Hydrogenase enzymes capable of catalyzing proton reduction to produce H2 have generated a considerable interest due to increasing motivation in finding sustainable carbon free energy sources. A considerable amount of research has been focused on producing synthetic structures mimicking the hydrogenase catalytic site, but the activity seen in hydrogenase enzymes in aqueous near neutral pH has yet to be replicated. It is now clear that the protein structure surrounding the H-cluster enables the high activity by fine tuning characteristics of the catalyst, but the structure and complexity of hydrogenase enzymes makes it difficult to predict exactly how the secondary coordination sphere affects catalysis. This work looks at incorporating both synthetic molecular catalysts and hydrogenase mimics into peptide scaffolds to improve the activity for photo-driven H2 production in aqueous solutions. The first chapter of this dissertation shows a de novo heme binding peptide improving the activity of cobalt protoporphyrin IX (CoPPIX) upon coordination inside a four-helix bundle. The peptide bound CoPPIX exhibited a 5.5-fold increase in anaerobic and an 8.3-fold increase in aerobic activity compared to free CoPPIX, while also showing dramatic increases to stability and solubility. In the second chapter, this work is expanded by using a randomly mutated cytochrome b562 library to identify beneficial attributes for downstream implementation of an ideal coordination site. A high-throughput assay was developed to measure H2 production using WO3/Pd deposited on a glass plate for a colorimetric first-pass screen. This assay successfully measured H2 production from CoPPIX bound cytochrome b562 in the periplasm of cells and identified a possible mutant showing 70% more H2 production compared to the wildtype. The third chapter incorporated a hydrogenase mimic into a four-helix bundle using a semi-synthetic strategy yielding a 3-fold increase in activity due to catalyst encapsulation. The method created will allow for easy modifications to the synthetic catalyst or peptide sequence in future work. The systems developed in this work were designed to facilitate the identification and implementation of beneficial characteristics for future development of an optimal secondary coordination sphere for a peptide bound molecular catalyst.

The use of enzyme-catalyst interfaces is underexplored in the field of biocatalysis, particularly in studies on enabling novel reactivity of enzymes. For this thesis, the HaloTag® protein tagging platform was proposed as a bioconjugation method for a pinacol coupling reaction using lipases, as a model for novel reactivities proceeding via ketyl radical intermediates and hydrogen-bonding-facilitated redox attenuation. After an initial lipase screening of 9 lipases, one lipase (Candida rugosa) was found to perform the pinacol coupling of p-anisaldehyde under standard conditions (fluorescein and 530nm light, 3% yield). Based on a retrosynthetic analysis for the photocatalyst-incorporated HaloTag® linker, the intermediates haloamine 1 and aldehyde 6 were synthesized. Further experiments are underway or planned to complete linker synthesis and conduct pinacol coupling experiments with a bioconjugated system. This project underscores the promising biocatalytic promiscuity of lipases for performing reactions proceeding through ketyl radical intermediates, as well as the underdeveloped potential of incorporating bioengineering principles like bioconjugation into biocatalysis to overcome kinetic barriers to electron transfer and optimize biocatalytic reactions.

Heliobacteria are an anaerobic phototroph that require carbon sources such as pyruvate, <br/>lactate, or acetate for growth (Sattley, et. al. 2008). They are known for having one of the <br/>simplest phototrophic systems, the central component of which is a Type I reaction center (RC) <br/>that pumps protons to generate the electrochemical gradient for making ATP. Heliobacteria <br/>preform cyclic electron flow (CEF) with the RC in the light but can also grow chemotropically in <br/>the dark. Many anaerobes like heliobacteria, such as other members of the class Clostridia, <br/>possess the capability to produce hydrogen via a hydrogenase enzyme in the cell, as protons can <br/>serve as an electron acceptor in anaerobic metabolism. However, the species of heliobacteria <br/>studied here, H. modesticaldum have been seen to produce hydrogen via their nitrogenase <br/>enzyme but not when this enzyme is inactive. This study aimed to investigate if the reason for <br/>their lack of hydrogen production was due to a lack of an active hydrogenase enzyme, possibly <br/>indicating that the genes required for activity were lost by an H. modesticaldum ancestor. This <br/>was done by introducing genes encoding a clostridial [FeFe] hydrogenase from C. thermocellum<br/>via conjugation and measuring hydrogen production in the transformant cells. Transformant cells <br/>produced hydrogen and cells without the genes did not, meaning that the heliobacteria ferredoxin <br/>was capable of donating electrons to the foreign hydrogenase to make hydrogen. Because the <br/>[FeFe] hydrogenase must receive electrons from the cytosolic ferredoxin, it was hypothesized <br/>that hydrogen production in heliobacteria could be used to probe the redox state of the ferredoxin <br/>pool in conditions of varying electron availability. Results of this study showed that hydrogen <br/>production was affected by electron availability variations due to varying pyruvate <br/>concentrations in the media, light vs dark environment, use acetate as a carbon source, and being <br/>provided external electron donors. Hydrogen production, therefore, was predicted to be an <br/>effective indicator of electron availability in the reduced ferredoxin pool.

Coronavirus disease 2019 (COVID-19), an illness caused by severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), has been responsible for significant social and economic
disruption, prompting an urgent search for therapeutic solutions. The spike protein of the virus
has been examined as an immunogenic target because of its role in viral binding and fusion
necessary for infection of host cells. Previous studies have identified a recombinant protein
(denoted as S1) that has been shown to potentially induce a neutralizing antibody response by
mimicking the structure of the SARS-CoV-2 spike protein. We have produced the S1 in plants
using agroinfiltration, a plant transformation technique whereby plasmid-containing
Agrobacterium tumefaciens is injected into Nicotiana benthamiana plants, resulting in transfer of
the desired gene from bacteria to plant cells. S1 was expressed to high levels within 5 days of
infiltration, and Western blot analysis showed recognition of the S1 by an anti-S1 antibody.
ELISA results exhibited increased binding activity to anti-S1 with increasing concentrations of
S1, indicating their specific interaction. This ongoing study will demonstrate the potential of a
plant-produced S1 as a vaccine, therapeutic, and diagnostic tool against COVID-19 that is not
only effective, but also cost-efficient and scalable in comparison to conventional mammalian cell
culture production methods.

The successful reduction of CO2 and protons by a light-induced cobalt porphyrin/cytb562 hybrid metalloenzyme in water is reported. Incorporation of the porphyrin into a protein scaffold results in increases in CO and H2 production over naked porphyrin. Rational point mutations to the CoPPIX binding site of cytb562 modulate production, indicating possible further improvements in catalytic activity.

Typical eukaryotic organelles use membranes formed by lipid bilayers in order to compartmentalize their functions within the cell. However, cells also contain membraneless organelles formed by intrinsically disordered proteins (IDPs) via liquid-liquid phase separation. The organelles form localized compartments that separate their contents from the environment.1 Here, this mechanism is used to generate artificial membraneless organelles that comprise a chemical reaction. An IDP, DEAD-box helicase (Ddx4), was bioconjugated to an enzyme, horseradish peroxidase (HRP), through the use of a bifunctional chemical linker, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), in order to examine if the enzyme could be incorporated in droplets and whether its activity would be affected. The conjugation of HRP-SMCC (43.4 kDa) to Ddx4 (25.6 kDa) was successful: SDS-PAGE analysis confirmed the presence of a product that was within the range of a full conjugate.

The highly specialized telomerase ribonucleoprotein enzyme is composed minimally of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) for catalytic activity. Telomerase is an RNA-dependent DNA polymerase that syntheizes DNA repeats at chromosome ends to maintain genome stability. While TERT is highly conserved among various groups of species, the TR subunit exhibits remarkable divergence in primary sequence, length, secondary structure and biogenesis, making TR identification extremely challenging even among closely related groups of organisms.

A unique computational approach combined with in vitro telomerase activity reconstitution studies was used to identify 83 novel TRs from 10 animal kingdom phyla spanning 18 diverse classes from the most basal sponges to the late evolving vertebrates. This revealed that three structural domains, pseudoknot, a distal stem-loop moiety and box H/ACA, are conserved within TRs from basal groups to vertebrates, while group-specific elements emerge or disappear during animal TR evolution along different lineages.

Next the corn-smut fungus Ustilago maydis TR was identified using an RNA-immunoprecipitation and next-generation sequencing approach followed by computational identification of TRs from 19 additional class Ustilaginomycetes fungi, leveraging conserved gene synteny among TR genes. Phylogenetic comparative analysis, in vitro telomerase activity and TR mutagenesis studies reveal a secondary structure of TRs from higher fungi, which is also conserved with vertebrates and filamentous fungi, providing a crucial link in TR evolution within the opisthokonta super-kingdom.

Lastly, work by collabarotors from Texas A&M university and others identified the first bona fide TR from the model plant Arabidopsis thaliana. Computational analysis was performed to identify 85 novel AtTR orthologs from three major plant clades: angiosperms, gymnosperms and lycophytes, which facilitated phylogenetic comparative analysis to infer the first plant TR secondary structural model. This model was confirmed using site-specific mutagenesis and telomerase activity assays of in vitro reconstituted enzyme. The structures of plant TRs are conserved across land plants providing an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

Continuing and increasing reliance on fossil fuels to satisfy our population’s energy demands has encouraged the search for renewable carbon-free and carbon-neutral sources, such as hydrogen gas or CO2 reduction products. Inspired by nature, one of the objectives of this dissertation was to develop protein-based strategies that can be applied in the production of green fuels. The first project of this dissertation aimed at developing a controllable strategy to incorporate domains with different functions (e. g. catalytic sites, electron transfer modules, light absorbing subunits) into a single multicomponent system. This was accomplished through the rational design of 2,2’-bipyridine modified dimeric peptides that allowed their metal-directed oligomerization by forming tris(bipyridine) complexes, thus resulting in the formation of a hexameric assembly.

Additionally, two different approaches to incorporate non-natural organometallic catalysts into protein matrix are discussed. First, cobalt protoporphyrin IX was incorporated into cytochrome b562 to produce a water-soluble proton and CO2 reduction catalyst that is active upon irradiation in the presence of a photosensitizer. The effect of the porphyrin axial ligands provided by the protein environment has been investigated by introducing mutations into the native scaffold, indicating that catalytic activity of proton reduction is dependent on axial coordination to the porphyrin. It is also shown that effects of the protein environment are not directly transferred when applied to other reactions, such as CO2 reduction.

Inspired by the active site of [FeFe]-hydrogenases, the second approach is based on the stereoselective preparation of a novel amino acid bearing a 1,2-benzenedithiol side chain. This moiety can serve as an anchoring point for the introduction of metal complexes into protein matrices. By doing so, this strategy enables the study of protein interactions with non-natural cofactors and the effects that it may have on catalysis. The work developed herein lays a foundation for furthering the study of the use of proteins as suitable environments for tuning the activity of organometallic catalysts in aqueous conditions, and interfacing these systems with other supporting units into supramolecular assemblies.

Redox enzymes represent a big group of proteins and they serve as catalysts for

biological processes that involve electron transfer. These proteins contain a redox center

that determines their functional properties, and hence, altering this center or incorporating

non-biological redox cofactor to proteins has been used as a means to generate redox

proteins with desirable activities for biological and chemical applications. Porphyrins and

Fe-S clusters are among the most common cofactors that biology employs for electron

transfer processes and there have been many studies on potential activities that they offer

in redox reactions.

In this dissertation, redox activity of Fe-S clusters and catalytic activity of porphyrins

have been explored with regard to protein scaffolds. In the first part, modular property of

repeat proteins along with previously established protein design principles have been

used to incorporate multiple Fe-S clusters within the repeat protein scaffold. This study is

the first example of exploiting a single scaffold to assemble a determined number of

clusters. In exploring the catalytic activity of transmetallated porphyrins, a cobalt-porphyrin

binding protein known as cytochrome c was employed in a water oxidation

photoelectrochemical cell. This system can be further coupled to a hydrogen production

electrode to achieve a full water splitting tandem cell. Finally, a cobalt-porphyrin binding

protein known as cytochrome b562 was employed to design a whole cell catalysis system,

and the activity of the surface-displayed protein for hydrogen production was explored

photochemically. This system can further be expanded for directed evolution studies and

high-throughput screening.

All organisms need to be able to sense and respond to their environment. Much of this process takes place via proteins embedded in the cell membrane, the border between a living thing and the external world. Transient receptor potential (TRP) ion channels are a superfamily of membrane proteins that play diverse roles in physiology. Among the 27 TRP channels found in humans and other animals, TRP melastatin 8 (TRPM8) and TRP vanilloid 1 (TRPV1) are the primary sensors of cold and hot temperatures, respectively. They underlie the molecular basis of somatic temperature sensation, but beyond this are also known to be involved in body temperature and weight regulation, inflammation, migraine, nociception, and some types of cancer. Because of their broad physiological roles, these channels are an attractive target for potential therapeutic interventions.

This dissertation presents experimental studies to elucidate the mechanisms underlying TRPM8 and TRPV1 function and regulation. Electrophysiology experiments show that modulation of TRPM8 activity by phosphoinositide interacting regulator of TRP (PIRT), a small membrane protein, is species dependent; human PIRT attenuates TRPM8 activity, whereas mouse PIRT potentiates the channel. Direct binding experiments and chimeric mouse-human TRPM8 channels reveal that this regulation takes place via the transmembrane domain of the channel. Ligand activation of TRPM8 is also investigated. A mutation in the linker between the S4 and S5 helices is found to generally decrease TRPM8 currents, and to specifically abrogate functional response to the potent agonist icilin without affecting icilin binding.

The heat activation thermodynamics of TRPV1 are also probed using temperature-controlled electrophysiology. The magnitude of the gating enthalpy of human TRPV1 is found to be similar to other species reported in the literature. Human TRPV1 also features an apparent heat inactivation process that results in reduced heat sensitivity after exposure to elevated temperatures. The work presented in this dissertation sheds light on the varied mechanisms of thermosensitive TRP channel function and regulation.