Browsing by Author "Webb, Ian"

Now showing 1 - 10 of 11
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    Campylobacter jejuni Periplasmic Nitrate Reductase NapA as a Medium for Elucidating Molybdenum Cofactor Reactivity
    (2025-05) Metzger, Mikayla; Basu, Partha; Blacklock, Brenda; Webb, Ian; Pu, Jingzhi
    Periplasmic nitrate reductase NapA is found within many gram-negative bacteria and catalyzes oxygen atom transfer reactions. NapA utilizes nitrate as a terminal electron acceptor in place of oxygen in low-air conditions, such as in the human gastrointestinal tract. Within the scope of this thesis, NapA from Campylobacter jejuni is primarily focused due to its reputation in poultry husbandry and antibiotic resistance. Although nitrate reduction aids in pathogen survival, it is also a geochemically essential process. Many enzymes closely related to NapA are involved in geochemical cycling and contain the same active site ligand structure. The ligand within these enzymes is called molybdenum cofactor, or Moco. The structure of the enzyme is influential on reactivity of the molybdenum cofactor. The rate of catalysis, electron and proton transfer, and substrate preference can be affected by the amino acids surrounding Moco. Elucidating details of Moco reactivity aids in understanding bacteria in relation to human health and geochemical cycles. The primary tools throughout this research were site-directed mutagenesis and steady-state kinetics.
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    Contributions of the Presynaptic Protein Bassoon to Tau Pathogenesis and Neurodegeneration
    (2024-05) Patel, Henika Sanjaybhai; Oblak, Adrian; Lasagna-Reeves, Cristian; McKinzie, David; Kim, Jungsu; Webb, Ian; Murray, Melissa
    Neurodegenerative tauopathies, characterized by the aggregation of misfolded tau protein, pose a significant clinical and scientific challenge. A high-molecular-weight (HMW) tau species is known to be involved in spreading tau pathology. However, the nature and composition of this species remain elusive, hindering targeted interventions. There are four main chapters in this dissertation. The first chapter highlights the existing knowledge about tau and its role in neurodegenerative tauopathies and discusses the possible contribution of protein interactors in the pathogenesis of tau pathology. The second chapter investigates the association between pathological hallmarks and functional deficits in the aged PS19 tauopathy model. The findings indicate that a diverse spectrum of pathological tau species may underly different symptoms and that neuroinflammation might contribute to functional deficits independent of tau pathology. In the third chapter, we isolated and characterized the HMW tau species with seeding capabilities from the PS19 brains. Using unbiased quantitative mass spectrometry analysis, we identified Bassoon (BSN), a presynaptic protein, as a crucial interactor of the HMW tau seed. BSN overexpression exacerbated tau-seeding and toxicity both in vitro and in the Drosophila model of tauopathy. Conversely, the downregulation of BSN reduced tau spreading and overall disease pathology in the PS19 mice, indicating the important role of BSN in taumediated pathogenesis. In chapter four, we studied the disease-associated p.Pro3866Ala missense mutation in BSN and further evaluated the mechanisms through which BSN could induce toxicity and neurodegeneration. Using CRISPR-Cas9 technology, we developed a knock-in mouse model harboring the BSN P3866A missense mutation in the endogenous murine Bsn. We observed somatic BSN accumulation suggesting that the P3866A mutation might be enhancing the aggregation propensity of BSN and provide a conducive environment to promote tau aggregation. Furthermore, we observed dysregulation in protein degradation pathways, neuroinflammation, and enhanced synapse elimination by microglia. These findings underscore the pivotal role of BSN in providing a favorable environment for tau aggregation and influencing the properties of the tau seed, thereby contributing to neurodegenerative processes. Overall, our results indicate that targeting BSN could be a potential therapeutic intervention for neurodegenerative diseases.
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    Dueling Electrospray Implemented on a Traveling-Wave Ion Mobility/Time-Of-Flight Mass Spectrometer: Towards a Gas-Phase Workbench for Structural Biology
    (Elsevier, 2019-07-05) Webb, Ian; Morrison, Lindsay; Brown, Jeff
    The traveling wave trap cell of a commercial ion mobility mass spectrometer (IM/MS) was used as a gas-phase reactor for covalent chemistry by making a simple modification to a standard nanoelectrospray source. Reagents and analytes were generated from pulsed opposite polarity nanoelectrospray sources and isolated by their m/z prior to reaction. Covalent bond formation was first observed with the model peptide angiotensin I. The modification site was identified as the N-terminus of the peptide by collision induced dissociation (CID). The IM cell separated the covalent reaction product from the proton transfer product by their respective ion mobilities. Next, the effects of several trapping parameters, including the trap traveling wave height, the trap RF voltage, and the trap pressure, were evaluated. Decreasing traveling wave height and increasing RF voltage and pressure increased the number of proton transfer events from apomyoglobin to reagent anions. The 6+ charge state of ubiquitin generated from nanospray under native-like conditions was covalently modified in the gas phase through ion/ion reactions. Probing the reacted protein with CID led to the assignment of lysine 29 and arginine 54 as reactive nucleophiles accessible to the reagent. IM analysis of the unmodified native-like 6+ charge state revealed that the gas-phase structure of the protein in the trap was in its compact form. Overall, we introduce a promising method for three-dimensional structural characterization of biomacromolecules.
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    Electrochemical Tape-and-Paper-Based Sensor for the Quantification of Potassium
    (2023-08) Zhang, Tommy; Deiss, Frédérique; Webb, Ian
    Potassium levels in serum are used in the diagnosis of diseases involving cardiac arrhythmias, neuromuscular weakness, and chronic kidney diseases. These illnesses are becoming more prevalent, therefore, developing new potassium quantification methods would aid in advancing preventative care. Current methods of quantifying potassium mainly rely on the use of glass ion-selective electrodes which are costly, fragile, and requires frequent maintenance and recalibration. For faster and more accessible quantification of potassium, we are developing low cost, portable, and easy to fabricate electrochemical tape-and-paper-based devices. Our sensor bypasses the inconveniences of ion-selective electrodes and could ultimately serve as a point-of-care device to allow for regular monitoring or even home-use. Our sensing method relies on Prussian blue immobilized on the surface of electrodes as a potassium recognition element. Potassium ions intercalate into the Prussian blue lattice and subsequently changes the electrochemical characteristics of Prussian blue such as the redox peak potentials. These devices are highly robust, feature a limit of detection of 1.3 mM potassium and the response is linear to at least 100 mM, which contains the clinically relevant ranges required for diagnostics. Quantification was developed using cyclic voltammetry, demonstrated in Chapter 3. We observed changes in Prussian blue redox peak potentials at different concentrations of potassium and followed the expected Nernstian response. We investigated multiple methods of immobilizing Prussian blue onto the electrode surfaces to investigate stability and reproducibility in Chapter 4. Adsorption, in-situ synthesis, and carbon paste incorporation of Prussian blue was tested. Prussian blue-carbon paste devices had reproducibility issues and featured broad reduction peaks. In-situ synthesis of Prussian blue directly onto the surface of the electrodes also featured broad reduction peaks but the Prussian blue response was reproducible. The issue with in-situ synthesis was the stability of the Prussian blue layer, which was susceptible to degradation after repeated use of the device, which is required for evaluating the performance of the device. Although adsorption using Prussian blue in water had some reproducibility issues as well, this method led to the most stable Prussian blue layer, had distinct reduction peaks, and was simple to perform. Various solvents were used to dissolve Prussian blue in Chapter 5 to investigate methods of increasing device reproducibility when using adsorption. A few organic solvents were able to dissolve Prussian blue to form a stable solution with the goal of forming a more uniform Prussian blue layer and potentially improving consistency of the layer immobilization. While these alternative solvents were able to dissolve Prussian blue, they also damaged the graphite electrodes on the devices, which altered the electrochemical responses of the devices to the point where potassium quantification was no longer possible. Due to incompatibility between these alternative solvents and the devices, adsorption of Prussian blue in water continued to be used. Different modes of adsorption were explored and was optimized in Chapter 6. By altering the adsorption setup and allowing the Prussian blue particles to settle evenly onto a level electrode surface, device reproductivity increased substantially. To understand the applicability of the devices in real samples, interferent studies were performed in Chapter 7. Other cations such as Na+, Li+, Ca2+, Mg2+, and Ba2+ were not observed to enter the Prussian blue lattice in the cyclic voltammograms. Monovalent cations that share the same charge as K+ but have smaller ionic radius, Na+ and Li+, were able to decrease K+ sensitivity. Divalent cations that had a smaller ionic radius than K+ did not alter sensitivity. The exception was Ba2+, which also decreased K+ sensitivity. These results suggested that both ionic radius and charge of a species were important factors in impacting K+ intercalation into the Prussian blue lattice. Other interferents such as sulfates, phosphates, carbonates, urea, and lactic found in serum and sweat samples were tested. The presence of these interferents decreased the current intensity of the reduction peak of Prussian blue, which resulted in less definition in the peaks. For the future of this project, the effects of interferents found in serum and sweat must be investigated further. Additionally, reproducibility of the devices could be improved further if less harsh organic solvents are tested for adsorption, square wave voltammetry could be used for quantification to evaluate the viability of alternative voltametric techniques, and Prussian blue analogues could be implemented into the devices for quantification of other cations.
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    Electrochemical tape-and-paper-based sensors suitable for oral pH measurements
    (2021-08) Cherebin, Oreoluwa; Deiss, Frédérique; Webb, Ian; Laulhé, Sébastien
    Low oral pH (<5.5) has been shown to play an important role in dental erosion. The measurement of oral pH can be useful in preventative care, in aiding the dental caregiver in determining the likeliness of future dental cavities. The measurement of oral pH has become a popular area of research in an effort to develop a more quantitative method for the diagnosis of dental caries. We are developing an electrochemical tape-and-paper-based pH sensor for future applications in oral pH measurements. These tape-and-paper-based devices are low-cost, easy to fabricate, sterilizable, disposable and portable. The presence of intrinsic material defects of the painted graphite electrode generates oxo-groups which are electroactive. Some of these electroactive species, such as quinone, are pH-dependent and allow for the measurement of pH using cyclic voltammetry. There is shift in the potential of the redox peaks corresponding to the sensing species that can be correlated with the pH of a sample. We optimized the assay with a conditioning oxidative potential pre-treatment step and an ionic adjuster to carry out the pH measurements. We characterized the devices in different buffer solutions, as well as commercial pH standards and establish calibration curves. The reproducibility of the electrochemical response of the devices was also successfully across multiple devices and users. Their shelf-life was demonstrated to be at least three months. The devices successfully measured the pH of beverage and mouthwash, and different formulations of artificial saliva. Their performance in the presence bacteria and in growth media was assessed. Some complex matrices such as growth media required some additional optimization. Towards this objective we fabricated and tested devices with various formulations of carbon paste for the painted working electrode. These flexible tape-and-paper-based devices are promising sensors for pH measurements in oral samples and potentially even for in vivo and in situ pH measurements.
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    Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions
    (2021-12) Carvalho, Veronica Vale; Webb, Ian; Manicke, Nicholas; Laulhé, Sébastien
    Gas-phase ion/ion covalent modification was coupled to ion mobility/mass spectrometry analysis to directly correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross-section (CCS) distributions were measured prior to ion/ion reactions to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Ubiquitin ions were electrosprayed from aqueous and methanolic solutions yielding a range of different charge states that were analyzed by ion mobility and time-of-flight mass spectrometry. Aqueous solutions stabilizing the native state of ubiquitin generated folded ubiquitin structures with CCS values consistent with the native state. Denaturing solutions favored several families of unfolded conformations for most of the charge states evaluated. Gas-phase covalent labeling via ion/ion reactions was followed by collision-induced dissociation of the intact, labeled protein to determine which residues were labeled. Ubiquitin 5+ and 6+ electrosprayed from aqueous solutions were covalently modified preferentially at the lysine 29 and arginine 54 residues, indicating that elements of secondary structure, as well as tertiary structure, were maintained in the gas phase. On the other hand, most ubiquitin ions produced in denaturing conditions were labeled at various other lysine residues, likely due to the availability of additional sites following methanol and low pH-induced unfolding. These data support the conservation of ubiquitin structural elements in the gas phase. The research presented here provides the basis for residue-specific characterization of biomolecules in the gas phase.
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    Machine Learning Facilitated Quantum Mechanic/Molecular Mechanic Free Energy Simulations
    (2023-08) Snyder, Ryan; Pu, Jingzhi; Naumann, Christoph; Webb, Ian; Deng, Yongming
    Bridging the accuracy of ab initio (AI) QM/MM with the efficiency of semi-empirical (SE) QM/MM methods has long been a goal in computational chemistry. This dissertation presents four ∆-Machine learning schemes aimed at achieving this objective. Firstly, the incorporation of negative force observations into the Gaussian process regression (GPR) model, resulting in GPR with derivative observations, demonstrates the remarkable capability to attain high-quality potential energy surfaces, accurate Cartesian force descriptions, and reliable free energy profiles using a training set of just 80 points. Secondly, the adaptation of the sparse streaming GPR algorithm showcases the potential of memory retention from previous phasespace, enabling energy-only models to converge using simple descriptors while faithfully reproducing high-quality potential energy surfaces and accurate free energy profiles. Thirdly, the utilization of GPR with atomic environmental vectors as input features proves effective in enhancing both potential energy surface and free energy description. Furthermore, incorporating derivative information on solute atoms further improves the accuracy of force predictions on molecular mechanical (MM) atoms, addressing discrepancies arising from QM/MM interaction energies between the target and base levels of theory. Finally, a comprehensive comparison of three distinct GPR schemes, namely GAP, GPR with an average kernel, and GPR with a system-specific sum kernel, is conducted to evaluate the impact of permutational invariance and atomistic learning on the model’s quality. Additionally, this dissertation introduces the adaptation of the GAP method to be compatible with the sparse variational Gaussian processes scheme and the streaming sparse GPR scheme, enhancing their efficiency and applicability. Through these four ∆-Machine learning schemes, this dissertation makes significant contributions to the field of computational chemistry, advancing the quest for accurate potential energy surfaces, reliable force descriptions, and informative free energy profiles in QM/MM simulations.
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    QM/MM Applications and Corrections for Chemical Reactions
    (2023-05) Kim, Bryant; Pu, Jingzhi; Naumann, Christoph; Vilseck, Jonah; Webb, Ian
    In this thesis, we present novel computational methods and frameworks to address the challenges associated with the determination of free energy profiles for condensed-phase chemical reactions using combined quantum mechanical and molecular mechanical (QM/MM) approaches. We focus on overcoming issues related to force matching, molecular polarizability, and convergence of free energy profiles. First, we introduce a method called Reaction Path-Force Matching in Collective Variables (RP-FM-CV) that efficiently carries out ab initio QM/MM free energy simulations through mean force fitting. This method provides accurate and robust simulations of solution-phase chemical reactions by significantly reducing deviations on the collective variables forces, thereby bringing simulated free energy profiles closer to experimental and benchmark AI/MM results. Second, we explore the role of pairwise repulsive correcting potentials in generating converged free energy profiles for chemical reactions using QM/MM simulations. We develop a free energy correcting model that sheds light on the behavior of repulsive pairwise potentials with large force deviations in collective variables. Our findings contribute to a deeper understanding of force matching models, paving the way for more accurate predictions of free energy profiles in chemical reactions. Next, we address the underpolarization problem in semiempirical (SE) molecular orbital methods by introducing a hybrid framework called doubly polarized QM/MM (dp-QM/MM). This framework improves the response property of SE/MM methods through high-level molecular polarizability fitting using machine learning (ML)-derived corrective polarizabilities, referred to as chaperone polarizabilities. We demonstrate the effectiveness of the dp-QM/MM method in simulating the Menshutkin reaction in water, showing that ML chaperones significantly reduce the error in solute molecular polarizability, bringing simulated free energy profiles closer to experimental results. In summary, this thesis presents a series of novel methods and frameworks that improve the accuracy and reliability of free energy profile estimations in condensed-phase chemical reactions using QM/MM simulations. By addressing the challenges of force matching, molecular polarizability, and convergence, these advancements have the potential to impact various fields, including computational chemistry, materials science, and drug design.
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    Structural Comparison of Denatured and Supercharged Proteins in Gas and Solution Phases
    (2025-05) Cain, Rebecca; Basu, Partha; Webb, Ian; Manicke, Nicholas; Naumann, Christoph; Larriba-Andaluz, Carlos
    Mass spectrometry has become a popular tool for studying large biomolecules due to its high sensitivity. Mass spectrometry techniques like ion mobility separation and fragmentation methods allow for top-down analysis to determine protein structures. As a gas phase technique, it is crucial to understand how ions fold or unfold in the gas phase without the support of solvents/water, as well as how they behave during the various energy and pressure changes experienced throughout the instrument. It is especially important to understand how protein gas phase ions behave and to monitor any major structural changes occurring since protein structure directly influences protein function. There is a plethora of structural studies that aim to retain native protein structure without additives or the disruption of native salt bridges and inter- and intra- molecular bonds. However, more work is needed on non-native proteins. This work analyzes denatured and supercharged proteins using different mass spectrometry techniques to compare the solution and gas phase structures. First, measuring the amount of unfolding under various denaturing and instrument conditions helps to identify how denatured protein ions behave in the gas phase without the presence of solvent. Next, comparing denatured structures using solution and gas phase crosslinking identifies specific structural changes as proteins unfold, as well as tracks how well protein denaturation retains solution phase structure in the gas phase. Finally, protein supercharging helps to enhance mass spectrometry analysis by shifting charge state distributions towards a lower m/z range. By comparing a supercharged protein’s ATDs and CCS values, we can measure any influence supercharging has on protein structure. Crosslinking supercharged proteins also allows for a comparison of structure and can help confirm how supercharging agents add charge to proteins. Similar structures between solution and gas phases help to substantiate mass spectrometry as a robust tool for proteomic studies. Structural differences between the two phases facilitate the interpretation of gas phase phenomena for mass spectrometry analysis and highlights how solvent influences protein structure.