Development of novel single-molecule analysis (SMA) system to measure real-time kinetics of low-affinity molecular interactions: a case study of Ras-e

Ras蛋白家族是一种小的gtpase，作为信号中枢，激活多种效应物，通过直接的蛋白-蛋白相互作用进行下游细胞信号传导。然而，Ras如何将信号转导到多个效应器仍然知之甚少。同时，由于在约25%的癌症中出现Ras突变，其生理意义被强调。我们的目标是阐明一种新的单分子分析（SMA）技术，该技术允许弱蛋白质-蛋白质相互作用，例如活性Ras （gtp负载）与其多种效应物的相互作用。以前的大多数ras -效应物相互作用研究使用的是“体”技术，包括等温滴定量热法（ITC）、停止流动法、荧光极化法、表面等离子体共振法（SPR）和生物层干涉法（BLI），相互作用过程的细节被总体平均效应模糊了。从这个角度来看，SMA是独特而极具吸引力的，因为它允许观察实时结合动力学和竞争事件。然而，传统的SMA并不适合研究低亲和力蛋白质-蛋白质相互作用，因为样品需要高度稀释才能在单分子水平上解决每个相互作用事件。一般来说，KD值需要在30 nM左右或以下（Selvin和Ha， 2008，单分子技术：CSHL出版社）。该项目旨在打破这一限制，通过建立一种新的SMA系统，使用定制的严格稳定的TIRF显微镜研究ras -效应物相互作用，允许长期成像记录相对罕见的相互作用事件。该项目的成功完成将揭示RAS信号传导机制的基本原理，并将为更广泛的研究低亲和力蛋白-蛋白相互作用的研究带来方法论上的宝贵资源。

Ras family of proteins are small GTPases that act as a signalling hub, activating multiple effectors to carry out downstream cellular signalling via direct protein-protein interactions. However, how Ras transduces signals to multiple effectors remains poorly understood. Meanwhile, its physiological significance is underlined due to the appearance of mutated Ras in about 25% of all cancers. Our aim is to elucidate a novel single molecule analysis (SMA) technique which allows for weak protein-protein interactions such as those of active Ras (GTP-loaded) with its multiple effectors to be studied.Most of the previous Ras-effector interaction studies used "bulk" techniques, including isothermal titration calorimetry (ITC), stopped-flow, fluorescence polarisation, surface plasmon resonance (SPR), and biolayer interferometry (BLI), and the details of the interaction processes were blurred by population averaging effects. From this point of view, SMA is unique and highly attractive as it allows the observation of real-time binding kinetics and competition events. However, conventional SMA is not best suited to study low-affinity protein-protein interactions as the samples need to be highly diluted to resolve each interaction event at a single-molecule level. Generally speaking, KD values need to be about 30 nM or below (Selvin and Ha, 2008, Single molecule techniques: CSHL Press).This project aims to break this limit by establishing a novel SMA system to study Ras-effector interaction using a custom-made rigorously stabilised TIRF microscope allowing long-term imaging to record relatively rare interaction events.Successful completion of this project will reveal the fundamental principle of RAS signalling mechanism and will bring a methodologically valuable resource for the wider community allowing for investigation of low-affinity protein-protein interaction studies.