Coordination Geometries in Metallobundles Enforcing Oxidative and Hydrolytic Catalysis and Designing Biomaterials for Use As Antimicrobials
Date of Award
Doctor of Philosophy (PhD)
Makhlynets, Olga V.
Biochemistry | Biochemistry, Biophysics, and Structural Biology | Life Sciences
De novo protein design permits discovery of intricate folds and functions emulating natural enzymes in simpler, yet robust model constructs. Helical bundles serve as premier scaffolds to incorporate diverse reactivities from oxidative, reductive, to hydrolytic transformations observed in much grander O2-utilizing metalloproteins such as radical-generating ribonucleotide reductases and catalases. One notable family of de novo proteins is the Due Ferri or DF series of four-helix bundles providing a dinuclear site for metal incorporation and are amenable for tailoring active site reactivity. As reactive oxygen species and radical-based intermediates are prevalent and necessary for steering life-essential processes through reactive radicals stabilized by metals, we were curious to understand how the nature and number of metal ions influence sequestration of such species. We investigated the influence metal has on semiquinone stabilization in DF bundles, a minimalist model representing active sites of more complex natural diiron and dimanganese proteins. Coordination sphere was modified in the original DF single chain version to incorporate a 2-His-1-carboxylate facial triad, a single metal binding motif, mirroring morphology of mononuclear non-heme enzymes. We discovered breaking symmetry of the metal coordination site leads to a stable construct with one site exhibiting tight affinity for metal, and this single metal binding construct Uno Ferro single chain stabilizes semiquinone radical anions equally efficient as a two-metal binding version. We envision a robust and easy to modify DFsc and UFsc family of proteins would be versatile tools for gaining mechanistic insights of metalloenzymes. One application of DF constructs we've pursued is to create a hydrolase bridging a heteronuclear metallosite. Phosphoester substrates were introduced to DFsc and UFsc to characterize hydrolase features. Defining the structural and functional properties of hydrolases in DF proteins would facilitate creation of catalysts for bioremediation purposes. We further apply minimalist design approaches to redesign natural proteins displaying no inherent catalytic properties (or showing marginal activity) for a chosen chemical transformation. Calmodulin and myoglobin were selected as model proteins to introduce enzymatic properties rationally designed using non-rigorous set of computational tools to modify the substrate pocket for evolution. We use HSQC-NMR to guide the directed evolution process to arrive at beneficial mutations which enhance reactivity of generated enzymes. Our minimalist design approach also demonstrates the practical application of our calmodulin designs to reveal an acid-base promoted catalysis in conversion of therapeutics to their metabolically active forms. Only a single precisely positioned amino acid is needed to promote conversion of an antirheumatic prodrug leflunomide bearing an isoxazole ring for proton abstraction to the active form teriflunomide. Furthermore, we dissected possible defense mechanisms microorganisms utilize to survive habitats under oxidative stress. A reported diiron catalase conserved exclusively in Mycobacterium tuberculosis (Mtb) was characterized to determine if the protein acts as a dimanganese catalase as we observed the coordination ligands are similar to nonheme catalases. Therefore, we sought to identify metal preference of this four-helix bundle and establish possible mechanisms utilized by Mtb to disproportionate toxic oxygen metabolites. Conversely, a possible defense strategy by humans to promote bacterial clearance in cases where invasive bacteria trigger human innate immune responses was examined. Hemoglobin is found to be antimicrobial generating cytotoxic radicals to sustain microbicidal action. We considered myoglobin involved in similar peroxidatic processes could be reactive in presence of pathogen associated molecular patterns, specifically lipopolysaccharides (LPS), to produce radical cations. Hence, myoglobin was characterized in a mixture of different proteases and LPS for peroxidase activation. Lastly, design of antimicrobial hydrogels is described. Our hydrogel design is based on self-assembly of peptides induced by silver or copper complexation and does not incorporate permanent crosslinking agents. Short peptides of alternating polar and nonpolar amino acids affixed by an unnatural amino acid pyridyl alanine which coordinate metal form a strong hydrogel. The hydrogel is self-healing and can be delivered in syringe format for application in wound healing therapy.
Yoon, Jennifer, "Coordination Geometries in Metallobundles Enforcing Oxidative and Hydrolytic Catalysis and Designing Biomaterials for Use As Antimicrobials" (2021). Dissertations - ALL. 1424.