DescriptionThis dissertation describes the work towards phosphorylation-based proteolytic logic circuitry, computational design and structure of flexible protein assemblies, and the computational strategies and methods of photocontrollable proteins with photoswitchable molecules.
The Introductory chapter describes the current state of computational protein design with its regards to designing flexible protein assemblies, the work towards enzyme design and its limitations, and the design of proteins with noncanonical amino acids. It continues as a preface for the importance and use for the development of protease-based biological circuitry, adaptable protein assemblies, and light-controllable proteins.
The second chapter describes the work towards a highly dynamic phosphorylation-based split-protease switch and its integration into current state-of-the art proteolytic switches. Through amino acid substitutions and both experimental and computational optimizations, we have produced a switch with a wide range of sensitivities for use as an in vitro or in vivo device. Further, we have integrated its functionality into similar devices, creating a novel phosphorylation and rapamycin detecting AND and OR Gate.
The next chapter details the computational strategy and design of a modular protein assembly composed of previously characterized protein moieties. Leveraging evolved protein-protein interactions and symmetry, we designed multiple protein pairs capable of rapid assembly once mixed. From our atomistic models, we develop a coarse-grain model on which we ran Brownian dynamics simulations, demonstrating topologies and structures we later visualized under microscopy.
The fourth chapter reviews the current state-of-the-art in computational design of photocontrollable proteins. This focused review on design primarily covers the azobenzene moiety and its derivatives, alongside various computational strategies in which photoswitchable proteins have been designed and analyzed.
The final chapter details a pair of computational methods for the design photocontrollable proteins with azobenzene-derived non-canonical amino acids. One method details a design protocol of protein-protein interactions with an azobenzene-based crosslinker, with corresponding results, while the second describes the design of enzyme control with azobenzene-based non-canonical amino acids.
Together, this work comprises a collection of developments in the fields of computational biology and protein design.