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

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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.