NMR-Guided Directed Evolution

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




Ivan Korendovych


Catalytic amyloids;Directed evolution;Peptide fibrils;Protein engineering

Subject Categories

Chemistry | Physical Sciences and Mathematics


The efficiency, selectivity, and sustainability benefits offered by enzymes have been amazed chemists to consider biocatalytic transformations to complement traditional synthetic routes. With an increased demand for efficient and versatile synthetic methods combined with novel discovery of engineering tools have prompted innovations in biocatalysis, especially the development of new enzymes for desired transformations. Over the past three decades, there has been an impressive expansion of catalytic repertoire of enzymes to include practically useful transformations not known to the biological world. The continuing discovery and enhancement of novel enzymatic functions have provided an enormous opportunity to connect the chemistry of the biological world to that invented by humans for synthetic purposes. This thesis primarily focuses on the discovery and development of a novel tool for protein engineering to guide directed evolution. Even in small proteins the potential of directed evolution is inherently limited by the sequence space. Although many computational strategies have been developed to limit the search space, all of them heavily rely on a priori structural, functional, and/or bioinformatic information, which may simply not be available for the protein of interest. We show for the first time that mutagenic hot spots can be identified proactively by simple NMR experiment. Guided by NMR method, we have been successful to evolve two unrelated protein scaffolds into Kemp eliminases with a notable improvement in enzymatic efficiency over the starting design. From the practical point of view, our results drastically expand the possibilities for evolving enzymes to promote new chemical transformations. From the fundamental standpoint, our results for the first time prospectively validate the recent paradigm-shifting work that links protein dynamics and evolution of enzyme function. Minimalist, de novo designed peptide fibrils have previously been shown to self-assemble to form amyloid-like catalysts for hydrolysis of activated esters. This inspired us to explore whether catalytic amyloids can replicate the native functions of natural enzymes. In this thesis, several rationally designed seven-to-nine amino acid-residue peptide sequences have been reported, which promote CO2 hydration in presence of zinc. The most active peptide promoted CO2 hydration with a specific activity that is on par with some of the naturally occurring carbonic anhydrases: our results show for the first time that a synthetic system could beat an enzyme in its own game. This observation notably contributes to the debate that functional enzymes were evolved from short, self-assembling peptides. In addition, catalytic amyloids have an additional advantage over the enzymes that they can handle harsh conditions to help promote CO2 fixation and capture.


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