Baker, DavidNaylor, Donal2023-09-272023-09-272023Naylor_washington_0250O_26096.pdfhttp://hdl.handle.net/1773/50704Thesis (Master's)--University of Washington, 2023This study employs de novo protein design to augment protein mechanostability, a critical characteristic in bioengineering, biotechnology, and therapeutic development. The enhancement of protein mechanostability may find significant applications in areas such as modulating biomaterial elastic properties for a range of biomedical applications or controlling the stiffness of cellular environments to study the mechanobiology of cell differentiation. This work opens new avenues for the development of robust biomaterials and improved cellular therapeutics. Two design rounds were conducted, each employing computational design methods and subsequent experimental validations. The first round utilized inpainting and diffusion methods, revealing consistent results for inpainting-derived proteins and varied results for diffusion-derived ones. As assessed by atomic force microscopy, all designed proteins, despite varying success, fell short of matching or surpassing the mechanostability of the natural proteins which served as structural templates for their design. The second round, a targeted redesign of high rupture force proteins, led to less consistent results overall but one standout protein showed significant increase in rupture force, accompanied by notable structural changes that confirmed an underlying mechanistic hypothesis at the core of protein mechanostability. This work underscores the potential of de novo protein design in enhancing protein mechanostability, paving the way for applications requiring stable proteins.application/pdfen-USnoneBiochemistryComputational chemistryBiological chemistryThesis