Protein Mechanics and Engineering at the Single Molecule Level

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Elastomeric proteins are key elastic elements in a wide range of mechano-chemical machinery in living systems, and also constitute structural materials of superb mechanical properties. Understanding how such elastomeric proteins are designed to function under force is not only important in elucidating fundamental biophysical principles underlying various biological processes, but may also pave ways to using these proteins for the bottom-up construction of advanced materials for applications in materials science and biomedical engineering. We propose to combine single molecule force spectroscopy with protein engineering, molecular dynamics simulations and traditional biophysical techniques to rationally engineer protein-based fluorogenic force sensors to quantify stretching forces experienced by individual protein in biomaterials, engineer novel smart elastomeric proteins and use them to construct protein-based dynamic biomaterials whose mechanical properties can be dynamically tuned using external stimuli, and develop novel force spectroscopy-based strategies to elucidate the folding mechanism of proteins of knotted topologies. Specifically, we will engineer a library of protein-based fluorogenic force sensors that can be used to quantify the stretching force experienced by individual protein in biomaterials optically. The knowledge of such molecular scale forces will help bridge the gap between single molecule mechanics and mechanics of biomaterials, and help tailor elastomeric proteins at the molecular level to engineer biomaterials with desirable mechanical properties for specific biomedical applications. Making use of ligand binding-induced folding as a means to control the folded and unfolded states of intrinsically disordered proteins, we will develop a general method to engineer dynamic protein biomaterials that can change their mechanical properties in response to environmental stimuli. Such protein-based dynamic biomaterials may open up possibilities for using protein hydrogels as model biological structures and in tissue regeneration. Moreover, we will also use single molecule force spectroscopy and molecular dynamics simulations to elucidate the folding-unfolding mechanism of a knotted tRNA methyltransferase TrmD, and shine light on the mechanism by which knotted proteins overcome topological barrier to fold into their complex functional structures.