The Cryo-EM structure of mechanically unfolded proteins

About the project

About the Project A 2027 Crick Joint PhD project with Alessandro Costa and Sergi Garcia-Manyes. Project background and description Alongside biochemical signals and genetic considerations, mechanical forces are rapidly emerging as a master regulator of human physiology, impacting functions as diverse as embryo development, skin integrity, immuno-response or cardiac contractility. While the effect of force in cell/tissue biology has been substantially explored, how force affects the underpinning molecular constituents is comparatively less understood – often preventing mechanistic understanding of the macroscopic manifestations. To directly regulate function, external and persistent mechanical forces imposed by the extracellular matrix need to cross long distances to eventually reach the cell nucleus and activate gene expression. To sense and transmit mechanical forces, cells have evolved specific proteins that can unfold and refold under force. Most of these proteins, such as talin, nesprin or titin, are in form of long polyproteins composed of long chains of individual domains. This structure enables storage and release of large amounts of energy as each domain stretch and recoil when exposed to mechanical stress. Single-molecule force spectroscopy provides exceptional force and spatial resolution, allowing detailed characterisation of the conformational dynamics of individual proteins under force and their associated timescales. These experiments revealed that when denatured with force, proteins unfold following a completely different pathway from that sampled in thermodynamic denaturation, as they stretch along their end-to-end length. Importantly, upon unfolding, proteins expose previously cryptic sites that can undergo new post-translational modifications and binding of new proteins, functionally crucial in cellular mechanosignalling pathways. Structural biology tools to solve structures of protein under physical strain are yet to be developed, which limits our understanding of force-unfolded proteins that have a central role in mechanobiology. The proposed PhD project aims to fill this gap. The student will integrate single-molecule nanomechanical manipulation, microfluidics and cryo-EM to impose control stretching under shear flow, aiming to capture structures of force-stretched protein forms, providing molecular insights into the basis of mechanotransduction. The PhD project will combine the expertise in single molecule mechanics of the Garcia-Manyes lab with the cryo-EM of the Costa. Specifically, the project aims to (1) characterise, using single molecule technology, the mechanical stability of each individual domain in different polyproteins, including talin, filaminA and nesprin1. We will then (2) design an experimental pipeline to expose biotinylated proteins tethered from both ends to mechanical force when coupling the EM grids with a microfluidic device. Next, (3) use in silico reconstitution approaches to map the relative orientation of structured domains we will reconstruct the full polyprotein under different conditions of mechanical stress, and correlate the position of unfolded protein segments with the force-spectrometry experiments. We will finally (4) interface our EM-structures with fluorescence microscopy to reveal the structure of protein complexes formed between a mechanically stretched protein and its binder, starting with the talin/vinculin and the nesprin/FHOD1 pairs as a benchmark. The PhD student will receive training in molecular biology, single-molecule manipulation, cryo-EM and image processing. Candidate background This project would suit candidates with a background in Biochemistry, Biophysics or Physical Chemistry and an interest in protein structure, protein mechanics and mechanobiology. Lab-specific question Why does mechanical unfolding occur in a physiological context and why is it important in the cell?

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