验室开创了BRET的生物学用途，并继续开发基于BRET的传感器（迄今为止已设计并验证了70多种不同的传感器/测定），探测蛋白质-蛋白质相互作用以及蛋白质构象变化、运输和信号传导。要求的BRET兼容读板仪将用于许多NSERC支持的（发现赠款）项目，包括使用BRET。我们目前的应用程序中，这说明了广泛的可能的应用程序的文书，这些项目中的四个。项目1旨在开发和使用基于BRET的新方法来监测G蛋白偶联受体（GPCR）信号传输的时空传播。将以亚细胞分辨率真实的时间监测GPCR效应物的易位和活性。在项目2中，基于BRET的检测将用于研究有丝分裂期间控制细胞表面蛋白质组变化的机制，方法是监测细胞周期调节的各种细胞器之间的蛋白质亚细胞运输。控制T细胞受体相关Lck激酶活性的结构决定因素将在项目3中进行评估，使用分子内基于BRET的传感器监测与CD 3结合后激酶的构象重排。项目4旨在表征SUMO化在控制与癌细胞衰老和迁移相关的蛋白质-蛋白质相互作用中的作用。为此，将开发一种基于BRET的测定法，以监测小分子对波形蛋白和UBC 9的SUMO化状态的影响，以及它们对蛋白质-蛋白质相互作用和活性的影响。学生，博士后研究员和高素质的人员将参与每个项目，使他们能够学习如何设计，执行和解释BRET结果，除了获得他们所解决的生物学问题的见解。由于BRET原理类似于荧光共振能量转移（FRET）和其他基于邻近性的测定，如蛋白质互补，蛋白质连接和邻近依赖性生物素鉴定（BioID），学员将接触到这些创新和备受追捧的方法。鉴于现有仪器已被最大限度地使用，NSERC支持的其他项目需要购买一台新的BRET阅读器。新一代仪器更高的灵敏度和吞吐量将使这些项目得以有效实现。

This application to the Research Tools and Instruments Grants program is for the acquisition of a multimodal plate reader adapted for high sensitivity and medium throughput bioluminescence resonance energy transfer (BRET) measurements. Manipulation of the naturally occurring BRET has enabled the design of sensors detecting various cellular events in real time. The laboratory of the principal applicant pioneered the biological use of BRET and continues to develop BRET-based sensors (over 70 distinct sensors/assays have been designed and validated so far), probing protein-protein interactions, as well as protein conformational changes, trafficking and signaling. The BRET-compatible plate reader requested will be used in many NSERC-supported (Discovery Grants) projects that include the use of BRET. We present four of these projects in the current application, which illustrates the wide spectrum of possible applications of the instrument. Project 1 aims at developing and using new BRET-based approaches to monitor the spatio-temporal propagation of signal transmission by G protein-coupled receptors (GPCR). The translocation and activity of GPCR effectors will be monitored at subcellular resolution in real time. In project 2, BRET-based assays will be used to investigate the mechanisms controlling the changes in the cell surface proteome during mitosis by monitoring the cell cycle-regulated subcellular trafficking of proteins between various organelles. Structural determinants controlling the activity of the T cell receptor-associated Lck kinase will be assessed, in project 3, with the use of intramolecular BRET-based sensors monitoring the conformational rearrangement of the kinase upon binding to CD3. Project 4 aims at characterizing the role of SUMOylation in controlling protein-protein interactions associated with the senescence and migration of cancer cells. For this purpose, a BRET-based assay will be developed to monitor the effects of small molecules on the SUMOylation state of vimentin and UBC9, and their impact on protein-protein interactions and activity. Students, post-doctoral fellows and highly qualified personnel will be involved in each project, allowing them to learn how to design, perform and interpret BRET results in addition to gaining insights into the biological questions that they address. Because the BRET principles are similar to those of fluorescence resonance energy transfer (FRET) and other proximity-based assays, such as protein-complementation, protein-ligation and proximity-dependent biotin identification (BioID), the trainees will be exposed to these innovative and highly sought after approaches. Given that the existing instrument is already used at its maximal capacity, the additional NSERC supported projects necessitate the acquisition of a new BRET reader. The greater sensitivity and throughput of the new generation instruments will allow the efficient realization of these projects.