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Viroporins are a class of viral membrane proteins that play diverse roles in the viral infection cycle. Viroporins are known to play roles in ion transport, inducing membrane curvature, as well as participating in a variety of protein-protein interactions. However, there is little known about the oligomeric state of viroporins. The lack of quantitative data on the oligomeric state of viroporins is due to key analytical challenges associated viroporins, including that they are 1) hydrophobic, and require a membrane mimetic for analysis, 2) small, making viroporins unamenable to some structural biology techniques such as cryo-EM, and 3) contain intrinsically disordered regions, which also poses challenges for structural characterization. To overcome these analytical challenges, this dissertation outlines the application of the unique combination of native mass spectrometry (MS) and nanodiscs for the oligomeric state determination of viroporins. The unique combination of native MS and nanodiscs can be used to determine the oligomeric state and lipid specificities of membrane proteins (such as viroporins) while embedded within a lipid bilayer using mass defect analysis. This dissertation reviews how to perform mass defect analysis, provides examples of how mass defect analysis has been applied in the past, as well as providing tips for overcoming some of the technical limitations associated with mass defect analysis. This dissertation also outlines a structure-activity relationship study performed to identify novel charge reducing agents for native MS. Charge reducing agents can be useful for the preservation of fragile complexes, such as viroporins, during native MS. Here, we found improved charge reducing agents that were effective on a wide range of analytes. We also uncovered the chemical principles governing charge reduction, which may provide a basis for the development of even better novel charge reagents in the future. The first viroporin characterized with nati

Pipet aperture-based electrophysiological (EP) sensors have emerged relatively recently as powerful bioanalytical tools. Taking advantage of the ligand specificity and inherent high sensitivity of membrane proteins, pipet aperture-based EP sensors can transduce target analyte binding events into measurable electrical signals. A variety of analytes including neurotransmitters and hormones that traditional fluorescence or electrochemistry methods are ineffective for detection can be quantitatively measured, facilitated by biologically “paired” receptor membrane proteins. Additionally, such sensors intrinsically provide high spatial resolution due to the pipet apertures that can be fabricated on µm scales, enabling real-time cellular secretion measurements. Despite these strengths, applications that aim to utilize such sensors have been limited, predominately due to low efficiency in sensor preparation. First, the preparation of some sensor components, such as membrane proteins, is typically laborious and time-consuming, resulting in a low success rate and a long timescale of sensor assembly and functionalization. Second, artificial lipid membrane platforms for membrane proteins have low stability, leading to early rupture and short sensor longevity. This requires reproduction of sensors of the same type to achieve measurements that could have been performed with fewer but more stable individual sensors. Therefore, improving sensor preparation efficiency could significantly broaden applications of EP sensors. This dissertation investigates these two aspects of sensor preparation efficiency and demonstrates two ways to apply such sensors in measuring small molecules and studying peptides. To stabilize the membrane, methacrylate polymerization with various initiation methods was adopted (Chapter 2). To increase membrane protein preparation efficiency, in vitro transcription and translation was utilized to overcome limitations in traditional protein expression and purific

Micro- and nano-sized glass pipettes have garnered significant attention from researchers due to their numerous advantages, including ease of fabrication, low cost, tunable geometry, and modifiable surface properties. These favorable characteristics have facilitated a wide range of applications in the fields of analytical chemistry, material science, and cellular biology. This dissertation presents three innovative probe designs that facilitate the analysis of challenging samples. First, we demonstrate the utilization of nanopipettes as probes in scanning ion conductance microscopy (SICM) to collect topographical images of live platelets near their resting state. This approach enables the imaging of live human platelets without subjecting them to excessive mechanical stimulation. Second, we demonstrate the reconstitution of a functional ligand-gated ion channel, the eGFP-Kir6.2 protein, into a polymer scaffolding-stabilized black lipid membrane (BLM) suspending across the opening of a surface modified glass micropipette. Our results reveal that the reconstituted ion channel retains its ability to undergo conformational changes and remains sensitive to its ligand. Furthermore, we establish the groundwork for an innovative expansion of the ion channel probe (ICP) sensing platform using double-barrel theta pipettes. We describe the fabrication process of these pipettes and propose a protocol for constructing a dual ion channel-functionalized double-barrel ICP system capable of concurrently quantifying two analytes. Additionally, we explore the potential of combining scanning ion conductance microscopy with the ICP system for simultaneous topographical and chemical mapping. Third, we present a rapid and cost-effective method to enhance the sensitivity of native mass spectrometry (MS) and charge-detection mass spectrometry (CD-MS) by surface modifying micropipettes for nanoelectrospray ionization (nESI). Additionally, we conduct a thorough investigation into the mechanis

Bioconjugation is essential in many fields such as biotechnology, medical diagnostics, and drug development. It refers to the process of attaching a synthetic molecule or biomolecule to a biological entity such as a protein, nucleic acid, lipid or carbohydrate. One common approach for bioconjugation involves the use of reactive functional groups on biomolecules. Aryl diazonium ion chemistry to target tyrosine residues on proteins is a major focus of this dissertation. Scarcity of tyrosine helps with the selectivity and it is also an active amino acid in the protein protein interaction surfaces. A chemical probe classified as a Triazabutadiene “TBD”; a masked aryl diazonium ion was advanced for its reactivity and used in selective bioconjugation of proteins. The TBD has also undergone additional modifications to create protected TBDs that have increasing potential in the field of targeted delivery. Protected TBDs, which can be activated in a pH-triggered manner, were used to study the alkaline mosquito larval midgut. By acting as molecular spies, these probes can provide valuable information about the localization and function of midgut proteins, which are crucial for digestion, immunity, and other physiological processes. Covalent modification of these proteins through targeted bioconjugation can disrupt normal larval development and prevent further insect population growth. Therefore, gaining knowledge about midgut proteins can aid in the design of effective and specific larvicides. Cysteine bioconjugation is another common protein bioconjugation technique which relies on native cysteines as well as genetically introduced cysteines on proteins. The story of the development of a modular bioconjugation probe for cysteine bioconjugation is also an emphasis in this dissertation. The UV cleavability associated with the novel probe can serve general purposes such as the release of a cargo which can be a drug, fluorophore or a pull down moiety. As mentioned in this disser

DYRK1A is a protein kinase and target for drug design. It was first discovered in neurological tissues and linked to neurological disease, particularly Down Syndrome and Alzheimer’s disease, where few treatment options are available. This has led to numerous studies seeking inhibitors of DYRK1A as new drugs for disease treatment, since inappropriate DYRK1A expression and activity contributes not only to Down Syndrome and Alzheimer’s disease, but also to other diseases including Parkinson disease, Pick’s disease, cancer and HPV (human papilloma virus).Development of new treatment strategies for these diseases is badly needed and development of specific inhibitors of DYRK1A provides a promising new avenue. Presented herein are studies designed to uncover mechanism in DYRK1A inhibition and provide insight for improved drug design. Crystallographic studies of DYRK1A in both inhibited and unliganded forms are presented. Structures with tight binding inhibitors DYR533, DYR542, DYR636 were determined and revealed their unique binding motifs, which include a new hydrogen bonding network. Regions of the inhibitors that do not contribute to binding were also identified, leading to suggestions for additional inhibitor improvements. Structures without ligand and with an ATP analog were also determined, which provide additional insight into drug efficacy. Inhibition studies under cellular conditions were also undertaken, revealing additional mechanistic insight.

Natural cell membranes are diverse and complex amphipathic barriers with thousands of different lipids that play multiple physiological functions. How membrane protein structure and function are affected by different lipids in the cell membrane is understudied. Furthermore, the lipid bilayer composition can change with age, disease, and diet;how these changes affect membrane protein structure and their link to disease and pathology are unknown. Measuring how different lipid interactions affect membrane protein and peptide structure is challenging because they require a hydrophobic lipid environment to be stable. To address this challenge, this dissertation details methods to assemble lipid bilayer mimetics called nanodiscs with multiple lipids that are resolvable by native mass spectrometry (MS). This allows us to retain noncovalent interactions and measure the oligomeric state of incorporated peptides or proteins in intact mixed lipid nanodiscs. Furthermore, these methods are applied to the characterization of gene therapy delivery systems termed adeno-associated viral (AAV) capsids.Previously, native MS of nanodiscs was limited to one or two lipid systems because of the added polydispersity in the mass spectra caused by the addition of a new lipid. To resolve nanodiscs with multiple incorporated lipids, we chose lipids that were integer or fraction multiples of each other, so that their m/z peaks overlap constructively. Using this mass resonance approach, we resolved two lipid nanodisc systems with sterols, monosialotetrahexosyl-ganglioside (GM1), and cardiolipin. Then, we assembled and resolved model mammalian, bacterial, and mitochondrial nanodiscs with up to 4 different phospholipids, which were useful for characterizing polydisperse lipid interactions with LL-37, which is a potential therapeutic for antibiotic resistant bacteria. Moving to more complex lipid systems, we investigated nanodiscs made from commercially available natural lipid extract that provide

I have examined the structure, mechanism, and functional relevance of two DNA-binding proteins in this study. The first of these proteins is SgrAI. It is a type II restriction endonuclease that recognizes and cleaves certain sequences, known as primary and secondary sites. Depending on the type of recognition site SgrAI is bound to, SgrAI has a dimeric form or a filamentous form. The dimeric form of SgrAI is an inactive structure which can cleave only primary site sequences slowly. On the other hand, when it is in the filamentous form, its cleavage rate increases dramatically for both primary and secondary sites. In the absence of primary site sequences, SgrAI will not form filaments. In addition, by substituting the first or second base pair of the primary recognition site, the secondary recognition site is formed. This suggests that interaction between SgrAI and these two base pairs plays an important role in filamentation regulation. The mechanism by which this regulation occurs, however, is as yet unknown. In previous studies, the structure of SgrAI bound to the primary site and one type of secondary site was solved. Cryo-EM was also used to determine the filamentous structure of SgrAI bound to primary site. According to structural and kinetic studies, substitution of the second base pair of the primary site can change the energy between the inactive (T state) and active (R state) states in a way that favors the T state and, as a result, prevents the formation of filamentous structures. However, these studies did not provide any information on the substitution of the first base pair. DNA is cleaved by SgrAI using a two-metal ion mechanism. The presence of both metal ions is required for optimal cleavage rates. In the previously solved dimeric structures, only one of these atoms was found at the correct position. The other one was located about 4A away from its predicted position. The displacement of this ion in dimeric form probably contributes to the slow DNA c

Cancer treatments often target singular oncoproteins in attempt to block one hallmark of cancer and stop tumor growth and progression. However, although these treatments are often initially effective, many cancer patients develop resistance and experience relapse due to redundant signaling or tumor heterogeneity. To avoid resistance mechanisms, cancer treatments must inhibit multiple hallmarks of cancer, and therefore multiple oncogenic signaling programs. One possible way to achieve this is to target nodes of convergence of oncogenic signaling, in which multiple oncogenic pathways depend on one protein for expression or signal proliferation. These nodes can be found within the central dogma processes that drive all cellular function. In this dissertation, this strategy is used to discover inhibitors of nodes involved in transcription, translation, and protein folding of oncogenes and oncoproteins. Targeting nodes of convergence may yield inhibitors that evade resistance mechanisms that plague current cancer therapies and provide superior clinical outcomes for cancer patients.

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