Browsing by Department "Chemistry"

The majority of consumer electronic devices, electric vehicles, and aerospace electronics are powered by lithium ion batteries because of their high energy and power densities. Commercially available lithium ion batteries consist of electrodes, separators and current collectors fabricated in multilayer rolls that are packaged in cylindrical or rectangular cases. The size and shape of the package as well as the composition of the electrode has a significant impact on the battery life and design of the products they power. For example, the battery life and shape of portable electronics such as cell phones or laptops, is governed by the volume that is dedicated to the battery. In the case of electric vehicles, decreasing the size and weight of the battery while increasing capacity is an engineering challenge that affects vehicle range and cost. Therefore, the of my dissertation consists of the development of a novel 3D printable lithium ion battery nanocomposites and the integration of conductive metal nanomaterials into conventional lithium ion anodes. Here, we report the development of PLA-anode, cathode, and separator materials that enable 3D printing of complete lithium ion batteries with a low-cost FFF printer for the first time. The most common 3D printing polymer polylactic acid (PLA) is an insulator. However, our work demonstrates that 3D printed PLA can be infused with a mixture of ethyl methyl carbonate, propylene carbonate, and LiClO4 provides an ionic conductivity of 2.3 x 10−4 S cm−1 which is comparable to that of polymer and hybrid electrolytes (10−3 to 10−4 S cm−1). It was found that up to 12-30 volume % of solids, depending on the filler morphology, could be mixed into PLA without causing it to clog during 3D printing. It was also found that not only is electrical conductivity crucial to the performance of a 3D printed lithium ion battery, but efficient electrical contact to the active materials is as well. To that effect, we investigated the effect of aspect ratio of silver-copper core-shell nanowires on the performance enhancement of a commercially fabricated graphite lithium ion anodes. Currently, carbon is the most common conductive filler used in commercial lithium ion battery anodes. We hypothesize that a more conductive, high aspect ratio would improve the performance of a lithium ion battery. We examined the effect of exchanging carbon with CuAg nanowires as the conductive filler in graphite lithium ion batteries. We tested 4 different aspect ratios and found that not only does aspect ratio matter, diameter and length have profound effect on capacity and energy of the anode at the same volume percent as carbon conductive filler.

In the last two decades, the field of homogeneous gold catalysis has been

extremely active, growing at a rapid pace. Another rapidly-growing field—that of

computational chemistry—has often been applied to the investigation of various gold-

catalyzed reaction mechanisms. Unfortunately, a number of recent mechanistic studies

have utilized computational methods that have been shown to be inappropriate and

inaccurate in their description of gold chemistry. This work presents an overview of

available computational methods with a focus on the approximations and limitations

inherent in each, and offers a review of experimentally-characterized gold(I) complexes

and proposed mechanisms as compared with their computationally-modeled

counterparts. No aim is made to identify a “recommended” computational method for

investigations of gold catalysis; rather, discrepancies between experimentally and

computationally obtained values are highlighted, and the systematic errors between

different computational methods are discussed.

Currently there is a dearth of analytical techniques for studying protein-ligand interactions on the proteomic scale. Existing techniques, which rely on various calorimetry or spectroscopy methods, are limited in their application to the proteomic scale due to their need for large amounts of pure protein. Recently, several mass spectrometry-based methods have been developed to study protein-ligand interactions. These mass spectrometry-based methods overcome some of the limitations of existing techniques by enabling the analysis of unpurified protein samples. However, the existing mass spectrometry-based methodologies for the analysis of protein-ligand binding interactions are not directly compatible with current mass spectrometry-based proteomics platforms.

Described here is the development and application of a new technique designed to detect and quantify protein-ligand binding interactions with mass spectrometry-based proteomic platforms. This technique, termed SPROX (Stability of Proteins from Rates of Oxidation), uses an irreversible covalent oxidation labeling reaction to monitor the global unfolding reactions of proteins to measure protein thermodynamic stability. Two variations of the SPROX technique are established here, including one variation that utilizes chemical denaturant to induce protein unfolding and a second variation that utilizes temperature to denature proteins. The SPROX methodology is tested on five proteins including ubiquitin, ribonuclease A, bovine carbonic anhydrase II, cyclophilin A, and calmodulin. Results obtained on these model systems are used to determine the method's ability to measure the thermodynamic parameters associated with each protein's folding/unfolding reaction. Results obtained on calmodulin and cyclophilin A are used to determine the method's ability to quantify the dissociation constants of protein-ligand complexes.

The primary motivation for the development of the SPROX protocols in this work was to create a protein-ligand binding assay that could be interfaced with conventional mass spectrometry-based platforms. Two specific SPROX protocols, including a label-free approach and an oxygen-16/18 labeling approach, are developed and demonstrated using the thermal SPROX technique to analyze ligand binding in a model four-protein component mixture consisting of ubiquitin, ribonuclease A, bovine carbonic anhydrase, and cyclophilin A. The thermal SPROX technique's ability to detect cyclosporin A binding to cyclophilin A in the context of the model mixture is shown using both labeling approaches.

An application using the SPROX technique combined with a multi-dimensional protein identification technology (MudPIT)-based proteomics platform is also described. In this application, which utilized an isobaric mass tagging strategy, 325 proteins in a yeast cell lysate are simultaneously assayed for CsA-binding. This study was also used to investigate the protein targets of an already well-studied immunosuppressive drug, cyclosporin A. Two of the ten protein targets identified in this work are known to interact with CsA, one through a direct binding event and one through an indirect binding event. The eight newly discovered protein targets of CsA suggest a molecular basis for post-transplant diabetes mellitus, which is a side effect of CsA in humans.

Metallosupramolecular polymers are increasingly of interest for functional and degradable polymeric materials. In these materials, the metal-ligand bonds often bear an external mechanical load, but little is yet understood about the nature of mechanically-triggered reactions of metal-ligand bonds and how that reactivity influences the mechanical limits of the material. This dissertation presents a poly(cyclooctene) polymer bearing 2,6-bis(1′-methyl-benzimidazolyl)pyridine (Mebip) ligands on sidechains, which provides easy incorporation into polymer backbones and sidechains, binding to a large variety of metal species, and facile synthesis with sites for future study substituent effects. This platform is employed in proof-of-concept studies comparing the crosslinking behavior of iron(II) trifluoromethanesulfonate and copper(II) trifluoromethanesulfonate. It was found through small molecule spectroscopic studies that both metal species bind in the desired 2:1 MeBip:metal stoichiometry for crosslinking. When these small molecule complexes are polymerized as crosslinkers in gel and solid networks, though the extent of crosslinking is found to be similar, the copper(II)-crosslinked networks exhibited a faster relaxation than the iron(II)-crosslinked networks. Further, under high strains, the copper(II)-crosslinked networks exhibited significantly higher extensibility. This work lays the foundation for further investigations of the effect of metal-ligand bonding on force-coupled properties of materials.

Biological electron transfer (ET) reactions are typically described in the framework of coherent two-state electron tunneling or multi-step hopping. Yet, these ET reactions may involve multiple redox cofactors in van der Waals contact with each other and with vibronic broadenings on the same scale as the energy gaps among the species. In this regime, fluctuations of the molecule and its medium can produce transient energy level matching among multiple electronic states. This transient degeneracy, or flickering electronic resonance among states, is found to support coherent (ballistic) charge transfer. Importantly, ET rates arising from a flickering resonance (FR) mechanism will decay exponentially with distance because the probability of energy matching multiple states is multiplicative. The distance dependence of FR transport thus mimics the exponential decay that is usually associated with electron tunneling, although FR transport involves real carrier population on the bridge and is not a tunneling phenomenon. Likely candidates for FR transport are macromolecules with ET groups in van der Waals contact: DNA, bacterial nanowires, multi-heme proteins, strongly coupled porphyrin arrays, and proteins with closely packed redox-active residues. The theory developed here is used to analyze DNA charge-transfer kinetics, and we find that charge transfer distances up to 3-4 bases may be accounted for with this mechanism. Thus, the observed rapid (exponential) distance dependence of DNA ET rates over distances of ≲10 Å does not necessarily prove a tunneling mechanism.

Molecular structures that direct charge transport in two or three dimensions could help to enable the development of molecule-based electrical switches and gates. As a step toward this goal, we use theory, modeling and simulation to explore DNA three-way junctions (TWJs). Molecular dynamics (MD) simulations and quantum calculations, indicate that DNA TWJs undergo dynamic interconversion among “well stacked” conformations on the time scale of nanoseconds, a feature that makes the junctions very different from linear DNA duplexes. The studies further indicate that this conformational gating would control charge flow through these TWJs, distinguishing them from conventional (larger size scale) gated devices. Simulations also find that structures with polyethylene glycol (PEG) linking groups (“extenders”) lock conformations that favor CT for 25 ns or more. The simulations explain the kinetics observed experimentally in TWJs and rationalize their transport properties compared to double-stranded DNA. Furthermore, we redesigned DNA TWJs that have equally coupled output pathways for charge. The TWJ was also designed to switch between the two conductive states in responsive to an applied electric field.

Computationally aided drug discovery confronts the problem to balance performance and computation cost. Earlier study reveals that a less-expensive docking approach is not reliable to estimate the protein-ligand affinity in exploring drug candidates targeting CARM1 (coactivator-associated arginine methyltransferase 1). However, more accurate binding free energy calculation based on molecular dynamics sampling is not affordable in a high throughput screening. A truncated MD method was developed and can be used to estimate the binding free energy with similar accuracy with the full-system MD methods, while reducing the computation cost ten fold. Thus, this truncated MD method is feasible in a high throughput screening towards drug discovery.

Recent work has shown that hyperpolarized magnetic resonance spectroscopy (HP-MRS) can trace in vivo metabolism of biomolecules and is therefore extremely promising for diagnostic imaging. The most severe challenge this technique faces is the short signal lifetime for hyperpolarization, which is dictated by the spin-lattice (T1) relaxation. In this thesis we show with theory, simulation and experiment that the long-lived nuclear spin states in chemically equivalent or near equivalent spin systems offer a solution to this problem. Spin polarization that has lifetime much longer than T1 (up to 70-fold) has been demonstrated with pulse sequence techniques that are compatible with clinical imaging settings. Multiple classes of molecules have been demonstrated to sustain such long-lived hyperpolarization.

Perovskite photovoltaics has attracted tremendous attention recently due to the advance in the device performance. However, it is still challenging to effectively commercialize the perovskite technology due to several issues including current-voltage hysteresis, stability, complicated device architectures, etc. In this dissertation, we use the additive to tailor the properties of the functional layers in perovskite photovoltaic devices, aiming to engineer the interface, film morphology, carrier dynamics and film crystallization process. By using the additive engineering approaches, our goal is to achieve high-performance perovskite photovoltaics with reduced hysteresis, improved stability, versatile processing methods and simplified device architectures.

Perovskite solar cells usually employ p-i-n device architectures and TiO2 is a typical n-type semiconductor widely used in perovskite solar cells. However, perovskite/TiO2 interface is not preferable for the photo-excited carrier collection due to the energy band misalignment, conductivity mismatch, etc. In chapter 2, additive was used to tailor the properties of TiO2 and enable improved interface for perovskite solar cells. With Nb5+ as additive in TiO2, the conductivity of TiO2 and interface band alignment were simultaneously improved. Consequently, high-performance perovskite solar cells were successfully obtained with reduced hysteresis by using the Nb-TiO2.

In addition to the interface, we explored the impact of morphology and carrier dynamics of perovskite films on solar cell performance. In chapter 3, NH4SCN and PbI2 were used as additives to tune the morphology and charge carrier dynamics of perovskite films. Using NH4SCN additive could significantly enlarge the grain size of the polymorph perovskite films while using PbI2 additive could increase charge carrier lifetime of perovskite films. It was found that the open-circuit voltage and fill factor of perovskite photovoltaics were correlative with charge carrier lifetime while short-circuit current density of perovskite photovoltaics were correlative with grain sizes. Using both PbI2 and NH4SCN simultaneously could synergistically improve the quality of perovskite films and performance of perovskite solar cells.

Based on the understanding from chapter 3, a room-temperature process was developed to deposit high-quality perovskite films by using PbI2 and methylammonium thiocyanate (MASCN) as additives in chapter 4. Due to the synergistic effects of the additives, room-temperature-processed perovskite films with micron-size grains and microsecond-range carrier lifetime were successfully obtained for high-performance devices. More importantly, we established the correlation between the crystal grain size in resultant perovskite films and the precursor aggregate size in precursor solutions. The correlation suggested that the perovskite grain sizes from solution process depended on the precursor aggregate sizes.

Following the understanding built in chapter 3, we used the additive engineering method to impact the performance of ETL-free perovskite solar cells. In chapter 5, we found out that the photo-excited carrier injection at the interface was significantly inhibited without the assistance of an ETL, which would compromise the collection of the photo-excited carriers. By using PbI2 as additive to tune the carrier lifetimes in perovskite films, it was found out that increased carrier lifetimes in perovskite films could effectively counterbalance the inferior interface without ETLs and enabled high performance for ETL-free perovskite solar cells. By using perovskite with microsecond carrier lifetime, ETL-free perovskite solar cells were successfully realized with performance comparable to that of ETL-containing perovskite devices. Such results offer the opportunity for the perovskite devices with simplified device architecture.

Molecular dynamics (MD) simulations is an invaluable tool to investigate chemical and biological processes in atomic details. The accuracy of MD simulations strongly depends on underlying force fields. In conventional molecular mechanics (MM) force fields, the total energy is divided into bond energy, angle energy, dihedral energy, electrostatic interactions and van der Waals interactions. Each of these energy terms is parameterized by fitting to either experimental data or quantum mechanical (QM) calculations. In this dissertation, our aim is to develop accurate force fields for small molecules, water and proteins fully from QM calculations of small fragments. In the framework of conventional MM force fields, we calculated both transferable and molecule-specific atomic polarizabilities of small molecules by electrostatic potential fitting. Atomic polarizabilities are the key physical quantities in induced dipole polarization model. Molecular polarizabilities recovered from our atomic polarizabilities show good agreement with those obtained from QM calculations. We believe the main limitation of conventional MM force fields is the limited form of its Hamiltonian. Going beyond conventional MM force fields, we adopt the many-body expansion method and residue-based systematic molecular fragmentation (rSMF) method to start afresh building force fields for water and proteins, respectively. We used electrostatically embedded two-body expansion as the Hamiltonian of bulk water. QM reference of electrostatically embedded water monomer and dimer at the level of CCSD/aug-cc-pVDZ are parameterized by neural network (NN). Compared with experimental results, our water force fields show good structural and dynamical properties of bulk water. We developed rSMF to partition general proteins into twenty amino acid dipeptides and one peptide bond. The total energy of proteins is the combination of the energy of these small fragments. The QM reference energy of each fragment is parameterized by NN. Our protein force fields compare favorably with full QM calculations for both homogeneous and heterogeneous polypeptides in terms of energy and force errors.

Single-molecule spectroscopic (SMS) measurements have revolutionized biological science due to their ability to directly observe exactly one molecule in the crowd. This single molecule observation removes the ensemble average, revealing molecular heterogeneities. However, traditional SMS techniques fail to study single molecules in the solution phase for a long observation time, because the molecules rapidly diffuse away from the small focal spot. Accordingly, it is typically required to isolate the molecules from the native solution to study time-dependent dynamical behaviors, by either tethering the molecules to a stationary surface or confining the molecules in a small space based on physical principles. This isolation of molecules barricades in-situ and in-vivo single-molecule research. To address this gap, real-time feedback single particle tracking (RT-3D-SPT) has been developed, with the ability to directly monitor individual freely diffusing particles in the solution phase less disruptively. Real-time tracking is realized by estimating the particle’s position using photon information and applying active feedback to keep the particle in a small detection center. This set of techniques is largely divided into two classes, each with its limitations. The first class of RT-3D-SPT techniques spatially separates the emission of the particle using a set of detectors. The signal variation detected by these detectors can be used to estimate the particle’s position in real-time. The second class of RT-3D-SPT uses a spatiotemporally patterned laser excitation to illuminate the particle. The detected photon arrivals can therefore be used to estimate the particle’s position within the dynamic laser excitation pattern. A feedback control actuator such as a scanning mirror or a piezoelectric stage is driven to compensate for the particle’s diffusion in real-time, keeping the particle in the focal volume. However, both methods have limited ability to track dim and fast diffusing objects, such as single molecules. Moreover, very few of these optical configurations provide simultaneous contextualization in three dimensions yet fail to observe rapid processes happening in the surrounding of the tracked objects.In this dissertation, we present a new real-time 3D tracking and imaging method to directly observe fast biological and chemical processes. These processes include the rapid protein adsorption onto nanoparticles when the nanoparticles are exposed to biological fluids. This adsorbed protein layer, called the protein corona, alters nanoparticles’ biological identity and their fate in vivo. Therefore, it is important to understand this critical roadblock in the biological application of engineered nanoparticles. Herein, we first introduce the construction of this microscope, called 3D real-time ultrafast local microscopy (3D-RULM), with its ability to track fast diffusing and lowly emitting objects while rapidly imaging the surroundings concurrently. Next, we show this RT-3D-SPT method can be applied to quantitatively characterize individual nanoparticle protein corona in situ particle by particle, with single protein sensitivity at signal-to-background ratios down to 1%. Finally, to expand this method to smaller NP-protein systems, we have further implemented a Galvo mirror as a control actuator with a response time five times faster than the currently used piezoelectric stage, opening the possibility to study transient NP-protein interactions and other fast biological phenomena in situ and in vivo.

The design of synthetic transformations that proceed selectively in the presence of multiple sites of similar chemical reactivity has been a long-standing challenge. Cross-coupling and C–H functionalization processes have gathered significant attention over the past few decades as a result of their ability to rapidly generate molecular complexity. Despite these advances, a chemist’s ability to judiciously, selectively, and predictably perform these synthetic transformations has not yet been fully realized. Herein disclosed are the investigations into new methods for the Suzuki-Miyaura and Hofmann-Löffler-Freytag reactions that proceed with predictable selectivity.

Tactical advances have resulted in the development of selective, serial, and exhaustive cross-coupling transformations of alkyl pinacol boronic esters with polyhalogenated (hetero)arenes. These Suzuki-Miyaura processes facilitate access to a wide variety of alkyl-substituted aromatic compounds, specifically 2-alkylpyridines, which constitute an important class of functional molecules.

Conceptual innovations have established sulfamate esters as a promising manifold for the radical-mediated functionalization of remote, aliphatic C–H bonds. New methods for the synthesis of sulfamate esters have facilitated access to these valuable functional motifs, thereby allowing their use in Hofmann-Löffler-Freytag reactions. Accordingly, the selective, sulfamate ester-guided chlorination of remote C(sp3)–H bonds has been developed. This transformation proceeds with unique site-selectivity and furnishes γ-functionalized masked alcohol derivatives.

The α-alkylation of N-sulfonyl hydrazones via in situ-derived azoalkenes provides an umpolung approach to ketone α-alkylation that has considerable potential with regard to catalysis and the direct incorporation of functionality not amenable to the use of enolate chemistry. Herein, the first Cu(I)-catalyzed addition of Grignard reagents to in situ-derived N-sulfonyl azoalkenes is described. This method is remarkable in its ability to deliver highly sterically hindered compounds that would be difficult or impossible to synthesize via traditional enolate chemistry, including those having up to three contiguous quaternary centers. This method is compatible with a wide variety of α-halo tosylhydrazones, including cyclic and acyclic α-halo tosylhydrazones as well as those derived from both ketones and aldehydes. Also, herein, the first asymmetric organocatalytic sulfenylation of in situ-derived nitrosoalkenes leading to chiral nonracemic α-sulfenylated ketones is described. The transformation proceeds in an umpolung fashion, relative to enolate/azaenolate methods, and uses simple thiols, thereby obviating the need for elecrophilic sulfur reagents. Moreover, excellent ee's were obtained starting from a variety of α-chloro oximes, including cyclic and acyclyic systems. Chiral nonracemic sulfur containing compounds are important both biologically, and in synthetic context through their use as chiral auxiliaries, ligands for metal catalysis, and organocatalysts. Also, herein, the addition of cuprates to α,β-epoxy tosylhydrazones is described. The transformation is operationally simple and efficient and has the unusual feature of giving high syn selectivity, which is opposite of that produced by a simple SN2-type epoxide opening reaction. This method compatible with α,β-epoxy tosylhydrazones with additional α-substitution, which provides access to aldol-like products that would be impossible to make using traditional enolate chemistry. Moreover, this method is compatible with a wide variety of both cyclic and acyclic α,β-epoxy tosylhydrazones, and produces dr's of >20:1.

While a number of different proteomic, genomic, and computational approaches exist for the characterization of drug action, each of the experimental approaches developed to date has both strengths and weaknesses. Currently, there is no one "perfect" assay for drug mode-of-action studies. A protocol that could assay all the proteins in the proteome for both direct and indirect binding interactions of drugs would greatly facilitate studies of drug action. Recently, the SPROX (stability of proteins from rates of oxidation) technique was developed as a chemical modification- and mass spectrometry-based strategy for detecting protein-ligand interactions by monitoring the change in thermodynamic stability of proteins upon ligand binding. This is accomplished by monitoring the denaturant dependent oxidation of globally protected methionine residues. The SPROX technique has been interfaced with bottom-up proteomics methods to allow for the proteome-wide analysis of protein-ligand interactions. However, the strategy has been limited by the need to detect and quantify methionine containing peptides in the bottom-up proteomics experiment.

The work in this dissertation is focused on evaluating the current SPROX protocol, developing modifications to improve proteome coverage, and applying the SPROX platform to two different drug mode-of-action studies. Three main strategies were employed to improve protein coverage. First, a chemo-selective isolation of un-oxidized methionine containing peptides was employed to enrich for methionine containing peptides, and it was found to produce a ~2-fold improvement in proteomic coverage. Second, a pre-fractionation strategy involving the use of isoelectric focusing was employed to decrease sample complexity prior to LC-MS/MS analysis and it was found to generate a ~2-3 fold improvement in proteomic coverage, however when combined with the methionine enrichment strategy the improvement was ~6-fold as the benefits of both were additive. Third, a tryptophan modification strategy was developed that could ultimately expand the number of useful peptides in proteome-wide SPROX experiments to include those that contain tryptophan. Also, investigated was the use of several different mass spectrometer systems (including a bench-top quadrupole and orbitrap system and two different quadrupole time-of-flight systems) in the SPROX protocol. The results of these studies indicate that there is a significant advantage in proteome coverage when faster mass spectrometers are used. The use of high energy collision dissociation (HCD) in the orbitrap system was also more advantageous than the use of collision induced dissociation (CID) in the Q-ToF systems. Regardless of the mass spectrometer used, the major source of error in the SPROX experiment was found to be the random error associated with the LC-MS/MS analysis of isobaric mass tagged peptides. This random error was found to yield a false discovery rate of between 3 and 10% for "hit" peptides in the SPROX experiment.

The above improvements in the SPROX protocol were used in two protein-ligand binding experiments. One set of experiments involved studies on two small molecules with a specific anti-cancer phenotype in human colon cancer cells. These studies identified 17 proteins as potential "hits" of these two small molecules. After preliminary validation of these proteins, approximately 50% were eliminated as false positives and one protein, p80/nucleophosim, showed consistent data indicating a destabilizing interaction with both small molecules. The destabilization is indicative of an indirect interaction with the small molecules that would be mediated through a protein-protein interaction network. In another set of experiments the breast cancer drug, tamoxifen, and its main, active metabolite, 4-hydroxy tamoxifen, were assayed for binding to the proteins in a yeast cell lysate to better understand its adverse effects on yeast cells. The results of these studies identified ~80 proteins as potential "hits" of these two drugs. After preliminary validation of these proteins, approximately 30% were eliminated as false positives and one protein, SIS1, type II Hsp40, showed consistent data indicative of a direct binding interaction.

Recently, several mass spectrometry-based proteomics techniques have been developed for the large-scale analysis of thermodynamic measurements of protein stability. This has created the possibility of characterizing disease states via differential thermodynamic stability profiles. Described here is the application of the Stability of Proteins from Rates of Oxidation (SPROX) technique to characterize mouse models of disease. The mouse models studied here are of normal aging and two genetically induced Parkinson’s Disease (PD) models.

Thermodynamic stability profiles were generated for 809 proteins in brain cell lysates from C57BL/6 mice at age 6- (n=7) and 18-months (n=9). The biological variability of the protein stability measurements was low, and within the experimental error of the SPROX technique. Remarkably, the large majority of the 83 brain protein hits were destabilized in the old mice, and the hits were enriched in proteins that have slow turnover rates (p<0.07). Furthermore, 70% of the hits have been previously linked to aging or age-related disease.

One of the PD mouse models involved characterizing the protein interactions induced by mutated leuceine-rich repeat kinase 2 (LRRK2) at a pre-symptomatic time point (3 months old). The models used were a control, overexpressed wildtype LRRK2, and overexpressed R1441G mutated LRRK2 (n=2 for all models). Comparative analyses on thermodynamic stability profiles of ~470 proteins revealed relatively few differences. In fact, the observed hit rate in each comparative analysis was close to that associated with the biological variability of the mice. However, four protein hits, dihydropyrimidinase-related protein 2, eukaryotic translation initiation factor 4A2, Rap1 GTP-GDP dissociation stimulator 1 and myelin basic protein, were identified with consistent thermodynamic stability in multiple mice within a biological state and as hits in multiple comparisons suggesting they are the most likely to be true positives.

The second PD mouse model studied was one in which the human α-synuclein protein, containing the known PD mutation A53T, was overexpressed. To characterize the disease progression of PD induced by this mutation, mice were sacrificed at 1 month (n=4), 6 months (n=4) and when they became symptomatic at 10-16 months (n=3). Thermodynamic stability profiles were generated for >850 proteins at each time point. The relative stabilities of these proteins were assayed in a series of comparative analyses involving mice at the different time points and the normally aged mice from above. In total 244 peptides were found to be differentially stabilized during PD progression. A subset of 52 peptide hits was identified to be of particular interest. Of these 52 peptides 22 were identified with early disease progression, 5 peptides showed late disease progression, 5 peptides reported a gradual difference in stability over disease progression and 20 peptides indicated no disease progression trend. More than 90% of the 32 peptides indicating a trend in disease progression showed progression related destabilization.

The results of this thesis help validate the use of thermodynamic stability measurements to capture disease-related proteomic differences in mice. Furthermore, these results establish a new biophysical link between the hit proteins identified and their role in aging, LRRK2 protein interactions, and PD progression.

Melanin is a biological pigment that is ubiquitous in nature and generally produced within melanosomes, specialized organelles. Typically, melanin is categorized into two distinct classes, based on color and molecular precursor: eumelanin (brown-black) and pheomelanin (yellow-red). Whereas much is known regarding the molecular precursors to the two pigments, an understanding of their resulting molecular structure remains elusive. Despite this lack of knowledge, several functions are attributed to the pigments, including photoprotection and photosensitization. Epidemiological data for skin and ocular cancers have observed an increased incidence for increased relative concentrations of pheomelanin. Furthermore, eumelanin is generally identified as photoprotective and antioxidant, whereas pheomelanin is generally identified as photoreactive and pro-oxidant. This thesis describes the photophysical properties of the naturally-occuring melanin pigments and presents new insights into their roles within the context of skin and ocular cancers.

Photoemission electron microscopy provides a unique opportunity to probe the complex photoproperties of melanins contained within intact melanosomes isolated from tissues of bovine and human eyes. Photoionization threshold potentials characteristic of eumelanin and pheomelanin have been determined and are used to investigate the molecular architecture of the pigments within the melanosome. Furthermore, a novel approach to photoemission electron microscopy is used to obtain the first direct measurements of the absorption coefficients from intact melanosomes.

Human iridal stroma melanosomes are comprised of both eumelanin and pheomelanin in various ratios according to iris color; dark brown and blue-green iris melanosomes are characterized by a eumelanin:pheomelanin ratio of 14.8 and 1.3, respectively. Despite the significant difference in the overall pigment composition, a common eumelanin surface photoionization threshold is obtained for both melanosomes. This data indicates that within the melanosome, the phototoxic pheomelanin pigment is encased by eumelanin. This structure mitigates the adverse photochemical properties of pheomelanin. However, damage to the eumelanic exterior and or significant reduction in the amount of eumelanin present could compromise the protective ability of eumelanin, providing mechanisms for exposure of pheomelanin and consequently contributing to oxidative stress.

The absorption spectra of intact melanosomes of varying melanin compositions were determined over the spectral range from 244 to 310 nm. The absorption spectra of eumelanic melanosomes are similar regardless of monomer composition or embryonic origin. Furthermore, the absorption spectra of melanosomes containing a mixture of pigments were similar to those containing pure eumelanin, arguing that the absorption properties of the melanosome are maintained regardless of increased pheomelanin composition. Therefore, the correlation between epidemiological data and the eumelanin:pheomelanin ratio is not predicted to be a reflection of the melanosome's decreased ability to attenuate biologically relevant wavelengths, but instead is predicted to be a reflection of the different photoreactivities of the melanin pigments contained within.

The development of electronic textiles, which have many potential healthcare and consumer applications, is currently limited by a lack of energy storage that can be effectively incorporated into such devices while having sufficient energy density, power density, and durability to perform well. The overall goal of this work was to improve the energy density and potential for use in electronic textile applications of a nanostructured composite of few-walled carbon nanotubes, manganese oxide, and reduced graphene oxide. Two approaches towards improving the desired properties by controlling the macroscopic structure of the composite were pursued: one, to make fiber or wire-shaped electrodes via wet-spinning in aqueous chitosan solutions (10% acetic acid), and the other, to make composite films with controlled porous structures using nitrocellulose as a sacrificial filler material. Both approaches yielded the desired macroscopic structures. The composite fibers were non-conductive due to the insulating nature of manganese oxide and its positioning on the surface of the fibers. Composite fibers of few-walled carbon nanotubes and reduced graphene oxide made by the same method were found to have good volumetric capacity, rate capability, stability and flexibility. Nonintuitively, electrochemical performance of composite films declined with increasing porosity due to a decrease in conductivity, highlighting the importance of balancing the interplay between properties important to device performance when designing controlled structures of complex materials.

The phylum Apicomplexa encompasses multiple obligate intracellular parasites that pose significant burdens to human health including the causative agents of malaria, toxoplasmosis, and cryptosporidiosis which infect millions of humans and cause hundreds of thousands of deaths each year. During their complex life cycles, apicomplexan parasites coordinate the function of specific proteins to both evade the host immune system and thrive under stressful conditions. Notably, Toxoplasma gondii has been found to harbour multiple polyketide synthase (PKS) genes by bioinformatic analysis, suggesting they can produce secondary metabolite polyketides. While secondary metabolite biosynthetic gene clusters (BGCs) have been known in Apicomplexa for over two decades, limited studies on these enzymes have been completed to date and there have been no characterized products, leaving a void in our understanding of the role of these enzymes in parasite biology. Therefore, characterization of these proteins may aid in our ability to target these biosynthetic enzymes as sources of potential therapeutic candidates in Apicomplexa.While protists are underexplored for biosynthetic potential, research points to this kingdom as an untapped potential for new chemical space. T. gondii for instance possesses multiple putative PKS biosynthetic gene clusters (BGCs) however there have been no secondary metabolite products elucidated thus far. Therefore, our work explores a T. gondii PKS, TgPKS2, and investigates the architecture, predicted structures, and activity of multiple domains within this synthase. Subsequently, Chapters 2 and 3 describes our initial studies on TgPKS2 including hydrolysis activities of acyltransferase (AT) domains, mutagenesis studies, and a first of its kind self-acylation activity of acyl carrier protein (ACP) domains in a modular type I PKS. Chapter 4 further emphasizes the unique attributes of TgPKS2, delving into a never before characterized chain release mechanism, while Chapter 5 compares TgPKS2 transacylation activity to well-characterized bacterial and fungal systems. Combined, these chapters describe our work to biochemically explore TgPKS2, discover the role it plays within the T. gondii life cycle, and further our work to elucidate the metabolite(s) produced by this synthase. Altogether, this research lays the ground work for exploring other apicomplexan and eukaryotic polyketide synthases and significantly increases our knowledge of the biochemical properties of these unique proteins.

Biological signaling via DNA-mediated charge transfer between high-potential [4Fe4S]2+/3+ clusters is widely discussed in the literature. Recently, it was proposed that for DNA replication on the lagging strand, primer handover from primase to polymerase α is facilitated by DNA-mediated charge transfer between the [4Fe4S] clusters housed in the respective C-terminal domains of the proteins. Using a theoretical-computational approach, I established that redox signaling between the clusters in primase and polymerase α cannot be accomplished solely by DNA-mediated charge transport, due to the unidirectionality of charge transfer between the [4Fe4S] cluster and the nucleic acid. I extended the study by developing an open-source electron hopping pathway search code to characterize hole hopping pathways in proteins and nucleic acids. I used this module to analyze protective hole escape routes in cytochrome p450, cytochrome c oxidase, and benzylsuccinate synthase. Next, I used the module to analyze molecular dynamics snapshots of a mutant primase, where the Y345C mutation (found in gastric tumors) attenuates charge transfer between the [4Fe4S] cluster and nucleic acid, which in turn, could disrupt the signaling process between primase and polymerase α. In another protein-nucleic acid system, I found that charge transfer in the p53-DNA complex plays an important role for p53 to differentiate Gadd45 DNA and p21 DNA in metabolic pathway regulation. Using density functional theory calculations on molecular dynamics snapshots, I found that hole transfer (HT) from Gadd45 DNA to the proximal cysteine residue in the DNA-binding domain of p53 is preferred over HT from p21 DNA to cysteine. This preference ensures that the p21 DNA remains bound to the transcription factor p53 which induces the transcription of the gene under cellular oxidative stress. This dissertation concludes with a study that demonstrates similar electron conductivities between an artificial nucleic acid, 2'-deoxy-2'-fluoro-arabinonucleic acid (2’F-ANA), and DNA. Compared to DNA, 2’F-ANA offers the additional benefit of chemical stability with respect to hydrolysis and nuclease degradation, thereby promoting its use as a sensor in biological systems and cellular environments.

This project is predicated on the ability of the boranophosphate modification of siRNA to increase its therapeutic applicability for gene silencing in in vitro and in vivo systems. It has been shown that the boranophosphate (BH3-PO3) can overcome many of the limitations that are traditionally found when using RNAi, namely nuclease stability. The synthesis of siRNA modified with 5'-(alpha-P-borano)-nucleoside triphosphates (NTP) analogs alone and in combination with 2'-deoxy-2'-fluoro nucleoside triphosphate analogs were performed and optimized. It was found that normal RNA transcriptions showed the highest yield with higher NTP concentrations and shorter incubation times. Boranophosphate modified RNA and 2'F/borano modified RNA transcription yield was optimal at lower NTP concentrations and extended incubations. The boranophosphate NTPs and RNA were characterized with high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance, indicating successful synthesis of NTPalphaB and 2'F NTPs. PAGE and mass spectrometry analysis were performed to ensure full-length transcription of the modified siRNA molecules. The effects of these modifications were explored with respect to the biophysical properties of the modified homoduplex and heteroduplex siRNA. The techniques used in this work included hybridization affinity assays (melting temperature), secondary structure determination (circular dichroism), nuclease stability assays, and assessment of the lipophilicity of the modified siRNA by determining partition coefficients.

Modification of siRNA with boranophosphate and 2'fluoro/borano modified NTPs appears to have caused the homoduplexes and heteroduplexes to adopt a more B form-like helix that had lower Tm compared to unmodified RNA. The stability of the siRNA transcript to enzymatic hydrolysis by Exonuclease T was on the order of 2'fluoro/borano> normal = boranophosphate. Boranophosphate modification increased the stability of the transcript to enzymatic hydrolysis by the endonuclease RNase A, compared to both normal and 2' fluoro modified siRNA. Overall, the 2' fluoro/borano modified siRNA showed the greatest biological stability. Modification of the siRNA with increasing percentages of boranophosphates resulted in increasing lipophilicity of the molecule up to 60-fold, compared to both normal and 2' fluoro RNA.

A method to site-specifically modify the boranophosphate siRNA using T4 RNA ligase was also investigated. Finally, the siRNA in this work was tested in several in vitro systems, yielding promising results for the usage of boranophosphate siRNA for use against human viruses and cancers. It was shown that in for in vitro systems for human papillomavirus gene expression (HeLa, SiHa, and W12E) and luciferase expression (B16F10 cells), boranophosphate modified siRNA can specifically downregulate gene expression, and in the case of human papillomavirus, can downregulate cell growth.

Natural products belonging to the lipid II-binding family act as potent antimicrobial agents by disrupting cell wall biosynthesis via sequestering the late-stage intermediate lipid II. However, the emergence of resistance mechanisms and poor bioavailability have hindered the utility of these molecules as promising therapeutic intervention strategies to combat pathogenic bacterial infections. Gaining a deeper understanding of structural components and biosynthetic pathways can lead to the creation of second-generation derivatives to improve bioactivity and pharmacological properties. To explore this superfamily, we have used bioanalytical, biochemical, synthetic, computational, and enzymatic approaches that have been applied to three distinct projects. The first includes efforts to characterize the relationship between structural feature and bioactivity for the lipid II-binding CDA (calcium dependent antibiotic), malacidin. Through a series of minimally complex analogs, we determined non-proteinogenic amino acids and the N-acyl fatty acid moiety are essential for bioactivity. For the second project, we investigated a conserved mechanism of action for phylogenetically-related natural products within the lasso peptide subfamily. This work led to the discovery of a novel class I lasso peptide, arcumycin, and we validated a conserved mechanism of action for Actinobacteria-produced lasso peptides in targeting lipid II biosynthesis. Our last project sought to elucidate the mechanism of lipoinitiation for the ramoplanin family of molecules. Through a series of bioactivity assays, we found the transfer to the acyl carrier protein (ACP) in a fatty acyl-AMP ligase (FAAL)-dependent manner determined the specificity of lipids selected in the biosynthetic process. Collectively, through each project we have gained a deeper understanding of the structural elements and biosynthetic pathways of lipid II-binding antimicrobials.