Doctor of Philosophy, Miami University, 2022, Chemistry and Biochemistry

This work investigated the impact of polymer conjugation on the activity and stability profile of a lipase enzyme. An industrially important enzyme, Candida antarctica lipase B (CalB), was conjugated with different functional polymers, including those with hydrophilic, hydrophobic, cationic, and anionic characters. The study demonstrated enhanced CalB activity when conjugated with hydrophilic and cationic polymers against a hydrophobic substrate, p-nitrophenol palmitate (p-NPP), due to the possible non-covalent interaction between the polymer and the substrate. On the contrary, conjugating with hydrophobic polymer showed significant activity inhibition, likely due to binding to the catalytic site. Hence, CalB conjugation with this diverse range of polymers demonstrated the significance of polymer composition, polymer-substrate interactions, and protein-polymer interactions in deciding protein catalytic performance. With this in mind, this dissertation also showed the manipulation of these protein-polymer interactions to tune a protein's catalytic performance in different pH environments. In addition, this thesis also explored work focusing on combating coronavirus. In one project, the receptor-binding domain of spike protein of SARS-CoV-2 was modified with different polymers to explore the sensitivity of interactions between the modified RBD and ACE2 protein, which may be beneficial in the downstream development of inhibitors targeting the RBD-ACE2 interaction. In another project, spike binding peptide (SBP1) was immobilized into a covalent crosslinked network system to develop a novel peptide-functionalized network to capture the spike protein of coronavirus. The development of such functionalized network could provide us materials with potential antiviral properties, which can provide us the opportunity to mitigate coronavirus spread. Hence, this thesis work highlighted different functional consequences of protein-polymer interaction ranging from tuning activity a (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2023, Food Science and Technology

Consumer demands for ethically sourced and environmentally friendly food products have led to development efforts to replace animal-based proteins with plant-based alternatives. However, plant-based protein ingredients can be limited by their functional and sensory properties, and thus processing techniques to improve these properties must be explored. Enzymatic hydrolysis has been suggested to improve key functional properties, such as solubility, but the research methodology in this area is questionable and hydrolysis does not fully address sensory deficits in plant-protein ingredients, notably bitterness. In this dissertation, commercial extruded snack products containing soy protein hydrolysates were used as a model to quantify bitterness and test the viability of reformulation with flavor maskers or alternatively processed proteins to improve off-flavor. This study revealed that commercial flavor maskers are not effective at reducing bitterness in products containing soy hydrolysate, but soy protein hydrolysates made by different manufacturers with different processing methods proved a viable replacement with improved off-flavor. In search for conclusive evidence that enzymatic hydrolysis results in improved functionality, a review of literature was conducted. This review concluded that enzymatic hydrolysis process may result in the formation of insoluble aggregates, which in most studies are removed by centrifugation or filtration during processing, thus artificially increasing the reported solubility values for plant-protein hydrolysates. The phenomenon of hydrolysis induced aggregation was confirmed for protein isolates from soy and as well as a pulse protein alternative to soy, chickpea, which were hydrolyzed by Flavourzyme and Alcalase. Analysis of physical and structural properties of the hydrolyzed proteins revealed that hydrolysis led to protein destabilization, causing hydrogen-bond mediated aggregation during thermal enzyme inactivation. The knowledge (open full item for complete abstract)

Doctor of Philosophy, Miami University, 2023, Chemistry and Biochemistry

Proteins can misfold in a crowded cellular environment due to physiological, pathological, and environmental stress. The fate of such misfolded proteins to either refolding by chaperones or degradation by the proteasome is determined by a protein-protein complex comprising the molecular chaperone Hsp70 and the E3 ubiquitin ligase CHIP. The decision to shunt proteins to the refolding or degradative pathways is known as the triage decision, and sufficient mechanistic details are still lacking. We hypothesized that the ensemble of different folding states of a single misfolded protein could be represented by a library of model proteins, each with a varying degree of thermodynamic stability. We termed the novel library of proteins we devised as “Folding Sensors”. Structure-guided protein engineering, computational modeling, biophysical techniques, and structural biology were utilized in this process. According to thermal denaturation data, the spectrum of folding sensors showed varying degrees of quantitative thermal stability at a given temperature. The crystal structure of the most stable folding sensor closely resembled the structure of the TPR domain, which served as the initial basis for the folding sensors, while also forming a disulfide bond as expected. We further speculate that Hsp70 recognized this variant as a natively folded state protein due to its' low affinity towards Hsp70. We further discovered that Hsp70 tightly binds to the moderately stable folding sensors. We further observed that CHIP differentially ubiquitinated substrates based on the thermal stability and the folding state. The process was chaperone dependent. The folding sensor mimicking a natively folded protein was ubiquitinated the least while the variants with more unfolding showed more ubiquitination. Altogether we developed folding sensors with varying thermodynamic stability to serve as a model to study chaperone-mediated triage by the Hsp70/CHIP. This preliminary study provided insights (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2023, Food Science and Technology

Ultra-shear technology (UST) presents a promising way to preserve stable protein liquid foods. This dissertation investigated the effect of pressure, shear, temperature, and their interactions during UST on quality attributes and microbial inactivation. First, the impact of UST process parameters on milk was evaluated by subjecting to UST at 400 MPa/35 and 65°C. Untreated, high-pressure processed (HPP; 400 MPa/40°C/0 and 3 min) and thermal treated (72°C/15 s) milk served as controls. HPP did not cause particle size reduction but increased the viscosity up to 3.08 mPa·s compared with 2.68 mPa·s for untreated milk. 35°C UST reduced the particle diameter from 3511.76 nm (raw milk) to 291.45 nm and prevented creaming. To compare microbial safety of UST and HPP, cell suspension of Lactobacillus brevis (1.6×1010 CFU/mL) and spore suspension of Bacillus cereus (3.2×108 CFU/mL) were subjected to UST at 400 MPa/40 and 70°C. Thermal (0.1 MPa-70°C-0/5min) and HPP (400 MPa-40 and 70°C-0/5min) experiments were performed. HPP at 400 MPa /70°C/0 min resulted in 8.4 and 2.3 log reductions of L. brevis and B. cereus, respectively. After 70°C UST, L. brevis and B. cereus reduced by 7.1 and 1.6 logs, respectively. Different valve geometries, viz., ultra-shear valve, needle valve, and tubular valve, were evaluated. Ultra-shear valve produced inactivation of 2.0 and 7.1 logs for L. brevis at 40 and 70°C respectively, which was higher than needle valve and tubular valve. The ability of UST to obtain homogenous plant-dairy protein blends with varying protein and fat levels was evaluated. Milk-pea dispersions of 3 protein ratios viz., milk:pea 1:0.5, 1:1, and 1:3 were prepared. UST was performed at 400 MPa/40 and 70°C. HPP at 400 MPa/25±2°C/0 min and thermal treatment at 72°C/15s were conducted. Pea-dairy dispersions with 3 fat levels - Raw milk+Pea, Skim milk+Pea, Cream+Pea protein were prepared and UST-treated at 400 MPa/70°C. Discovery HR3-hybrid rheometer was used to determine visc (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2018, Molecular Medicine

Rhodopsin mutations are the leading cause of autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease. Rhodopsin is the light receptor in rod photoreceptors that plays a central role in phototransduction and rod photoreceptor health. A majority of rhodopsin mutations cause misfolding and aggregation of the apoprotein opsin. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood; and the pathogenesis of adRP caused by misfolded opsin remains unclear. Physical interactions between wild-type opsin and misfolded opsin mutants have been proposed to underlie the autosomal dominant phenotype in the literature. The misfolding mutants have been characterized biochemically and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand rhodopsin aggregation, disease pathogenesis, and evaluate therapeutic strategies. To better understand misfolded rhodopsin aggregation, a Forster resonance energy transfer (FRET) assay was developed to monitor interactions between fluorescently tagged opsins expressed in live cells. The FRET detergent assay employed discriminated between properly folded opsin oligomers and misfolded opsin aggregates. Using the FRET method established, Complete and partial misfolding mutants were characterized to reveal variability in aggregation properties, showing the current rhodopsin mutant classification system to incompletely describe the possible rhodopsin interactions. The complete misfolding mutants examined behaved similarly: forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and not responding to the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. The partial misfolding mutants reacted similarly to the pharmacological chaperone 9-cis retinal, displaying i (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2018, Physiology and Biophysics

Single-chain insulin (SCI) analogs have long provided a valuable model for structure-function studies of insulin. Recently, a biologically active SCI containing a six-residue connecting (C) domain (inserted between the B- and A chains of two-chain insulin) demonstrated marked resistance to thermal degradation. Such a design has potential to circumvent the cold-storage requirements of current insulin therapeutic formulations which hinder their distribution, storage, and use in developing regions. An understanding of how this SCI scaffold can be engineered to confer desired biophysical and pharmacological properties would enable rational design, development, and clinical implementation of SCIs. This Thesis leverages principles of protein folding, structure, dynamics, stability and function to modulate SCI properties. Following an introduction of insulin analog design with focus on how SCIs could address a global need for ultra-stable formulations (Chapter 1), a synthetic single-chain [des-B29,des-B30]-insulin platform was utilized to model an oxidative intermediate in the folding of wild-type insulin. This system enabled comparative studies of an unstable TyrB16->Pro variant, which exhibited a severe folding defect (Chapter 2). Chapters 3-4 contain studies of two novel biologically active SCIs: SCI-a and SCI-b. These analogs exhibited remarkable resistance to thermal inactivation; SCI-a had native-like duration of hormone action in diabetic rats. Collaborative X-ray crystallographic studies of SCI-a revealed a novel zinc-free T6 hexamer (Chapter 3) whereas NMR and molecular dynamics studies of SCI-b provided insight into its mechanisms of enhanced stability and receptor binding (Chapter 4). Studies of variant SCIs containing amino-acid substitutions at position A14 (Chapter 5) suggested that the reverse hydrophobic effect, a general biophysical principle uncommonly observed, was operative. This principle could be leveraged to enhance SCI resistance to chemical degrada (open full item for complete abstract)

Doctor of Philosophy, University of Toledo, 2008, Chemistry

DNA replication and repair is one of the most important cellular processes, since preserving the integrity of the DNA genome is essential to all forms of life. Many proteins are involved in the DNA replication process, and their interaction ensures that the DNA is duplicated and repaired in a coordinated and efficient manner. Bacteriophage T4 is a very good model to study DNA replication, since it encodes all the proteins required at the replication fork, proteins which have been extensively characterized. However, how these proteins interact and coordinate the replication process is still largely unknown. One of these interactions that appears to govern the rate and efficiency of the lagging strand synthesis occurs between the 5' to 3' exonuclease RNase H and the single-stranded DNA-binding 32 protein. The interaction between these two proteins is the focus of this work. RNase H and the 32 protein, as well as a number of mutants and truncations, were cloned, expressed and purified. These proteins were then used to form different variants of the RNase H + 32 protein complex, which were characterized through biophysical and structural studies. A crystal structure was obtained for the RNase H + 32-B truncation. This structure, along with the results obtained from the biophysical experiments, provides valuable information on how these two proteins interact to coordinate the lagging strand DNA replication. c Finally, the study of the interaction between RNase H and the 32 protein from bacteriophage Rb 69, a phage related to bacteriophage T4, was also initiated.

Doctor of Philosophy, University of Toledo, 2008, Chemistry

The organization and coordination of DNA replication machinery at the replication fork is important for accurate, efficient DNA synthesis in all organisms. The initial organization of the replication fork is vital for initiating lagging strand replication, while the regulation of proteins involved in Okazaki fragment processing is important for generating a complete daughter DNA strand. These DNA replication and repair proteins recognize DNA in a structure-specific manner, thus the recognition of these particular DNA structures promote the formation of certain protein-DNA and protein-protein complexes that are essential for DNA replication and repair to occur. Organisms such as the Bacteriophage T4 (T4) and Aeropyrum pernix (Ape) are model systems for use in the study of binary and ternary complexes that form during DNA replication and repair. Reassembling the replication fork would allow the determination of the mechanism used to synchronize replication on both the leading and lagging strands. Proteins (helicase assembly protein and single-stranded DNA binding protein) from T4 were used to study the complexes involved in initiation of lagging strand replication. The protein-protein interactions between the helicase assembly protein (59 protein), single-stranded DNA binding protein (32 protein), and truncations of the 32 protein have been investigated (ITC, DSC, DLS, native gels, crystallography). 59 protein had a moderate interaction with 32 protein (KD = 3.7 μM) and with 32-B (KD = 3.6 μM). DNA-protein interactions between the 59 protein and fork DNA substrate (with and without 32 protein) have been studied (fluorescence). X-ray data was collected on a truncation of the 32 protein (32-B). Models of the 59 protein-32-B complex have been elucidated (SAXS, SANS). Ape proteins (proliferating cell nuclear antigen, DNA polymerase B, DNA ligase, and flap endonuclease-1) were characterized to study Okazaki processing. Subunits (Ape0162, Ape0441, and Ape2182) from the het (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2011, Pharmacy

The objective of this dissertation was to design small molecule drug candidates for different disease targets by understanding the energetics and dynamics of their binding protein/enzyme/receptor partners. Protein-protein interactions are intrinsic to virtually every cellular process such as transcription regulation and signal transduction, and inappropriate protein-protein interactions may lead to human diseases such as cancer. These interactions commonly rely on a few key residues (“hot spot residues”) and single point mutations of “hot spot” residues could cause disruption of theses protein complexes. Hence, small molecule antagonists, which interfere mainly with critical amino acid contacts, could have significant outcomes on disruption of binding equilibrium of protein/protein complex. By utilizing this concept, we have designed IL-6 inhibitors to disrupt interactions between IL-6 and gp130 (chapter 2, 3 and 4). Traditional drug discovery begins by identifying the protein target related to disease and finding a lead compound, a potential drug that bears the desired physical and biological features from a library of known chemical compounds. This limits the search space from the beginning and makes new drug discovery (new chemical structure) a very difficult task. However, as the cellular and molecular mechanisms behind many diseases are increasingly understood, new avenues for rational drug development emerge. This can be complemented by structure based drug design methods that utilize three dimensional structure of the target protein. Recent advancements in computational techniques and hardware have helped researchers using in silico methods to a speedy lead identification and optimization. Large virtual chemical libraries are now available for screenings that lead to discovery of small molecule inhibitors of HIV-IN and LEDGF interactions (Chapter 5 and 6). Protein/receptor structures are not static in the body; they often bear plasticity by accommodating ch (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2008, Biophysics

The most controversial step in the study of the mechanism of action of insecticidal crystal toxins is that of insertion of the toxin into insect brush border membranes. Conflicting models of insertion of toxin can be categorized into two groups; ones that propose that only certain alpha helices of domain I insert into insect brush border membrane vesicles (BBMV) (Umbrella, Penknife and Serial Receptor Binding models) and others that propose that most of the toxin inserts into BBMV (Aronson, Buried Dragon and Unchanged Structure models). Protease protection studies of cysteine mutations from all domains of the toxin showed protection of most of the toxin (a 60 kDa form) similar to the wild type Cry1A toxin, when inserted into insect brush border membranes. Studies on steady state fluorescence measurements of these cysteine residues when bound to artificial vesicles or natural brush border membrane vesicles (BBMV), and fluorescence energy transfer measurements in labeled artificial vesicles suggested that residues from all the domains of the toxin inserted into the membrane. Residues in the loop 2 of Domain II of the toxin that played a vital role in the insertion of the toxin into insect BBMV were identified. Examination of receptor binding and insertion of mutants of these residues have shown that insertion of Cry1Ab into the membrane is dependent on specific residues at positions in this loop. Absence of phenylalanine or a closely related amino acid such as tryptophan at position 371 allowed initial binding of the toxin to the receptor but compromised the insertion of the toxin into insect membrane, thereby confirming that irreversible binding step of the toxin-BBMV interaction is the critical step in the mode of action of the toxin where Domain II is a major candidate mediating the step. Fluorescence blue shift studies into artificial and natural membranes also indicated a difference in the partitioning of the toxin into artificial and natural membranes; thereby s (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2006, Plant Biology

F-box protein-mediated proteolysis is crucial for the circadian clock progression. ZEITLUPE (ZTL) is the only F-box protein so far identified in plants that regulates the circadian clock function. Little is known about the mechanism by which it is involved in the regulation of the circadian clock. The purpose of this study has been to investigate how ZTL activity and expression is regulated, what the functions of the ZTL domains are, and what are the factors that genetically interact with ZTL to maintain the proper function of the circadian clock. In this study, we first characterized an Arabidopsis suspension culture that contains a fully functional clock. Then, we used this cell culture to analyze the expression of ZTL. We found that ZTL expression is regulated at posttranscriptional step and the degradation of ZTL is circadian phase-specific. The phase-specific degradation of an F-box protein provides an additional level of feedback regulation in the interlocked feedback regulatory loops of the circadian clock. The LOV domain of ZTL is believed to function in light sensing. Recent in vitro data also indicate that LOV domains from other proteins are able to self-dimerize. In this study, we provide additional evidences to show that LOV domain of ZTL can interact with full length ZTL in vivo. High levels of LOV expression in Arabidopsis plants result in dramatic reduction of endogenous ZTL protein likely accounting for the longer periods phenotype of the transgenic plants. We also show that LOV interact with FKF1 another ZTL family member that acts in the clock-controlled long day flowering pathway to promote flowering. Therefore, high levels of LOV is likely to inhibit FKF1 activity or to promote FKF1 protein degradation, leading to late flowering phenotype of the plants that overexpress LOV. From a genetic screen for mutants that enhance or suppress the ztl-1 long-period phenotype, we recovered seven ztl-1 enhancer lines. The initial mapping effort locates one of (open full item for complete abstract)

Doctor of Philosophy, Miami University, 2004, Chemistry and Biochemistry

Reactive oxygen species (ROS) are produced in human metabolic processes and uncontrolled ROS are detrimental to human health. In vitro chemical assays show that dietary tannins have potent ROS scavenging activity. My research is designed to use model systems to reveal interactions between tannins and factors (pH and protein) found in the human digestive tract where tannins work as biological antioxidants. Pentagalloyl glucose (PGG), bovine serum albumin (BSA), and NaIO4 were chosen as the model tannin, protein and oxidant, respectively. PGG was prepared from tannic acids via a methanolysis reaction. [14C]PGG was synthesized from [U-14C]-D-glucopyranose and tri-O-benzylgallic acid as a radiochemical tracer. When PGG was oxidized by NaIO4, the formation of different oxidation products was controlled by the reaction pH. PGG oxidation produced an o-semiquinone radical intermediate, which formed polymeric products at low pH (e.g. pH 2.1). The o-semiquinone radical ionized at pH > 5 and was labile to being further oxidized to o-quinone. Reaction pH similarly affected the formation of oxidation products of epicatechin16 (4→8) catechin (procyanidin) or epigallocatechin gallate (EGCG), suggesting that these three different tannins followed the same oxidation mechanism. When PGG was oxidized in the presence of BSA at various pH values, BSA promoted the formation of quinone but inhibited the formation of polymeric products. In addition to affecting PGG oxidation, BSA interacted with PGG under oxidizing conditions and formed oxidized PGG-BSA complexes. A radiochemical method was devised to quantitate oxidized PGG-BSA complexes. The complexes were treated with sodium dodecyl sulfate (SDS) to remove any non-covalently bound PGG and were co-precipitated with BSA by trichloroacetic acid (TCA) for radiochemical assessment. The molar ratio of PGG and BSA determined the solubility of oxidized PGG-BSA complexes. Soluble complexes were formed at low molar ratios (e.g. PGG/BSA=1). At hig (open full item for complete abstract)

Doctor of Philosophy, Miami University, 2025, Chemistry and Biochemistry

Chloroplasts are organelles involved in photosynthesis and require thousands of proteins to function. More than 95% of these proteins are encoded by the nucleus and synthesized in the cytosol as precursor proteins. The chloroplasts' double-membrane architecture necessitates the implementation of complex protein import mechanisms to facilitate the transportation of nuclear-encoded proteins to the thylakoid. Proper routing and sorting of thylakoid-targeted proteins is essential for assembling the photosynthetic apparatus within the thylakoid membrane during chloroplast biogenesis. Once proteins reach the chloroplast stroma, they employ one of four protein sorting mechanisms to target the thylakoid membrane or lumen. The chloroplast Twin Arginine Translocation (cpTat) system is an essential and evolutionarily conserved system that transports fully folded proteins across the thylakoid membrane in chloroplasts, as well as the cytoplasmic membrane of eubacteria and archaebacteria. The cpTat system is distinct from other protein transport systems because it utilizes the proton motive force as the sole energy source to transport proteins across the thylakoid membrane by forming a transient pore. Two key components of the system, cpTatC and Hcf106, facilitate the binding of the substrate signal peptide and the recruitment of the pore-forming component Tha4. As the mechanical features of this system are still largely unknown, understanding how these components interact will provide important clues for solving the transport mechanism of the cpTat system. The core component of the system, cpTatC, has a much longer N-terminal soluble domain compared to the predicted lengths for bacterial and algal TatC proteins. This dissertation focuses on the functional characterization of the N-terminal extension of the cpTatC protein through Cysteine (Cys) scanning disulfide crosslinking assays. We observed close contact between the N-terminal extension of cpTatC and the precursor mature do (open full item for complete abstract)

Doctor of Philosophy, Miami University, 2023, Chemistry and Biochemistry

Carboxy-terminus of Hsp70-interacting protein (CHIP) is an E3 ligase and a co-chaperone that plays a crucial role in protein quality control in eukaryotic cells. It promotes ubiquitination which is important in many cellular processes. However, misregulation in the ubiquitination cascade can lead to cellular dysfunction resulting in cancer and neurodegenerative diseases. Therefore, the ubiquitination cascade has become an attractive target for therapeutic interventions. Thus, the detection of ubiquitination is important to better understand the mechanism and explore the roles of cascade enzymes. Many efforts have been made in the field to detect ubiquitination using various techniques including fluorescence, spectrophotometry, chemiluminescence, and radioactive tracers. The most common method to detect ubiquitination is western blotting. However, western blotting is time-consuming, labor-intensive, and difficult to obtain fine-grained time resolution. We present the use of bio-layer interferometry (BLI) to rapidly assay ubiquitination in real time as a viable alternative to western blots. The newly developed assay tested the autoubiquitination of CHIP and CHIP-mediated ubiquitination of Hsc70. Using similar reagent concentrations to those used by western blots, the BLI assay produced instantaneous results with one-second time resolution. The assay opens avenues to explore other E3 ubiquitin ligase-mediated ubiquitination systems. In the second part of the dissertation, the focus was shifted to understanding the interactions of CHIP with Bcl2-associated athanogene 2 (Bag2) and CHIC2. In particular, Bag2 was identified to interact with CHIP to inhibit the ligase activity via its N-terminal domain (Bag2-NTD). However, the interaction between them was not fully understood. Based on the finding that leucine 57 and leucine 92 residues of Bag2-NTD are required for the interaction with CHIP, L57D, L92D, and L57D/L92D mutants were designed to extract st (open full item for complete abstract)

MS, University of Cincinnati, 2022, Arts and Sciences: Chemistry

Proteins are easily damaged by reactive oxygen species generated by oxidative stress. Advanced oxidative protein products (AOPP) and protein carbonylation are reliable biomarkers that can be used to estimate the degree of oxidant-mediated damage to proteins. In my current work, I focused on the identification of the detection limits for advanced oxidative protein products (AOPP) and protein carbonylation products generated by exposure to UVA and Fenton reaction-induced oxidative stress using E. coli cells as a model system. The generated AOPP and protein carbonylation were quantified in a dose-dependent manner following stress exposure. The commercially available AOPP assay kit used is a colorimetric-based method, while the protein carbonylation assay kit used has a fluorometric assay as the detection method. A chloramine standard curve was prepared to evaluate AOPP, with the detection limit found to be 0.5 µM. Protein carbonylation was determined with the fluorophore standard curve, and the detection limit was 0.63 nM. While UVA exposure generated both AOPP and carbonylated protein products, Fenton reaction-based oxidative stress produced carbonylated protein products but no AOPP indicating differential effects of various types of oxidative stress. Further studies can reveal the connections between the amounts and types of ROS generated by each stress and the resulting oxidative protein products.

Doctor of Philosophy (PhD), Ohio University, 2022, Mechanical and Systems Engineering (Engineering and Technology)

IRF3 dimerization is an important step during the innate immune signal transduction in the human body. However, abnormal dimerization of IRF3 has been linked to a number of diseases, including autoimmune diseases [46], diabetes [35], and cancer [73]. In this dissertation, I focus on understanding the molecular-level processes involved in the dimerization of two interferon regulatory factor 3 (IRF3) proteins. This investigation begins with a fundamental study in which I explore the role of water-mediated interactions in the association of the hydrophobic domains of the protein. I examine the role of hydrophobicity, flexibility, and density of the flexible side chains in systematic simulations using a highly simplified model. Next, I study molecular processes involved in IRF3 protein dimerization. For this study, I first calculate free energy along pathways associated with the association of two IRF3 chains and then determine the most stable dimeric configuration. I extend this investigation to determine how the stable dimeric configuration of IRF3 changes with its phosphorylation state by studying phosphorylation at six different serine residues. This work shows that phosphorylation of different residues of IRF3 protein can significantly change the stable dimeric configuration. Finally, I estimate the protein-ligand binding affinity via Markov state analysis applied to fully atomistic simulations. I apply this technique to study the binding affinities of six different ligands for the ATP binding site of Glycogen synthase kinase-3β (GSK-3β). The binding affinity prediction from Markov state analysis is in strong agreement with the experimental results. My MD simulations show that the ligands that have a higher propensity of attaining a compact conformation due to the stacking of their aromatic groups also show stronger inhibition of GSK-3β activity.

Master of Science (MS), Ohio University, 2022, Chemistry and Biochemistry (Arts and Sciences)

Apoptosis is a form of controlled cell death that occurs as a normal part of an organism's growth and development. Apoptosis is often initiated as a result of external or internal stresses that the cell cannot overcome and thus becomes committed to die. Defects in a cell's apoptosis machinery can result in uncontrolled cell division, leading to cancer or auto-immune disorders. Our work focuses on studying the interactions between pro- apoptotic (pro-death) and anti-apoptotic (pro-survival) proteins involved in the intrinsic apoptosis pathway. Specifically, we are interested in developing molecules that inhibit anti-apoptotic BCL2 family proteins in order to facilitate cell death in diseases that result from dysregulated apoptosis mechanisms. The primary area of my research involves studying the interaction between the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2. Bax interacts with Bcl-2 via its helical BH3 domain, causing Bax to remain inactivated. In times of cellular stress, activated Bax becomes unbound from Bcl-2 and initiates the release of cytochrome c from mitochondria, leading to cell death via apoptosis. Our lab recently developed a series of molecules based on the small protein scyllatoxin (ScTx) as inhibitors of the Bax:Bcl-2 interaction. Notably, synthetic ScTx can be modified to target anti-apoptotic Bcl-2 proteins by replacing natural amino acids within the ScTx helix with Bax BH3 residues that are important for Bcl-2 recognition. 4 My studies have included establishing screens to co-crystalize Bcl-2 or Bcl-2 (G145A) (a mutant of Bcl-2 that resists interactions with other BCL2 BH3 domains) with ScTx-based BH3 domain mimetics or truncated Bax BH3 domains. In addition, we have conducted isothermal titration calorimetry (ITC) studies with ScTx-Bax structural variants in complex with Bcl-2. These studies have helped to elucidate structural requirements and energetic properties for targeting anti-apoptotic Bcl-2 with ScTx-Bax BH3 (open full item for complete abstract)

Master of Science, Miami University, 2021, Chemistry and Biochemistry

The Twin-Arginine Transport (TAT) system is used in prokaryotes as well as eukaryotic chloroplasts to transport folded proteins across membranes. Recently, proteins homologous to TAT system component proteins have been discovered in the mitochondria. Both mitochondrial TatB (mtTatB) and mitochondrial TatC (mtTatC) have been identified and localized to the mitochondria. TatA, a component of chloroplast TAT, has not been confirmed. It is possible that TatA dual localizes to the chloroplast and the mitochondria. This work aims to confirm the mitochondrial location of mtTatB and mtTatC as well as identify TatA in the mitochondria

PhD, University of Cincinnati, 2021, Arts and Sciences: Chemistry

Protein quality control is one of the key cellular activities in any living cell, as it provides folding assistance or degradation mechanisms to prevent undesirable off-pathway reactions such as misfolding or aggregates using molecular chaperones. Bacterial Caseinolytic proteases (Clp) promote the degradation pathways by unfolding and translocating tagged substrate proteins (SPs) using powerful ring-shaped AAA+ (ATPases Associated with diverse cellular Activities) motor component with a narrow central pore and later deliver the unfolded polypeptide chain into the peptidase chamber for ultimate destruction into small peptide fragments. ATP fueled conformational transitions of subunits generate a repetitive mechanical unfolding force at the central pore loops and apply this force onto transient residues of SP that reside at the central pore and promote the unfolding and translocation mechanisms. Some aspects of these processes are addressed in experimental and computational studies. Nevertheless, the effects of mechanical anisotropies, non-native contacts, and topological features of SPs on allosteric cycle coupled Clp-mediated unfolding and translocation of SPs into peptidase chamber still remain unclear. To answer these questions, I presented the following three studies using an implicit atomistic model and performed Langevin dynamics simulations coupled with targeted molecular dynamics (TMD): (1) non-conserved allosteric cycles featured Clp nanomachine mediated remodeling mechanisms of three knotted SPs with 3.1, 5.2 and 6.1-knot types. These simulations reveal knot sliding along the contour of the knotted protein traversed through a rugged conformational landscape. Translocation hindrance of knotted SPs results from the synergetic coordination between knotted topologies and non-native contacts. In contrast to homopolymers, transmission of tension along the peptide chain occurs very differently. Disruption or formation of divergent type contacts along with backbone (open full item for complete abstract)

Master of Science, The Ohio State University, 2020, Chemistry

Rop protein is a four-bundle dimeric protein that assists RNAI to form a kissing complex with RNAII. By engagement in the kissing complex, the DNA polymerase will fail to replicate from the ColE1 origin and consequently, the copy number of the ColE1 plasmids will be downregulated. Rop's monomer is a helix-turn-helix structure with a tight turn containing a Schellman motif. We have studied the stability and activity of the Rop protein with different mutations in the loop section of the protein, residues 29-32, Leu-Asp-Ala-Asp (LDAD) in wild-type. The Arg55 residue appears to make an ionic contact with Asp32 in the wild-type. We engineered four variants, LDAG, R55Q_LDAN, LGGAD, and R55Q_LGDAD and compared their stability and activity with AV_Rop, as the native Rop with LDAD sequence in its loop region. To measure their stability, first, all these four variants were cloned into a T7 expression vector, and after expression and purification, their thermal stability was measured by CD thermal scanning. The result showed that the variant R55Q_LGDAD was the most stable variant. LGGAD and LDAG were more stable than wild-type one but less than R55Q_LGDAD. Finally, the variant R55Q_LDAN was the least stable variant. To measure the activity of the variants, all the variants were cloned into p15a vector and using green fluorescent protein (GFP) expression from a ColE1 plasmid, their activity was measured. We also ran a 1 ns MD simulation to validate our simulation system for future longer runs. We have plan to calculate RMSD, RMSF, DSSP, SASA, HB, Ramachandran plot, and free energy surface of all variants under longer runs.