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Engineering proteins of novel mechanical properties : from single molecule to biomaterials Cao, Yi

Abstract

Elastomeric proteins are an important class of mechanical proteins that take care of the strength, elasticity and extensibility of tissues and biological machineries. Elastomeric proteins are also essential building blocks of materials with outstanding mechanical properties. However, it was largely unknown how elastomeric proteins achieve the remarkable mechanical stability until the advent of single molecule force spectroscopy techniques, such as single molecule atomic force microscopy (AFM). Single molecule AFM has enabled the direct characterization of the mechanical properties of elastomeric proteins at the single molecule level and led to the new promising research area of single protein mechanics and engineering. Combined with molecular dynamics simulation and protein engineering, single molecule AFM has provided rich information about the mechanical design of elastomeric proteins. This dissertation focuses on engineering proteins with novel mechanical properties and makes use of this new technique. It is demonstrated how a non-mechanical protein GB1, the B1 immunoglobulin (IgG) binding domain of protein G from Streptococcus, shows superb mechanical stability. We also investigated the effect of denaturant, guanidium hydrochloride (GdmCl), on the mechanical stability of GB1. It was found that the mechanical stability of GB1 decreases with the increase of GdmCl concentration. Using GB1 as a model system, we demonstrated two ways to enhance the mechanical stability of proteins: by metal chelation and by stabilizing protein-protein interactions. It is revealed that preferentially stabilizing the native state over the mechanical unfolding transition state of proteins is the key to achieve enhanced mechanical stability. We also showed two applications of engineered elastomeric proteins. One is the design of an artificial elastomeric protein with dual mechanical stability that can be regulated reversibly by protein-protein interactions. We introduced proline mutations to GB1 to make it mechanically labile and behave as entropic springs. Upon binding of the Fc fragment of IgG, the proline mutants of GB1 switched into a state of significant mechanical stability and can serve as shock-absorbers. The other application is the engineering of the first tandem modular protein based thermo-reversible hydrogel, which paves the way for engineering hydrogels with much improved physical properties that can be used as artificial extracellular matrices and tissue engineering materials.

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Attribution-NonCommercial-NoDerivatives 4.0 International

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