Computational Protein Design and Analysis: From Nature to Novel Light-Harvesting Systems

Addressing the global food and climate crises necessitates a deeper understanding of photosynthetic processes and the development of innovative photosynthetic biotechnologies. A key, yet largely untapped, area in photosynthetic biotechnology is the design of organisms capable of capturing a broader spectrum of photosynthetically active radiation through de novo designed proteins. While most organisms are limited to distinct spectral niches, it is theoretically feasible to engineer organisms to harvest the entire solar spectrum. This endeavour requires easily expressible and spectrally tuneable light-harvesting molecules, for which bilins are an exemplary candidate. Chapter 3 of this thesis addresses the transmembrane chlorophyll biosynthesis enzyme, chlorophyll synthase, as a demonstration of applying novel protein structure analysis tools to a transmembrane chlorophyll protein. Chapters 4 through 6 will then detail the design of novel bilin-binding proteins, and assembly of these proteins into a phycobilisome-like structure to achieve a light-harvesting protein with a relatively large spectral span. Specifically, chapter 4 outlines the use of the novel protein backbone generation tool, RFDiffusion All-Atom, and the sequence design tool, LigandMPNN, to generate de novo bilin binding proteins based on the CXRD motif of light-harvesting bilin binding proteins. Chapter 5 shows the use of ESMFold and LigandMPNN to design interfaces between monomeric bilin binding subunits to generate a mini phycobilisome-like structure, the structure of which was solved with cryo-EM to 3.3 Å resolution. Chapter 6 uses a LigandMPNN-AlphaFold-Rosetta relax pipeline to redesign one of the biliproteins designed in chapter 4 to improve its stability and fluorescence properties. This thesis contributes to the fundamental understanding of photosynthetic protein engineering and lays the groundwork for developing next-generation bio-solar energy conversion systems.