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Tyrosine phosphorylation is one of the key covalent post-translational modifications through which multicellular organisms communicate. Phosphorylation of tyrosine residues on proteins can modulate enzymatic activity and can create binding sites for the recruitment of downstream signaling proteins. Conservation of enzyme structure across the human kinome makes it difficult to design small molecules to selectively modulate kinase activity, which can help elucidate their roles in signaling pathways and in diseases. To circumvent this limitation, we have developed a split-protein method to control the activity of individual kinases by utilizing chemical inducer of dimerization (CID) proteins. The split-protein approach relies on the identification of viable fragmentation sites in a protein that can be used to generate ligand-gated control of protein activity. The major focus of this dissertation is the identification of new kinase and firefly luciferase fragmentation sites for temporal control of a specific kinase and for the study of protein-protein interactions, respectively. With a sequence dissimilarity-based approach and structure-guided analysis, we successfully identified new split-Src sites, the first split-Syk site, the first split-Abl-1aFL, and new firefly luciferase fragmentation sites utilizing the CIDs of rapamycin and/or abscisic acid. Temporal control of split-Abl-1a coupled with quantitative phosphoproteomics analysis aided in the understanding of Abl-1 cellular signaling. Both known and novel substrates were identified, and validation of novel phosphotyrosine targets like AFDN, AMOT, DDX3X, NCAPH present opportunities for studying and understanding unanticipated functions of Abl-1. In summary, this work describes the split-protein approach to selectively control and dissect the complex signaling processes of specific protein tyrosine kinases (PTKs) and for monitoring PPIs. The new tools developed could potentially be used to rewire signaling pathways a

Filament formation by enzymes has long been known, but only recently the functional diversity of enzyme regulation via filamentation is beginning to be understood. In this work, I use SgrAI as a model system to investigate filament formation. SgrAI is a "rare-cutting" sequence-specific type II restriction endonuclease from Streptomyces griseus, which recognizes and cleaves dsDNA in a Mg+2 dependent manner. SgrAI cleaves three primary site sequences that are 8-bp long. Interestingly, binding of any of these primary site sequences induces filament formation by SgrAI. Filamentation of SgrAI leads to enzymatic activation that results in increased DNA cleavage kinetics and expanded sequence specificity, allowing the binding and cleavage of not only the three canonical sites but also 14 additional secondary sites. Binding of primary but not secondary sites by SgrAI induces filament formation suggesting that the interaction of SgrAI with these two sites differs. To address this hypothesis I solved a 3.5 Å crystal structure of WT-SgrAI bound to secondary site DNA. Comparison of this structure to previously reported structures revealed no large-scale differences from the previously solved structures of SgrAI bound to DNA, however, this may be due to the anisotropic nature of the x-ray diffraction data and low resolution obtained. Filament formation results in enzyme activation, and because of this, we hypothesize that filamentation is critical for the biological role of SgrAI. To test this hypothesis I show a mutational study where we found that mutations that even slightly disrupted filament formation resulted in almost complete loss of the enzyme’s ability to protect against phage infection. Work done in the Horton lab where FRET experimental data was used to model and simulate reactions in vivo revealed that SgrAI has a slow association rate constant that is advantageous for sequestering the enzyme’s activity onto a specific substrate DNA. The computational modeling study

The research focuses on two different approaches towards drug discovery for neurodegenerative diseases Multiple Sclerosis (MS) and Amyotrophic Lateral Sclerosis (ALS). Neurodegeneration is a progressive deterioration of neural structures leading to cognitive or motor impairment. There is still no effective therapy for any of the most common neurodegenerative diseases and the available therapies only lower the rate of the disease progression or manage the symptoms. Although neurodegenerative diseases exhibit distinct clinical characteristics, they usually present aggregation of specific protein(s) along with neuroinflammation among others. The first investigated approach towards discovering therapeutics is the use of small molecules targeting the N-terminal domain of transactive response (TAR) DNA binding protein-43 (TDP-43), a critical factor in neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Alzheimer’s disease (AD) to disrupt the aggregation of TDP-43. Targeting the N-terminal domain (NTD) of TDP-43 by in silico docking, a small molecule called nTRD022 have been discovered binding to the NTD of TDP-43 that allosterically affects the RNA binding of the protein leading to disruption of RNA binding to TDP-43 and improving the muscle strength, as an ALS related phenotype, in Drosophila fly MS models which could potentially be further developed as ALS therapeutic. The second approach focuses on developing a systematic evolution of ligands by exponential enrichment (SELEX) to generate therapeutic aptamers. Aptamers are short nucleic acid ligands that are able to specifically recognize a target with high specificity and high affinity. They have been widely used in therapeutic and diagnostic applications for neurodegenerative diseases over the last two decades. However, due to their relatively recent development, there are not as many aptamers in clinical trials as expected. In this study, aptamers have been developed against autoantibodies ta

Prions modulate cellular phenotypes by adopting alternative folds called amyloid that change their function and act as templates to convert natively folded protein to the amyloid state. The initial appearance of an amyloid is rare but enhanced by the presence of other aggregated proteins. Previous work has suggested this enhancement arises from the ability of pre-existing aggregates to template amyloid assembly but fails to explain all experimental observations. Our studies in S. cerevisiae suggest an alternative role for pre-existing aggregates: the titration of chaperones, which normally act to disassemble nascent amyloid nuclei. Using the [PSI+]/[PIN+] dual prion system, we demonstrate that the presence of [PIN+] reduces the binding of the molecular chaperone Hsp104 to Sup35 amyloid, the determinant of the [PSI+] prion. In turn, this change reduces the fragmentation of Sup35 amyloid, promoting appearance of the [PSI+] prion. Notably, the combination of [PIN+] and reduced levels of Hsp104 can inhibit prion appearance and propagation for some conformational variants of Sup35 amyloid, providing further evidence of titration. Using conformational variants of [PIN+] known to promote different levels of [PSI+] formation, we demonstrate that differential chaperone titration explains differences in [PIN+] variant promotion of [PSI+] induction. Thus, [PIN+] acts as a decoy for chaperones and thereby promotes persistence of nascent aggregates.

Exploration of protein structure by its solvent-accessible surfaces has been widely exploited in structural biology. Amino acids most commonly targeted for covalent modification of the native folded proteins are lysine and cysteine. For the first time, the present study has leveraged ene-type chemistry targeting tyrosine residues to discriminate those solvent-exposed from those buried. We find that 4-phenyl-3H-1,2,4- triazole-3,5(4H)-dione (PTAD) can conjugate the phenolic group of tyrosine in a manner heavily influenced by the orientation and depth of the residue with respect to the protein surface. We developed a strategy to investigate protein structure by analyzing PTAD conjugations with free tyrosine, protein structure, and found it adaptable to a wide range of analytic technologies, including fluorescence, chromatography, and mass spectrometry. This study shows how the established tyrosine-specific bioconjugation chemistry can be used as an analytical tool to distinguish the conformational states of a protein where traditional structural approaches are limited. Among limitations of the traditional structural techniques, are studies of low complexity proteins, such as tyrosine rich FET proteins, with essential cellular functions in transcription and DNA repair, and mutations associated with some neurodegenerative diseases (Schwartz et al., 2015a). The N-terminal low complexity domains of FET proteins have the ability to drive the formation of non-membrane bound cellular organelles, also known as granular bodies, through a process referred to as phase separation. Low complexity proteins are typically intrinsically disordered or lacking in rigid secondary structure elements. This renders the most popular NMR and X-ray crystallography methods incapable of providing high-resolution structure data and elevates the potential for a new method of structural analysis to reveal a wealth of otherwise unattainable information.

Due to the error-prone nature of viral replication, antiviral drugs can select for drug-resistant strains. Understanding the determinants for drug resistance is useful for the preclinical evaluation of antivirals, and provides important insight into the biochemical nature of the target. The first part of this dissertation describes studies designed to investigate properties of antiviral drug resistance using a well-established drug target from the influenza A virus as a model protein. The second part of the dissertation mainly focuses on the validation of a novel drug target for a re-emerging virus using biochemical and viral replication assays. Influenza A viruses are pernicious human pathogens that lead to seasonal and pandemic outbreaks. Amantadine and rimantadine are two FDA-approved drugs that target a viral proton channel called M2. These two therapeutics are no longer used due to the emergence of drug resistance. The two principal mutations that give rise to resistance in circulating strains are the S31N and V27A found within the pore of M2. Structural characterization of M2 has guided efforts to target these individual drug resistant channels through the conjugation of the adamantane group, and it was shown that the best V27A inhibitor has in vivo activity, and that the best S31N inhibitor displayed favorable pharmacokinetic properties. Although the determinants of drug resistance for amantadine and rimantadine have been thoroughly investigated, similar studies have not been performed for inhibitors of the V27A and S31N variants. Due to the fine-tuned function and structure of M2, I have hypothesized that V27A and S31N inhibitors would have a high genetic barrier to drug resistance. Using representative V27A and S31N inhibitors, I performed serial passage experiments in cell culture to select for drug-resistant influenza A viruses. Sequencing of the M2 gene segment identified mutations in the channel that gave rise to resistance, which were confirmed using r