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

开发新型单分子分析 (SMA) 系统来测量低亲和力分子相互作用的实时动力学：Ras-e 的案例研究

• 批准号: 2736904
• 金额: --
• 依托单位: University of Leicester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2736904
• 关键词: Development; novel; single; molecule; analysis

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.

Ras蛋白家族是小GTP酶，其充当信号传导枢纽，激活多个效应子以通过直接蛋白质-蛋白质相互作用进行下游细胞信号传导。然而，Ras如何将信号转导至多个效应物仍然知之甚少。同时，由于在所有癌症中约25%出现突变Ras，因此强调了其生理意义。我们的目的是阐明一种新的单分子分析（SMA）技术，允许弱蛋白质-蛋白质相互作用，如活性Ras大多数先前的Ras-效应物相互作用研究使用“本体”技术，包括等温滴定量热法（ITC）、停流、荧光偏振、表面等离子体共振（SPR）和生物层干涉测量法（BLI）。并且相互作用过程的细节被群体平均效应模糊。从这个角度来看，SMA是独特的和非常有吸引力的，因为它允许观察实时结合动力学和竞争事件。然而，常规SMA并不最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以在单分子水平上解析每个相互作用事件。一般来说，KD值需要约为30 nM或更低（Selvin和Ha，2008，单分子技术：该项目旨在通过建立一种新型SMA系统来打破这一限制，该系统使用定制的严格稳定的TIRF显微镜来研究Ras效应器相互作用，长期成像，以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信号机制的基本原理，并将带来方法学上有价值的资源，为更广泛的社区允许调查低亲和力蛋白质相互作用的研究。