Doctor of Philosophy, Case Western Reserve University, 2024, Pharmacology

Experimental determination of the structure of any given protein is an essential step in understanding the function of that protein. Historically, structural experiments are conducted in a targeted manner where a specific protein of interest is selected. We apply this targeted approach in this dissertation in the context of studying the inhibition of a mycobacterial lipid flippase. While this targeted approach can be very potent, the sample preparation workflows often require removing the protein from the context of its endogenous environment. Therefore, we also present a complementary untargeted strategy that we utilized to simultaneously solve multiple protein structures directly from human tissue samples. Our strategy bypasses traditional sample preparation protocols and largely preserves the extracted proteins in a native environment. An in silico data processing approach is then utilized to separate and identify different proteins from one sample. Specifically, we apply this methodology to soluble lysate protein fractions from human brain and human mitochondrial samples. We successfully obtained five distinct structures from the brain sample. These proteins play roles in a range of functions including neurotransmitter recycling, energy homeostasis and axonal development. Our approach also obtained nine distinct mitochondrial enzymes involved in various metabolic pathways. In this sample we were also able to structurally differentiate multiple members of the same enzymatic family. Across both tissue types we successfully obtained protein structures in the presence of native cofactors and ligands. Taken together, we believe our untargeted approach can complement traditional targeted protein strategies and be used as a viable strategy for multi-protein “structural-omics” experiments.

Committee: Edward Yu (Advisor); Phoebe Stewart (Committee Chair); Sichun Yang (Committee Member); Derek Taylor (Committee Member); Robert Bonomo (Committee Member); Marcin Golczak (Committee Member) Subjects: Biology; Pharmacology

Doctor of Philosophy, The Ohio State University, 2024, Chemistry

Proteins are fundamental macromolecules in biology, serving as the building blocks of cells and tissues, while also playing crucial roles as enzymes, structural components, signaling molecules, and transporters, thus governing various essential biological processes. These versatile molecules contribute significantly to the maintenance, regulation, and functionality of living organisms, embodying the molecular machinery that drives and sustains life. The secondary structure of a protein, formed by local folding patterns like alpha helices and beta sheets, significantly influences its stability by establishing a backbone conformation. This structural arrangement not only determines the protein's stability but also plays a critical role in dictating its activity, as it forms the basis for the protein's specific shape, which is crucial for interactions with other molecules and functional roles within biological processes. Protein engineering techniques allow the modification of amino acid sequences to probe how alterations impact the stability and activity of a protein, providing insights into the importance of specific secondary structures. By selectively modifying or designing secondary structures, such as helices or sheets, protein engineers can assess their contributions to stability and activity, enabling the fine-tuning of protein properties for various applications in biotechnology, medicine, and beyond. In chapter one we reviewed literature to find out the importance of protein loop on stability and activity of proteins. We also focused on studies Rop, the model protein that we used in this dissertation. In chapter two we focused on probing the loop of the four-helix bundle protein Rop with LDAD sequence, exploring its impact on stability, activity, and structure through the creation of four libraries: NNK4, NNK5, R55Q NNK4, and R55Q NNK5. Our results revealed that contrary to the typical expectation longer loops destabilize proteins, in Rop, two 5-amino ac (open full item for complete abstract)

Committee: Thomas Magliery Dr. (Advisor); Rafael Brüschweiler Dr. (Committee Member); Marcos Sotomayor Dr. (Committee Member) Subjects: Biochemistry; Biology; Biophysics; Chemistry

Doctor of Philosophy, The Ohio State University, 2024, Biomedical Sciences

Proteinopathies are the leading causes of neurodegeneration and dementia, and they carry extremely poor prognoses while affecting millions of people worldwide. They are characterized by filamentous inclusions composed of unique proteins such as tau [seen in Alzheimer's disease (AD) or different frontotemporal dementias and tauopathies] or α-synuclein [seen in Parkinson's disease (PD) and multiple system atrophy (MSA)]. Yet, mechanisms governing aberrant filament assembly in the different proteinopathies still require description, eluding intense scientific scrutiny. Recent structural works detailed the atomic models of ex vivo-derived filaments from AD, PD, MSA, and most common tauopathies, among others. While highlighting the novel role of cryoelectron microscopy (cryoEM) and the much higher standard of rigor that the technique brought to the field, it also revealed that the proteinopathy filaments exhibit polymorphism and strict disease-specificity. Moreover, multiple unidentified, anionic co-factors were visualized, suggesting a potential role for such co-factors in disease-related aggregation. The work presented here leverages the power of cryoEM and multiple anionic co-factor driven in vitro aggregation models to draw insights into the mechanisms behind anionic co-factor driven aggregation. First, biochemical techniques aimed at quantifying tau aggregation – a key readout for in vitro assays – are detailed. Second, a comprehensive, structure-driven analysis of geranine G (a small anionic co-factor)-induced tau aggregation is detailed. Lastly, the same approach is extended to both anionic-surface induced aggregation models of both tau and α-synuclein, revealing common mechanisms that are mediated by specific, positively-charged residues. Together, these findings identify multiple sites on both proteins and implicate a common mechanism for triggers of amyloid formation.

Committee: Jeffrey Kuret (Advisor); Krishna Chinthalapudi (Committee Member); Sherwin Singer (Committee Member); Charles Bell (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, Case Western Reserve University, 2024, Pharmacology

Dynamin superfamily proteins (DSPs) are present in all organisms, mediating critical membrane remodeling events throughout the cell. Despite the decades of structural and functional studies across the superfamily, no structure has been determined of a DSP demonstrating the conformational changes required to transition from a cytosolic, solution state to a helical assembly. This gap in knowledge limits the field's understanding of the mechanisms required for regulation and functional assembly into membrane remodeling complexes. Dynamin-related protein 1 (Drp1) is the master regulator of outer membrane fission for mitochondria. Proper mitochondrial dynamics is essential for cellular health, and an imbalance in this cycle has been implicated in many diseases, from heart failure to prion-related neurodegeneration. Drp1 has been identified as a potential therapeutic target; however, the underlying mechanisms governing its regulation are largely unclear. Drp1 exists predominantly as a mixture of dimers and tetramers in solution, but the specific interactions that stabilize these solution forms and prevent the assembly of larger complexes are not known. Using cryo-EM, we have observed significant conformational rearrangements in native solution structures for both dimer and tetramer states when compared to existing DSP crystal structures. Additionally, we have identified a helical lattice which demonstrates unique GTPase domain assembly between adjacent filaments, providing insight into the mechanisms of constriction. Finally, we studied the impact of terminal tags on the structure and function of Drp1 and identified a newly appreciated intrinsically disordered region of regulation. Together, these observations provide insight into regulatory interactions that stabilize oligomer states and mediate the activation of assembly into a functional fission machinery.

Committee: Jason Mears (Advisor); Edward Yu (Committee Chair); Marcin Golczak (Committee Member); Rajesh Ramachandran (Committee Member); Beata Jastrzebska (Committee Member) Subjects: Biology; Biophysics; Molecular Biology; Pharmacology

Doctor of Philosophy, Case Western Reserve University, 2023, Pharmacology

Mitochondria form dynamic networks and need to maintain a delicate balance between fission and fusion to satisfy the cell's energetic and metabolic requirements. Fission is necessary to ensure mitochondria are properly distributed throughout the cell and to remove damaged mitochondrial components. The dysregulation of mitochondrial dynamics resulting in either an abnormally fragmented or interconnected mitochondrial network is associated with a variety of pathologies. Mutations in dynamin-related protein 1 (Drp1), the master regulator of mitochondrial fission, have been identified in patients presenting with severe neurological defects. Patient-derived fibroblasts exhibit hyperfused mitochondria, indicating mitochondrial dysregulation. Thus, these clinically relevant mutations impair Drp1 function, but the mechanism by which these mutations disrupt mitochondrial fission was undetermined. To address this lack of knowledge, the overarching objective of this research was to elucidate the specific Drp1 functional defects that are caused by these disease-associated mutations to better understand the relationship between impaired Drp1 function and disease. Drp1 self-assembles around the outer mitochondrial membrane (OMM) and, subsequently, hydrolyzes GTP which provides the mechanical force required to cleave apart the mitochondrion. The recruitment of Drp1 to the OMM is mediated in part by lipid interactions. A mitochondria-specific lipid, cardiolipin, promotes Drp1 self-assembly, enhances its GTPase activity, and is believed to facilitate membrane constriction. Disease-associated mutations in Drp1 are predominantly located within the GTPase and middle domains, which mediate its capabilities for hydrolysis and self-assembly, respectively. We have employed an ensemble of biochemical and EM-based techniques to investigate the impact of these mutations on the self-assembly, lipid recognition, and enzymatic capabilities of Drp1. Ultimately, we have shown that even mutatio (open full item for complete abstract)

Committee: Jason Mears (Advisor); Marvin Nieman (Committee Chair); Phoebe Stewart (Committee Member); Danny Manor (Committee Member); Edward Yu (Committee Member) Subjects: Biochemistry; Biomedical Research; Biophysics; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2023, Chemistry

Positive Strand RNA (PSR) viruses, such as coronaviruses and enteroviruses, cause serious health and economic threats worldwide, as seen with the COVID-19 pandemic. This has drawn attention to the importance of identifying new antivirals and molecular targets in RNA viruses. The multifunctionality of PSR genomes make them desirable targets for therapeutic intervention. Here, we present a class of antivirals that can inhibit SARS-CoV-2 replication in vitro by targeting conserved viral RNA structures at the 5'-end. Specifically, stem loops (SLs) 1, 4, 5a, and 6 of the viral 5'-region have shown a degree of binding with dimethyl amiloride molecules as determined by NMR structural analysis. These results open the door to potentially develop specific small molecules against SARS-CoV-2 and related coronaviruses. Upon investigating SL6, interesting structural dynamics features were observed at the budge region when exposed to different temperatures. From various Nuclear Magnetic Resonance (NMR) and single angle x-ray scattering (SAXS) experiments, experimental restrains were obtained in order to generate a 3D structure of SL6 using molecular dynamics simulations. In SARS-CoV-2, stem-loop 3, which contains the transcriptional regulatory sequence, was proven to bind to the host Unwinding Protein 1 (UP1) using electrophoretic mobility shift assay (EMSA), isothermal titration chromatography (ITC), and NMR, which possibly suggests that UP1 participates in the mechanism of transcription of sub-genomic RNA. In addition, another PSR virus, Enterovirus A71 (EV-A71), which is the etiological agent of the hand, foot, and mouth disease, has caused severe morbidity and high mortality rates in children for decades. Thus, understanding the mechanisms by which EV-A71 replicates within the cellular environment can bring to light efficient drug targets for viral inhibition. The 5'-untranslated region (5'-UTR) of the RNA genome is the control hub of viral replication and transcription in EV- (open full item for complete abstract)

Committee: Blanton S. Tolbert (Advisor); Fu-Sen Liang (Committee Chair); Thomas Gerken (Committee Member); Robert Salomon (Committee Member); Divita Mathur (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry; Virology

Doctor of Philosophy, The Ohio State University, 2022, Biochemistry Program, Ohio State

The field of gene editing, which offers opportunities to treat genetic mutations and diseases at the level of the gene itself, has garnered a great deal of interest in recent years, especially with the advent of the CRISPR-Cas9 system. However, the safety of gene editing agents must be maximized, and their off-target effects minimized, if they are to be deployed therapeutically in patients. Many well-characterized gene editing systems, such as CRISPR-Cas9, TALENs, and zinc finger nucleases, are programmable to target specific DNA sequences but rely on nuclease activity, which generates double-stranded DNA breaks. The presence of double-strand breaks in a living cell activates error-prone cellular repair pathways, which can cause serious off-target effects on the genome. The tyrosine recombinase Cre presents an alternative approach to gene editing. It recombines loxP DNA sites without requirements for external host proteins or energy sources, and without generating double-strand breaks. These features bode well for the simplicity and safety of Cre as a gene editing tool. However, one limitation is its substrate scope; its activity is fairly specific for its loxP substrate, and it cannot easily be programmed to recognize alternate DNA sequences. Furthermore, there are gaps in our understanding of how it controls its activity and the determinants of DNA selectivity and recombination efficiency. A deeper understanding of the structural and dynamic features underlying these processes will enhance the potential to target Cre toward therapeutically relevant DNA sequences. NMR spectroscopy, our tool of choice, offers the advantage of insight into both structural and dynamic features of biomolecules in solution. We begin with dual introductory chapters that contextualize the subsequent studies. Chapter 1 is a literature review of Cre recombinase structural biology – what has been learned in the past 25 years and where the field stands now. Chapter 2 is a literature rev (open full item for complete abstract)

Committee: Mark Foster (Advisor); Charles Bell (Committee Member); Rafael Brüschweiler (Committee Member); Michael Poirier (Committee Member) Subjects: Biochemistry

PhD, University of Cincinnati, 2022, Medicine: Molecular Genetics, Biochemistry, & Microbiology

Bone Morphogenetic Proteins (BMPs) are the largest subgroup of the Transforming Growth Factor ß (TGFß) superfamily, one of the fundamental protein signaling pathways in biology. BMPs are involved in regulating numerous biological functions, with a particular focus on development, immune modulation, cell homeostasis and wound healing. When dysregulated, aberrant BMP signaling can indue a host of developmental and autoimmune disorders, as well as many different cancers. Given the wide array of biological functions BMPs regulate, precise regulation of signaling is a key component of their biology. Mechanistically, these secreted dimeric signaling proteins function by forming complex with two type 1 and two type 2 serine/threonine kinase receptors on the cell surface to drive signaling by intracellular SMAD proteins. Accordingly, the regulation of these potent signaling molecules in the extracellular space is a vital area of study. The purpose of the work outlined in this thesis is to explore certain understudied mechanisms of extracellular modulation of BMP signaling. We particularly focused on studying these mechanisms not in isolation, but rather as they actually exist in nature, as part of a complex environment with many competing biomolecules. We present studies contrasting related protein antagonists with different function in an attempt to gain insight into the key components needed for BMP inhibition. In addition, we explore a newly discovered interaction between the BMP and Wnt signaling pathways, where BMP ligands may directly antagonize canonical Wnt signaling. Lastly, we describe a procedure for the production of artificial BMP heterodimeric signaling molecules using chains with differential activity with respect to receptor preference, antagonist targeting, and affinity to the extracellular matrix. These asymmetrical signaling molecules were then used to isolate the key components of biological function across multiple different experimental systems. Overal (open full item for complete abstract)

Committee: Thomas Thompson Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member); James Wells Ph.D. (Committee Member); Michael Tranter Ph.D. (Committee Member) Subjects: Biochemistry

Master of Engineering, Case Western Reserve University, 2022, Biomedical Engineering

Mucin-type O-glycosylation is an abundant yet understudied post-translational modification of proteins in metazoans, initiated by the polypeptide N-acetylgalactosamine-transferases (GalNAc-Ts) of which twenty are known in humans. O-glycosylation is involved in many biological processes including intracellular signaling and cell-cell interactions. GalNAc-T mutations contribute to several disease states such as cancer. Previous characterization of the GalNAc-Ts has revealed unique preferences for peptide sequence and prior substrate glycosylation. Herein, another paradigm by which the GalNAc-Ts select peptide targets is investigated: flanking substrate charge. Twelve GalNAc-Ts have been characterized against substrates containing different N-/C-terminal charges, demonstrating unique and overlapping charge preferences. Electrostatic models, elevated ionic strength, and molecular dynamics simulations reveal that GalNAc-Ts selectivity is indeed modulated via charge-charge interactions. Michaelis-Menten kinetics revealed significant variation in kinetic parameters, particularly Vmax. Overall, this work reveals that charge-charge interactions are another important but previously overlooked factor that uniquely modulates GalNAc-T specificity.

Committee: Thomas Gerken (Advisor); Mei Zhang (Advisor); Xinning Wang (Committee Member); Ryan Arvidson (Committee Member); Sam Senyo (Committee Chair) Subjects: Biochemistry; Biomedical Engineering

PhD, University of Cincinnati, 2022, Medicine: Molecular, Cellular and Biochemical Pharmacology

The Transforming Growth Factor Beta (TGFß) Family consists of over 30 secreted growth factor ligands, which signal using seven type I and five type II serine/threonine kinase receptors. Given the large number of ligands compared to the number of receptors, there is a significant overlap of multiple ligands utilizing the same receptors. However, one ligand, Anti-Mullerian Hormone (AMH), is the only ligand in the TGFß family with its own unique type II receptor, Anti-Mullerian Hormone Receptor 2 (AMHR2). AMH promotes Mullerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. However, during fetal growth, loss of function mutations in AMH or AMHR2 have been linked to the development of Persistent Mullerian Duct Syndrome (PMDS), where males are born with normal external male genitalia, but also the presence of a uterus and fallopian tubes internally. Alternatively, more recent studies have shown that AMH also plays a role in the development of Polycystic Ovary Syndrome (PCOS) in women. Moreover, the role of AMH in female fertility is becoming more widely accepted. As such, AMH has potential for use in fertility therapies. Prior to the work outlined in this thesis, there was little understanding of how AMH binds to AMHR2. Part of this gap stems from the fact that no structural information about AMH or AMHR2 was available. Therefore, in this work we, solved the structure of AMH bound to the extracellular domain of AMHR2 and characterized the molecular interactions between these proteins. Overall, this work highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, through mutational studies, we identified residues in both AMH and AMHR2 that are key to the AMH/AMHR2 interaction. In conclusion, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonis (open full item for complete abstract)

Committee: Thomas Thompson Ph.D. (Committee Member); Terry Kirley Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member); Jo El Schultz Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2022, Chemistry

Enterovirus 71 (EV71), represents a persistent threat to global health and economies, as outbreaks are reported in the United States and globally each year. Infections are self-limited; however, prolonged infection in the immunocompromised can lead to severe neurological disorders and, eventually, death. As of the time of writing, there are no FDA-approved treatments against this pathogen. Thus, there is an immediate urgency to determine the mechanisms regulating host-virus interactions. EV71 utilizes a type I IRES element to initiate viral translation in a cap-independent pathway by recruiting multiple host proteins through a poorly understood mechanism. This thesis seeks to define the molecular and specificity determinants regulating the formation of IRES-protein complexes that modulate cap-independent translation. Herein, we studied the interactions of the negative translation modulator AUF1 with the conserved stem loop II (SLII) IRES domain. In chapter 2 we demonstrate that AUF1 and its isolated RRMs bind to the SLII bulge motif via a monophasic thermodynamic transition, where the bulk of the thermodynamic stability is conferred by the first RRM. Building on this knowledge, in chapter 3 we screened a library of RNA-targeting small molecules against the SLII IRES domain and found a potent inhibitor (DMA-135) of EV71 translation and replication. A combination of biophysical and functional studies revealed that DMA-135 functions by inducing a conformational change on SLII which stabilizes the formation of the repressive SLII:DMA-135:AUF1 complex. In chapter 4, we validated the proposed mechanism of action by generating (DMA-135)-EV71 resistant mutants, where the suppressor mutations mapped to SLII. Biophysical studies revealed that the suppressor mutations changed the local RNA structure around the SLII bulge which impaired DMA-135 and AUF1 binding. In chapter 5, we gathered the knowledge obtained in all previous chapters to delineate a pipeline for the identificat (open full item for complete abstract)

Committee: Blanton Tolbert (Advisor); Fu-Sen Liang (Committee Chair); Robert Salomon (Committee Member); Thomas Gerken (Committee Member); Shane Parker (Committee Member) Subjects: Biophysics; Chemistry; Virology

Doctor of Philosophy, The Ohio State University, 2021, Chemistry

The structures adopted by biological macromolecules and macromolecular complexes are directly tied to their function. By better understanding the relationship between structure and function of biomolecules, lifesaving and life-changing interventions can be designed such as small molecule inhibitors of viral enzymes. Characterization of macromolecules with high-resolution structural techniques has greatly improved our fundamental understanding of how structures dictate function. High-resolution structural techniques including x-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance spectroscopy, however, have many challenges and so they require complementary techniques. Native mass spectrometry is one such technique that can be used to interrogate macromolecular assemblies and accurately determine molecular weight(s), oligomeric state(s), ligand-binding, and topology of the assembly. Native mass spectrometry has the advantage of not being limited by some of the common issues encountered by high-resolution techniques like molecular flexibility and sample heterogeneity that can limit resolution attainable or prevent structure determination altogether. This attribute is particularly valuable for the characterization of protein-nucleic acid complexes that have proven to be some of the more challenging complexes for high-resolution techniques. Throughout this work, native mass spectrometry is emphasized as a clear approach for examining differences in macromolecular assemblies that highlight the structural diversity of macromolecular systems which would otherwise not be evident. Chapter 3 describes identification of the physiological protein interface of a plant protein, BX1.This approach demonstrates the use of native mass spectrometry and covalent cross-linking mass spectrometry to solve a common issue with X-ray crystallography, namely artificial protein contacts formed during the crystallization process. Chapter 4 describes the investigation of (open full item for complete abstract)

Committee: Vicki Wysocki (Advisor); Venkat Gopalan (Committee Member); Karin Musier-Forsyth (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2021, Biochemistry

Understanding the structure and function of macromolecules and their performative complexes is indispensable when developing novel treatments for disease. Nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) provide powerful insights into macromolecular structure and dynamics and thereby harbor the potential for aiding the development of much needed therapeutics. The studies presented here describe the application of NMR spectroscopy and cryo-EM to characterize the structure and/or dynamics of three distinct biochemical entities with fundamental biological implications. These targets include cyclic peptides being developed as cancer therapies, a homo-oligomeric ring protein that serves as a model system for understanding allosteric regulation, and a recombinase that selectively binds and becomes activated to site-specifically cleave DNA. Cyclic peptides are capable of binding to challenging molecular targets (e.g., proteins involved in protein-protein interactions) with high affinity and specificity, but generally cannot gain access to the intracellular environment because of poor membrane permeability. In chapter 2, I describe my work to characterize a pair of conformationally constrained cyclic cell-penetrating peptides (CPP) containing a D-Pro-L-Pro motif, beginning with cyclo(AFΦrpPRRFQ) (where Φ is L-naphthylalanine, r is D-arginine, and p is D-proline). The structural constraints provided by cyclization and the D-Pro-L-Pro motif permitted the rational design of cell-permeable cyclic peptides of large ring sizes (up to 16 amino acids). This strategy was applied by my collaborators to design a potent, cell-permeable, and biologically active cyclic peptidyl inhibitor, cyclo(YpVNFΦrpPRR) (where Yp is L-phosphotyrosine), against the Grb2 SH2 domain, a key mediator in Ras activation. Multidimensional NMR spectroscopic and circular dichroism analyses revealed that the initial cyclic CPP as well as the Grb2 SH2 inhibitor assume a pr (open full item for complete abstract)

Committee: Mark Foster (Advisor); Thomas Magliery (Committee Member); Charles Bell (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2019, Systems Biology and Bioinformatics

X-ray-initiated hydroxyl radical protein footprinting (XF) coupled to mass spectrometry (MS) is a well-characterized method for investigating protein and nucleic acid conformational changes in vitro over large timescales ranging from microseconds to milliseconds. For the past two decades, XF has been applied to a variety of structural biology studies, and now, there is growing interest in utilizing XF to probe more complex biological systems such as large macromolecular complexes, protein aggregates, and live cells, which are not easily prosecuted using other protein footprinting strategies. These difficult XF studies require increased X-ray flux density and greater protein sequence coverage, and the goal of this dissertation is to develop a high-flux X-ray beamline and a synergistic footprinting reagent, which provide improved beam power and extend the resolution of XF, respectively. The chapters herein describe the scientific commissioning and benchmarking of a new, high-flux X-ray beamline, XFP (Biological X-ray Footprinting and Spectroscopy beamline), at NSLS-II and the adaptation of a novel footprinting methodology, the trifluoromethylation of proteins, to XF. Results demonstrated XFP is capable delivering 10 times the flux density of its predecessor X28C, and trifluoromethylated radicals (•CF3) and hydroxyl radicals (•OH) were shown to label distinct subsets of amino acid side chains. The high-flux beamline and X-ray-initiated •CF3 footprinting strategy discussed in this dissertation provide a foundation for novel XF experiments on challenging systems in the next coming decades. An example of a recent, high impact XF study in which I played an integral role in the successful completion of the project is given in the concluding chapter.

Committee: David Lodowski (Committee Chair); Chance Mark (Advisor); Adams Drew (Committee Chair); Kiselar Janna (Committee Chair); Tilton Chip (Committee Chair) Subjects: Biochemistry; Biology; Biomedical Research

PhD, University of Cincinnati, 2019, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Notch is a highly conserved signaling pathway in multicellular organisms that regulates fundamental cellular processes such as proliferation, differentiation, and cell fate determination. Notch has a litany of roles in human biology and pathologies associated with aberrant Notch include cancers, cardiovascular diseases and developmental disorders. The Notch transcription factor CSL mediates either transcriptional activation or repression of Notch target genes, depending on the context, by forming coactivator or corepressor protein complexes that reside on Notch target promoters and enhancers. Notch pathway components remain very attractive as therapeutic targets due to the widespread downstream effects of the pathway and the limited success of current treatments. However, the pursuit of small molecule modulators of Notch is complicated by the dual role of CSL as an activator and a repressor, and even moreso by the realization that coactivators and corepressors often bind to CSL via conserved motifs. Still, any hope of developing small molecules with complex specificity relies on a growing body of molecular insight provided by crystal structures of the CSL-coregulator complexes and detailed understanding of the binding determinants of the complexes. A recently identified putative CSL binding protein called L3MBTL3 belongs to the malignant brain tumor (MBT) family of proteins that use a variable number of MBT structural domains to recognize mono- and di-methylated lysine residues on histone tails and facilitate chromatin compaction and transcriptional repression. In this dissertation, I will describe the work done to determine this poorly understood protein's involvement in the Notch pathway. Chapter 1 will be an introduction to the Notch pathway, from the protein machinery to the biological impact and human health implications. Chapter 2 is a thorough structural and binding analysis of CSL protein complexes. This will be an extended version of a review that we have r (open full item for complete abstract)

Committee: Rhett Koval Ph.D. (Committee Chair); Andrew Herr Ph.D. (Committee Member); Carolyn Price Ph.D. (Committee Member); Thomas Thompson Ph.D. (Committee Member); William Miller Ph.D. (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, The Ohio State University, 2018, Biochemistry Program, Ohio State

Adenosine, guanosine, cytidine, and thymidine nucleotides are the building blocks of life and are arranged in distinct combinations to give unique genomic DNA sequences. These DNA encode for all biological molecules and processes and therefore the preservation of genomic integrity is essential for cell growth and viability. To do so the cell has evolved specialized enzymes for efficient and faithful DNA replication. However, genomic DNA is continually damaged by reactive agents occurring from host processes or those encountered through the environment. The resulting DNA damage can act as mutagens causing errors in DNA replication and ultimately leading to disease states or can cause blocks to DNA replication causing replication fork collapse and potentially cell death. To counteract the onslaught of DNA damage cells have evolved a multitude of DNA damage repair mechanisms that can directly revert damaged DNA bases back to the canonical bases, remove and replace single DNA bases (base excision repair), or remove and resynthesize segments of DNA containing damage (nucleotide excision repair and mismatch repair). Base excision repair (BER) in humans is initiated by a damage specific DNA glycosylase that recognizes and removes a single damaged base from DNA, resulting in a product abasic site. This action is followed by cleavage of the abasic site containing DNA strand by apyrimidinic/apurinic endonuclease 1 generating a single-nucleotide gapped DNA substrate with a deoxyribophosphate-aducted 5'-end (5'-dRP). This gap is then filled and the 5'-dRP removed by DNA polymerase ß (Polß) resulting in a nicked DNA substrate. Finally, this nick is ligated by DNA LigaseIII/XRCC1 to complete repair. Here I have investigated the structure and function of Polß to better define its role in DNA repair. Through time-resolved X-ray crystallography and pre-steady-state gel-based kinetics, I have identified and characterized a third divalent metal ion utilized in the synthesi (open full item for complete abstract)

Committee: Zucai Suo PhD (Advisor); Jane Jackman PhD (Committee Chair); Marcos Sotomayor PhD (Committee Member); Kotaro Nakanishi PhD (Committee Member) Subjects: Biochemistry; Biology

Doctor of Philosophy, Case Western Reserve University, 2018, Pharmacology

The complex processes of mitochondrial fission and fusion are opposing functions whose proper balance ensures optimal mitochondrial function in eukaryotic cells. Aberrant mitochondrial morphology where one of these mitochondria-shaping processes dominates the other are commonly found in diverse pathologies, highlighting the importance of maintaining appropriate rates of both fission and fusion. Both of these competing processes are mediated by members of the dynamin superfamily of membrane-remodeling GTPases. Scission of the mitochondrial membranes is carried out by an ancient member of this protein superfamily called dynamin-related protein 1 (Drp1). While its function is required for fission, Drp1 alone is unable to mediate this complex process, and requires interaction with one or more partner proteins of the mitochondrial outer membrane to ensure fission. Chordates express several such proteins whose genetic interaction with Drp1 has been proven to be crucial for maintenance of 2 appropriate mitochondrial morphology. These include mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), and mitochondrial dynamics proteins of 49 and 51 kilodaltons (MiD 49/51). Of these proteins, the first that was proposed to contribute significantly to the maintenance of mitochondrial morphology in man was Mff. Due to its relatively recent discovery, its specific role(s) in this function remain unclear. To address this lack of knowledge, the primary objective of these studies was to better understand the various factors that control the association of Drp1 and Mff, and to shed light on the regulatory mechanisms that underlie this interaction. We have shown that the interaction between Drp1 and Mff is mediated by the stalk of Drp1, and not the variable domain (VD) as was previously thought. We also demonstrated the utility of mitochondrial outer membrane-like scaffolding liposomes as a template for studying the interaction between Drp1 and its various membran (open full item for complete abstract)

Committee: Jason Mears (Advisor); Philip Kiser (Committee Chair); Rajesh Ramachandran (Committee Member); Derek Taylor (Committee Member); Edward Yu (Committee Member) Subjects: Biochemistry; Pharmacology

Doctor of Philosophy, University of Toledo, 2018, Chemistry

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) is notoriously difficult to treat due to its impervious cell wall. However, the biosynthetic pathways responsible for its protective barrier have proven to be an Achilles heel, as many TB drugs target the enzymes within these unique pathways. Based on this proven approach, we too have decided to target enzymes responsible for the construction and maintenance of the outermost lipid membrane, the mycomembrane, for drug development. The focus of this dissertation is on the structural, enzymatic, and inhibitory study of two Mtb mycomembrane lipid esterases. Both enzymes are secreted, essential, have similar protein folds and utilize classical catalytic triads, yet perform different types of chemistry. The Antigen 85 complex (Ag85) of Mtb catalyzes the acyltransfer of the mycolic acids (MA) to produce trehalose-dimycolate (TDM) or the mycolylarabinogalactan (mAG), two hallmark lipids of the unique mycomembrane. Three homologous mycolytransferases comprise the Ag85 complex (Ag85A, B, and C) and despite the term complex, act independently through a ping-pong catalytic mechanism. Based on early structural studies, an interfacial mechanism model was proposed that detailed substrate arrangement within Ag85s; however, numerous problems exist with this model. To address those concerns, we sought a crystal structure of the acyl-enzyme intermediate form of Ag85C; however MAs are highly insoluble. Therefore, Ag85C was successfully co-crystalized with tetrahydrolipstatin (THL), an FDA approved lipase inhibitor, which mimics core structural attributes of MAs. The Ag85-THL structure served as a basis for the modeling of the acyl-enzyme intermediate. Based on structural similarities to previously solved Ag85 structures, mutagenic studies, and computational simulations, Ag85s were shown to undergo structural changes upon acylation that limit substrate hydrolysis and promote substrate transfer. Based on these (open full item for complete abstract)

Committee: Donald Ronning (Committee Chair); Peter Andreana (Committee Member); John Bellizzi (Committee Member); Scott Leisner (Committee Member) Subjects: Biochemistry; Chemistry; Molecular Biology; Molecular Chemistry

Master of Science, Miami University, 2018, Cell, Molecular and Structural Biology (CMSB)

Enzymes, while incredibly complex, are limited in functionality to their twenty canonical amino acids. To shortcut this limitation, nature has evolved to utilize post translational modification (PTM) of protein structure to regulate cellular functions. Whether this be as essential cofactors or acting as regulators of enzyme activity, controlling cellular processes by PTMs are absolutely essential to the molecular-level events that coordinate and sustain life. Within a massive population of possible binding sites, enzymes responsible for PTMs manage to find their precise target through highly selective recognition sites. LplA has been shown to be a convenient tool for attaching small molecule precursors to its recognition site. The aim of this study is to modify the enzyme Lipoic acid ligase A (LplA) to effectively carve a tunnel through the backside of the enzyme. By carving a tunnel through the ligase, this work aims to broaden the substrate compatibility range of LplA for post translational modification of target proteins. Additionally in this study, crystal structures of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) will be solved and analyzed for evidence of S-nitrosylation. It is the goal of this study to gain a better understanding of how this PTM regulates heme binding to GAPDH.

Committee: Richard Page (Advisor); Michael Robinson (Committee Chair); Yoshi Tomoyasu (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2017, Pharmacology

“Vitamin A is the precursor of visual purple as well as the product of its decomposition. The visual processes therefore constitute a cycle.” This statement by George Wald in 1935 described in astounding simplicity the visual cycle and the recycling of the universal chromophore 11-cis retinal. Here, we present recent findings derived from genetic, biochemical and physiological studies that provide a more advanced understanding of the retinoid cycle and color perception. The energetics of cis-trans isomerization of 11-cis-retinal accounts for color perception in the wide range of our visible spectrum and explains how human photoreceptors can absorb light in the near infrared (IR). Structural homology models of visual pigments reveal complex interactions of the opsin protein moieties with 11-cis-retinal and how certain color blinding mutations can impair secondary structural elements of these G protein-coupled receptors (GPCRs). Studies of hydrogen/deuterium exchange also confirmed the dimeric state of human green cone opsin, and revealed the potential role of intracellular loop 2 at the dimer interface in opsin activity. Additionally, these 1 studies demonstrated an alternative chromophore exit site involving a Pro-Pro motif at transmembrane helix 4. Once all-trans-retinal is released from opsin, it is reduced by a retinol dehydrogenase in concert with its co-factor NADPH. This first reaction of the retinoid cycle results in reduced all-trans-retinol, which is then recycled back to 11-cis-retinal which serves as the light-sensitive chromophore for visual pigment molecules. Finally, we solved the structure of the Drosophila melanogaster photoreceptor dehydrogenase. The crystal structure of this enzyme provided insight into the first reaction step of the visual cycle and revealed the likely location of disease related mutations in the homologous human retinol dehydrogenase 12.

Committee: Krzysztof Palczewski PhD (Advisor); Jason Mears PhD (Committee Chair); Marvin Nieman PhD (Committee Member); Focco van den Akker PhD (Committee Member); Marcin Golczak PhD (Committee Member) Subjects: Analytical Chemistry; Biochemistry; Biophysics; Molecular Biology; Pharmacology