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The amino-acid composition of gelatin has been extensively studied, but until recently little attention has been paid to that of its precursor collagen, the natural protein of skin. Some of the analyses of collagen which have been reported were carried out by methods which are now considered unsatisfactory, and in no case have most of the major component amino-acids been determined in one sample of the protein. Further, most of the analyses have been made on coUagens which had been given an alkaline or enzyme treatment, or both, to remove the keratins, elastic fibres and reticular tissue which occur in close association with it, and there is a risk that such treatments will cause some modification of the collagen. For the present study, a sample of collagen has been prepared with the minimum of chemical treatment, since it was considered that the presence of small amounts of elastic fibres and reticular tissue would lead to less error than the treatments necessary to bring about their removal. Also, it seems doubtful whether it is possible to remove these proteins completely by any of the methods which have been suggested.

Early detection of malignant disease is crucial for timely diagnosis and effective medical intervention, which significantly increases survival rates and reduce financial burden on patients. Biomarkers are becoming increasingly important in detection of malignant diseases, because they can be employed for indicating diseases, predicting risks and monitoring the progression of diseases. In addition, biomarkers show up at early stages of diseases in human tissues and fluids (e.g., blood, urine and saliva), which shows great promise for early disease detection. In this dissertation, paper-based lateral flow strips (PLFSs) have been developed for the detection of disease biomarkers, including protein biomarkers and microRNA (miRNA) biomarkers from clinical samples and whole blood samples. Among most of the reported biosensors, PLFSs appear to be an effective tool for providing access to point-of-care (POC) applications, due to low cost, fast response, portability and ease of use. In addition, paper is compatible with biological samples, which allows its application in analyzing various biomolecules. However, conventional PLFSs exhibit insufficient sensitivity and poor interference resistance. In particular whole blood samples, which contain numerous interference biomolecules, substantially affect detection accuracy and specificity. In this dissertation, several strategies have been employed to solve the problems, including introducing PLFSs with ultrasensitive techniques, meanwhile modifying PLFSs with functional nanomaterials and paper accessory unit to reduce the interference biomolecules from human fluids. In summary, five chapters will be demonstrated: (1) Surface-enhanced Raman scattering (SERS) technique modified PLFSs for protein biomarker detection in clinical blood plasma samples. In this chapter, silica coated SERS nanoparticles (NPs) have been developed for improving Raman signal and detection sensitivity, at the same time, silica coating of the SERS NPs substantially improve the stability of the NPs in complex human fluids. As a result, the developed SERS-PLFS can realize direct detection of neuron-specific enolase (NSE) from clinical blood plasma samples of traumatic brain injury (TBI) patients. The test results of the SERS-PLFSs were compatible with those from the standard enzyme-linked immunospecific assay (ELISA) method; (2) Blood plasma separation unit (PSU) integrated PLFS for cancer protein biomarker detection from whole human blood sample. In this chapter, a paper based PSU was fabricated to efficiently retain red blood cells (RBCs) inside the unit to block their migration and meanwhile push the target protein contained plasma to the detection area of the PLFS. As a result, cancer protein biomarker-carcinoembryonic antigen (CEA) was successfully detected by the PSU-PLFS from whole blood samples; (3) Plasmonic chip and PSU integrated PLFS for protein biomarker detection from whole blood. In order to meet the high sensitivity demand, a gold nanopyramid array functionalized chip was integrated into a PLFS for amplifying Raman signal and ultrasensitive detection of TBI protein biomarker s-100β; while the PSU was utilized to reduce the RBCs interference from whole blood. As a result, compared with the result of the SERS-PLFS in Chapter 2, an improvement in LOD with two-order of magnitude was obtained; (4) Near-infrared fluorophores (NIRFs) functionalized PLFS for miRNA detection from blood plasma. In this chapter, NIRFs encapsulated silica nanoparticles were synthesized and incorporated into a PLFS for stroke biomarker miRNA-34 detection. Compared with a single fluorescent dye, the synthesized NIRF NPs encapsulated numerous fluorophores into one single silica nanoparticle, exhibiting amplified luminescent intensity. Moreover, the NIRF nanoparticles minimized the fluorescent background from biological matrices and test strip materials, which elevates signal-to-noise ratio and anti-interference capacity; (5) Duplex specific nuclease (DSN) based signal amplification strategy modified PLFS for ultrasensitive miRNA detection in blood plasma. In order to meet the high sensitivity demand of miRNA-34 measurement from the clinical blood plasma of stroke patient, the developed NIRFs-PLFS in Part 4 was further elevated with DSN modification for amplifying fluorescent signal. As a result, the DSN-PLFS exhibited an improvement in detection sensitivity with two-orders of magnitude in blood plasma.

This dissertation describes a line of research that addresses translational research questions related to the use of computerized simulation to affect the knowledge, beliefs, motivation and self-management behaviors of individuals with chronic disease. The specific research projects focus on type 2 diabetes (T2DM) and physical activity as exemplars of a prevalent chronic disease and an underutilized self-management behavior, respectively. We first describe a conceptual framework for the design of Consumer Health Informatics (CHI) applications. The design of an envisioned diabetes self-management application is described as an example of the application of design principles derived from this framework. Subsequent chapters describe tests of research questions related to this envisioned intervention. The second chapter describes the development and preliminary evaluation of the interface for the intervention described above. The estimation of simulated glucose curves for individuals with T2DM is described. Next, the formative evaluation of a paper-based prototype based on those curves and a novel method to measure individuals' outcome expectations are described. The third chapter describes a randomized experiment of a narrated simulation based on simulated glucose curves. This trial tested the question: can computerized simulations affect the beliefs and behaviors of individuals with T2DM? In this experiment participants' beliefs changed in accordance with the discrepancy between the presented evidence, and their prior beliefs, and in combination with the completion of a planning intervention, which resulted in significantly greater increase in physical activity. The fourth chapter describes a test of the question: can predictive models of the acute physiologic effects of behavior be individualized? In this study we compared different predictive modeling techniques and found that a mixed effects modeling approach improves in accuracy as the individual contributes more data. This result is foundational to the development of the next generation of our simulation-based intervention, and has implications for CHI as a field; these are discussed. The dissertation concludes with a review of the strengths and limitations of the work described, a discussion of the implications of this work for consumer health informatics and a brief discussion of the next steps in this line of research. [The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page: http://www.proquest.com/en-US/products/dissertations/individuals.shtml.]

Glycosyltransferases (GTs) are important proteins that are widely distributed in both prokaryotes and eukaryotes and play a crucial role in the biosynthesis of carbohydrates and glycoconjugates. They catalyze the formation of specific glycosidic linkages by transferring sugar moieties from activated sugars to a variety of biomolecules such as carbohydrates, lipids, proteins, or water. Progress in functional genomics technologies, such as DNA sequencing and proteomics, has allowed the identification of a large number of GT genes in several species. In general, functional genomics approaches, which include genomics, genetics, proteomics, and biochemistry, are used to determine the function of a gene (i.e., GTs). In biochemical approaches, the most direct way to assign a function to a gene is through direct testing of the enzyme activity of its product (carbohydrate) in vitro. However, in contrast to genomics/proteomics approaches, the biochemical approaches are the most difficult to adapt to high-throughput screening. The reason is that biochemical approaches require the use of isolated/purified proteins, which is labor-intensive and prone to the formation of undesired products resulting from background enzyme activity. Therefore, there is a need for the development of protein-based in vitro high-throughput platforms for determination of biochemical functions of proteins, including GTs, and their interactions with other proteins. To be advantageous, a protein-based high-throughput platform should have the following characteristics: i) the platform can be adapted to all GTs and synthases, ii) the detection method should be sensitive enough to demonstrate the formation of GT products, and iii) the platform should be simple and easy to implement or accessible to any laboratory. This work describes the development of a novel platform for screening of enzyme activities of GTs in vitro. It is called the in vitro GT-array (i-GTray) platform. This platform uses an in vitro cell-free expression system to produce tagged proteins directly from plasmid DNA and capture the tagged proteins on a solid surface, such as microplate wells, using a capture antibody (anti-tag antibody). Thus, i-GTray combines protein synthesis and purification with post-assay desalting paper spray-mass spectrometry (DPS-MS) analysis. DPS-MS has the capability to detect as low as 50 fmol amounts of transferase products.As a proof-of-concept, the i-GTray platform was used to investigate the activity of 21 putative fucosyltransferases (FUTs) members of the GT37 family (CAZy database) using five different acceptor substrates representing fucosylated plant CW polymers, such as xyloglucan (XyG), arabinogalactan-proteins (AGPs), and pectin (rhamnogalacturonan I and II (RG-I and RG-II)). This screening consisted in 288 assays (four microplates) and resulted in the identification of five rice XyG-FUTs, four rice AGP-FUTs, three Arabidopsis RG-I-FUTs, and one Arabidopsis RG-II-FUT. However, none of the 15-rice putative FUTs acted on RG-I and RG-II acceptors used in this study. The i-GTray platform was also used to investigate the specificity of protein interactions among rice GTs belonging to the GT43 and GT47 families, and the results obtained corroborated BiFC data. Data from both i-GTray and BiFC are in agreement and demonstrated the assembly of at least six OsGT43/OsGT47 complexes that represent the central core complexes of three xylan synthase complexes (OsXSCs).

Poplar (Populus sp.) is a promising biofuel feedstock due to advantageous features such as fast growth, the ability to grow on marginal land, and relatively low lignin content. However, there is tremendous variability associated with the composition of biomass. Understanding this variability, especially in lignin, is crucial to developing and implementing financially viable, integrated biorefineries. Although lignin is typically described as being comprised of three primary monolignols (syringyl, guaiacyl, p-hydroxyphenyl), it is a highly irregular biopolymer that can incorporate non-canonical monolignols. It is also connected by a variety of interunit linkages, adding to its complexity. Secondary cell wall formation requires the coordination of many metabolic pathways. Additionally, complex traits such as lignin are highly polygenic. While there are several methods to analyze lignin structure, 2D HSQC NMR is a powerful analytical tool that elucidates many structural traits of lignin simultaneously. This work examined the lignin structure of 409 unique Populus trichocarpa genotypes via HSQC NMR. Twelve lignin phenotypes were subsequently utilized for a genome-wide association study (GWAS) – a powerful approach for identifying loci contributing to natural phenotypic diversity. This deep phenotyping enabled GWAS to identify 756 candidate genes associated with at least one lignin phenotype. Many of these candidate genes have not been previously reported to be associated with lignin or cell wall biosynthesis. These results provide a valuable resource for gaining insights into the molecular mechanisms of lignin biosynthesis and offer new targets for future genetic improvement in poplar.

This master's thesis presents several instructional methods and techniques developed for each of eleven topics or subject areas in clinical chemistry: carbohydrate metabolism, lipid metabolism, diagnostic enzymology, endocrinology, toxicology, quality control, electrolytes, acid base balance, hepatic function, nonprotein nitrogenous compounds, and proteins. Behavioral objectives for instructor and students are written for each of these subject areas. Instructional methods and techniques used for presenting each of the areas include lectures, textbooks, programmed study, handout sheets, student reports or papers, discussions, and still projection media. (CS)

This dissertation outlines research quantifying the entering and exiting the quinone reaction chamber in NADH dehydrogenase or respiratory complex I. Respiratory complex I, the first complex in the respiratory electron transport chain. The respiratory electron transport is essential for all aerobic life. The methods used to quantify the entrance and exit process are geometric modeling, steered molecular dynamics, and singular value decomposition of the process. Five structures were analyzed: bacterial, yeast, ovine mt, mice mt, and human complex I. The structures reveal an almost 30 angstrom tunnel-like chamber for quinone binding in the core part of the enzyme, at the joint between the membrane and hydrophilic arms of the enzyme. The entrance of this quinone chamber located in ND1 subunit and has an apparent bottleneck of quinone/quinol passage. The first chapter introduces complex I and how transition state theory using diffusion kinetics gives an approximate maximum for the energy of crossing the bottleneck for quinone/quinol passage. Chapter 2 introduces the techniques used to quantify the difficulty of passage as well as methods to identify modes for collective confirmational changes for bottleneck opening. Chapters 3 and 4 are reproductions of the published papers based on this work. The appendices are reproductions of the supplemental information for those two papers.

Carbon fibers have unique properties that include high strength, low density and excellent chemical and thermal resistance. However, they have a low level of utilization because of their high price; typically around $30/kg for an entry level polyacrylonitrile (PAN) based carbon fiber. Low-cost carbon fibers derived from lignin are currently being investigated at the University of Tennessee, because using lignin as a precursor could significantly reduce production costs. Lignins obtained from the pulp and paper and the emerging biofuel industries have the potential to be used for carbon fiber production, however, they are typically unsuitable because of the high levels of impurity and variable thermal properties. This research study examines the potential of a novel organosolv process to provide high purity lignin for carbon fiber production. This fractionation separates woody and herbaceous bioenergy crops into their three main components: cellulose, hemicellulose, and lignin, each of which can be used within the biorefinery for the production of fuels or chemicals. In this program, organosolv derived lignin from both tulip poplar (Liriodendron tulipifera) and Alamo switchgrass (Panicum virgatum) were recovered and compared as starting materials for carbon fiber. The organosolv derived lignin was analyzed using several different methods to assess quality differences for potential carbon fiber manufacture. Their purities, chemical structures, consistencies, thermal, and carbonization properties were evaluated and lignin exhibiting optimal properties was used for fiber spinning and conversion to carbon fiber. Lignin exhibiting the best thermal performance was achieved by isolation at 150°C to 170°C with an acid concentration of 0.05 and 0.1 M H₂SO₄, and a fractionation time of 120 and 180 minutes. Organosolv fractionation conditions and their influence on the properties of lignin-based carbon fiber are presented in this thesis.

Over-exploitation of fossil fuels coupled with increasing pressure to reduce carbon emissions are prompting a transition from conventional petrochemical feedstocks to sustainable and renewable sourced carbon. The use of lignocellulosic biomass as a feedstock for integrated biorefining is of current high interest, as separation into its component parts affords process streams of cellulose, hemicellulose and lignin, each of which can serve as a starting point for the production of biobased chemicals and fuels. Given the large number of potential sources of lignocellulosic feedstocks, the biorefinery will need to adapt to the supplies available over a normal growing season. Of particular importance is the lignin fraction, as its conversion to chemicals and materials to allow economic viability of the operation. Previous work has demonstrated that organosolv fractionation effectively separates lignocellulosic biomass into its component parts. In this project, we investigated the use of organosolv technology for separating mixtures of lignocellulosic feedstocks to isolate pure lignin. Mixtures of switchgrass (Panicum virgatum), southern yellow pine (Pinus taeda L.), and hybrid poplar (Populus spp.) were separated using organosolv fractionation. Experiments were performed by heating the feedstock mixtures at 150oC in a 3.5 L flow-through reactor with a ternary, one-phase solvent mixture of methyl isobutylketone (MIBK), ethanol (EtOH) and water (H2O) in a wt% ratio of 16/34/50, and containing sulfuric acid as a catalyst. The impact of different process variables was examined by experimental design (‘Design of Experiments’) to minimize the number of experimental runs using a balanced approach in the response surface to maximize inference. The process variables included two different runtimes (60, 120 min), two different sulfuric acid levels (0.05, 0.15 M), and four different wt% feedstock ratios for switchgrass/pine/poplar ([10/10/80], [10/80/10], [80/10/10], [33/33/33]). After completion of the initial experimental matrix, four additional center-points were carried out using a 90 min runtime, and 0.1 M acid level to validate the results for each of the four feedstock ratios. The dependent factors were lignin yield, lignin purity, and cellulose purity. Response surface methodology (RSM) was used to evaluate the impact of the process variables and to determine optimization settings for the process.

Alzheimer’s Disease (AD) is the 6th leading cause of death in the United States and affects over 6 million Americans. Surprisingly, dementia-related deaths have increased by over 16% during the SARS-CoV-2 pandemic, making finding a cure more now important than ever. AD is characterized by two major pathological hallmarks: amyloid plaques, rich in the intrinsically disordered, aggregation-prone Amyloid-β (Aβ) peptide, and neurofibrillary tau tangles. The transmembrane protein that produces Aβ, the Amyloid-β Precursor Protein (AβPP), is cleaved by β- (BACE1) and γ-secretases. While much of the Aβ-focused therapeutic and academic efforts have targeted late-stage, insoluble Aβ fibrils, interest has shifted to the more toxic intermediate oligomers. These transient, rapidly interconverting oligomers are exceptionally challenging to study and therapeutically target, a fact made abundantly clear by the succession of devastating drug trial failures. AβPP can be alternatively processed by other lesser-known enzymes, such as α-secretase, to produce alternative peptidic fragments. One such fragment, the p3 peptide, is a C-terminal fragment of Aβ, and spans residues 17-40/42, the segment most attributed to Aβ’s amyloidogenicity. Despite this, p3 has traditionally been described as non-amyloidogenic and neuroprotective. Consequently, the biological and biophysical properties of p3 have been sparsely studied. The studies described in this thesis aim to provide an extensive in vitro characterization of p3, to better understand its role, if any, in AD. In Chapter 1, we aim to summarize and deconvolute the small pool of conflicting findings in the literature surrounding the p3 peptide. According to PubMed, since 1984, there have been 56,502 papers that mention Aβ, and only 921 that mention p3, an over 60-fold difference (Figure 2). Of the small pool of papers published discussing p3 since the mid-1980s, only a handful investigate the properties of the peptide, while most simply state that it is non-amyloidogenic, or that its production precludes the production of Aβ. Despite this, a few rarely discussed papers, primarily published in the 1990s and early 2000s, expose amyloidogenic properties of p3. Our work, discussed in Chapters 2 and 3, builds on these early studies. In Chapter 2, we employed advanced chemical biological techniques to assess whether p3 is truly non-amyloidogenic, as indicated by the literature. We found that p3 self-assembles to form oligomers and fibrils morphologically indistinguishable from Aβ, and that these resultant aggregates share confirmational similarities with Aβ. Additionally, we determined that the rate of p3 fibril formation is significantly faster than that of Aβ. These results highlight the solubilizing effect of the N-terminus of Aβ, and the importance of hydrophobic contacts in amyloid formation. In Chapter 3, we investigated the kinetic and biological consequences of mixing p3 and Aβ, to simulate the endogenous heterogeneity of amyloid aggregation in the brains of AD patients. We observed fibrillar colocalization of Aβ and p3, and enhanced aggregation propensity of Aβ upon introduction of p3. This enhancement in stable, insoluble, heterogenous fibril formation resulted in reduced cellular toxicity. We found that under fibril forming conditions, mixtures of Aβ and p3 produced unique oligomers not observed in the homogenous preparations. Additionally, fibril formation proved favorable even under oligomer forming conditions. The enhanced fibril formation resulted in suppression of toxicity and ROS production in both PC12 and SH-SY5Y cells. Additionally, we found that at an early timepoint, TAMRA-Aβ and TAMRA-p3 uptake was comparable, while at a later timepoint, internalization of labeled-peptide was nearly 6x higher for the TAMRA-Aβ treated cells. However, no augmentation of uptake was observed upon addition of unlabeled p3 into TAMRA-Aβ treated cells. In Chapter 4, we discuss challenges in the field of intrinsically disordered proteins (IDPs), and offer novel methods to improve reproducibility. We first propose the benefits of employing all mirror-image peptides to both rigorously control peptide quality, and to probe complicated mechanisms in aggregation and toxicity of Aβ and related peptides. Through comparing the uptake of L- and D-Aβ, we observed that cellular uptake of Aβ is highly stereospecific, indicating that Aβ uptake is likely a receptor-driven process. We also demonstrated “chiral inactivation”, a technique previously developed by the Raskatov lab to abolish toxicity of Aβ42, with the Aβ40 system, which we monitored with 1H NMR. This chapter also presents a structural study of the long-orphaned Pauling-Corey rippled β-sheet. Current knowledge on rippled sheets is limited to a small pool of studies that combined partial experimental structures with theoretical modeling. At the end of Chapter 4, we report a high-resolution crystal structure, in which racemic (L,L,L)- and (D,D,D)-triphenylalanine form dimeric antiparallel rippled sheets, packed into herringbone layers. Overall, the studies described herein highlight the challenges and controversies in probing IDPs, and a few ways to overcome them. Special attention is paid to p3, a peptidic fragment of Aβ previously described as non-amyloidogenic and innocuous. We urge the field of AD-related research to expand the Amyloid Cascade Hypothesis (ACH) in light of these findings, to account for alternative proteolytic fragments of AβPP, and their resultant biological properties.

The coronavirus disease 2019 (COVID-19) pandemic caused millions of deaths and serious economic disruptions, also boosted the unprecedented development of novel nucleic acid detection methods. Although polymerase chain reaction (PCR) is the golden-standard and has been widely used for practical nucleic acid detection, isothermal amplification strategies capable of rapid, inexpensive, and accurate nucleic acid detection also provide new options for large-scale pathogen detection, disease diagnosis, and genotyping. Here we are going to describe an assay development journey from a simple COVID-19 detection assay to a genotyping strategy and eventually to a droplet-based amplification-free assay.First, we described a highly sensitive multicomponent XNA-based nucleic acid detection platform that combines analyte preamplification with X10–23 mediated catalysis to detect the viral pathogen responsible for COVID-19. It is termed RNA-Encoded Viral Nucleic Acid Analyte Reporter (REVEALR), functions with a detection limit of ≤20 aM (∼10 copies/μL) using conventional fluorescence and paper-based lateral flow readout modalities. With a total assay time of 1 h, REVEALR provides a convenient nucleic acid alternative to equivalent CRISPR-based approaches, which have become popular methods for SARS-CoV-2 detection. The assay shows no cross-reactivity for other in vitro transcribed respiratory viral RNAs and functions with perfect accuracy against COVID-19 patient-derived clinical samples.Second, we explained how we design REVEALR into a novel genotyping assay that detects single-base mismatches corresponding to each of the major SARS-CoV-2 strains found in the United States. Of 34 sequence-verified patient samples collected in early, mid, and late 2021 at the UCI Medical Center in Orange, California, REVEALR accurately identified the correct variant. The assay, which is programmable and amenable to multiplexing, offers an important new approach to personalized diagnostics.Third, we talked about an improved REVEALR platform, termed digital droplet REVEALR (ddREVEALR), that can achieve direct viral detection and absolute quantitation utilizing a signal amplification strategy that relies on DNAzyme multiplexing and volume compression. Using an AI-assisted image-based readout, ddREVEALR was found to achieve 95% positive predictive agreement from a set of 20 nasopharyngeal swabs collected at UCI Medical Center in Orange, California. We suggest that the combination of amplification-free and protein-free analysis makes ddREVEALR a promising approach for direct viral RNA detection of clinical samples.Finally, we summarized the developed DNAzyme-based nucleic acid detection methods, offered some alternatives, compared the DNAzyme-based platforms with CRISPR based platforms, and gave insight on potential future directions to further elevate the REVEALR system.

Synthetic biology holds promise for adapting and modifying molecular systems found in nature toward improving human life. This has sparked a modern bioindustry with applications ranging from biomanufacturing pharmaceutical products to engineered microbes for supporting space exploration. In the last decade two biotechnological advances have revolutionized the field of synthetic biology: CRISPR/Cas-mediated genome editing and directed protein evolution. As these principles have matured, advances in automation and artificial intelligence have been quickly adopted as major tools toward the generation of new molecular biotechnologies.This thesis incorporates the use of specific genetic systems and broad principles of evolution toward engineering new biotechnologies. To adopt novel genetic systems in synthetic biology applications they must first be fully understood. Thus, an important interest underlying the applied aspect of synthetic biology is solving genetic and proteomic mechanisms of action through basic science. One way of doing so is to understand the effects that these genetic systems of interest have had on the natural evolution of the organisms that utilize them.Toward this interest, my first chapter aims to better understand the impact that diversity generating retroelements (DGRs), may have had on evolving vast morphological, physiological, or adaptive complexity observed within the cyanobacterial phylum. This first chapter develops the hypothesis that, in cyanobacteria, DGRs have evolved to accelerate the evolution of proteins of a distinct functional class, compared to the DGRs of all other prokaryotic organisms currently known. This further sets apart cyanobacteria as an incredibly unique and diverse taxonomic clade within bacteria and sets DGRs apart as an important player in the evolutionary success of cyanobacteria. A paper has been communicated on the work presented in my first chapter, detailed below. Aside from the potential evolutionary and ecological impact DGRs may have had within the cyanobacterial phylum they are also of interest for biotechnological applications. DGRs are a potential tool for continuous in-vivo, targeted hyper-diversification within a small region of a gene. This is a desirable tool for directed evolution efforts, as the field currently lacks a means for generating highly diverse random mutations in-vivo in targeted genomic loci. As such, in-vivo directed evolution schemes are generally not attempted, though the efficiency of in-vivo natural selection schemes may be much greater when compared to in-vitro screening of individual protein variants as a selection scheme.The remaining two chapters of my thesis deal with mining the marine environment for other molecular systems that hold significant promise for developing new biotechnologies including products that may aid in space exploration. Marine isolates of Bacillus subtilis have been shown to display a unique propensity for producing highly complex and highly bioactive secondary metabolites. Furthermore, their ability to form endospores that are highly resistant to extreme environmental conditions make them an ideal candidate for engineering toward bioremediation and drug production for space conditions.The majority of pharmaceuticals are set, conservatively, to expire within two years of the manufacturing date, after which the reported potency and pharmacokinetics are no longer guaranteed. Though there are efforts to study and extend pharmaceutical expiration dates, viability of a product will vary based on manufacturing lot, storage conditions, and packaging. The environs of spaceflight may also affect the decay of pharmaceuticals, though the extent remains unclear. As such, for longer crewed missions, the pharmaceutical supply at launch is unlikely to remain viable for the duration of the mission. However, replenishment becomes complicated on any space mission, and particularly on longer- duration missions, when resupply is difficult, and it is impossible to predict the specific medication needs of the crew. Thus, a platform for on-demand drug synthesis is necessary to ensure the success of long-term space exploration and colonization missions to Mars and beyond.Thus, my second chapter presents the first step in a three-step process known as an “Astropharmacy”, a peptide drug production platform that uses genetically engineered bacterial spores or a cell-free system to produce drugs on-demand. Not only would this platform allow astronauts to produce drugs according to their needs, but it would also have utility on Earth, with applications including bioterror prevention, orphan drug production, and drug production in remote areas where refrigeration is impossible. Whereas my second chapter deals with mining the marine environment for microbial hosts which have unique and favorable evolutionary characteristics for space, my third chapter seeks to isolate a microalgal marine protein and study its ability to be engineered further to meet different needs we may have on earth.Microalgae have been considered the most promising biological chemists for the mass production of biologically sourced fuels. Algal organisms may have adapted this ability as a way of regulating cell membrane curvature and hydrophobicity. One goal in biofuel-focused synthetic biology is to reverse-engineer pathways that lead to the production of different hydrocarbon molecules to increase the efficiencies, yields, and culture survivability. One of the newest discoveries that apply to this effort is fatty acid photodecarboxylase (FAP) – an enzyme which uses light to make fuel from acidic fats.My third chapter details the need for confirming homologs of the originally discovered enzyme. Can mutations that work in the original enzyme be successfully transferred to its homologs? If so, it would allow us to consider homologs for directed evolution strategies including chimeric gene splicing. It would also allow us to add molecular tools found in other organisms to the biocatalytic toolbox of enzymes that enable light-driven decarboxylation of fatty acid substrates.

Cyanobacteria are photosynthetic microbes that have shaped the very environment of the world we live in over several billion of years. This is, in large part, because of their ability to perform the chemically exclusive processes of photosynthesis and nitrogen fixation. To segregate these processes temporally, they have evolved an elegant and complex circadian clock that aligns their physiology with the solar day to maximize biological fitness. This clock keeps time through the action of a biochemical oscillator comprising just three proteins: KaiA, KaiB and KaiC, that, along with ATP, can recapitulate a 24-h pacemaking activity in vitro. This process is achieved by a negative feedback loop akin to a biochemical game of Rock, Paper, Scissors, whereby the phosphorylation state of the hexameric ATPase KaiC is controlled by the nucleotide exchange factor KaiA, the ability of KaiA to stimulate KaiC repression is controlled by the metamorphic protein KaiB, and the ability of KaiB to inactivate KaiA is controlled by KaiC phosphorylation state. This creates a repetitive biochemical cycle that takes just bout 24 hours to complete. Additionally, the output proteins SasA and CikA interact with the clock in various phases of the biochemical oscillation that influence their ability to activate the master circadian transcription factor RpaA and orchestrate circdain gene expression throughout the cyanobacterial cell.This thesis focuses on the relationship between KaiC and KaiB. In particular, on positive cooperativity that increases KaiB’s affinity for KaiC, or in other words, the process by which initial binding of KaiB to the KaiC hexamer enables more efficient recruitment of KaiB to the remaining 5 binding sites. While this effect is well documented when considering KaiB and KaiC on their own, in Chapter 2 we expand this concept in the in the context of the entire reconstituted clock system including output pathways that link biochemical oscillation to DNA binding by the transcription factor RpaA. In doing so, we uncovered the unexpected result that SasA expands the range of permissive KaiB concentrations for biochemical oscillation to occur. I showed that SasA does this by binding to KaiC analogously to how KaiB does, and then recruiting additional KaiB molecules through positive heterotropic cooperativity. Integrating a novel crystal structure of the interacting domains of KaiC and SasA with existing crystal structures of the KaiC hexamer, I identified SasA mutations that abrogate heterotropic cooperativity but have only minor effects on the output signaling function of SasA. Remarkably, cyanobacteria bearing these mutations have defective circadian rhythms, demonstrating that SasA’s ability to bolster KaiB recruitment is an evolved aspect of biochemical oscillation.Chapter 3 describes our structural analysis of phosphomimetic variants of KaiC that differ in their ability to bind KaiB. Because crystallography has failed to identify the structural basis of this discrimination previously, we employed cryo-electron microscopy to analyze KaiC particles as a structural ensemble frozen in ice. Each KaiC protomer is composed of two ATPase domains, termed CI and CII. The phase-determining phosphosites are located on CII, and KaiB binds to KaiC over 70 Å away to the ADP-bound form of CI. Our data corroborated previous studies that daytime KaiC, which does not bind KaiB, loses interactions amongst the CII domain protomers within the hexamer, while maintaining a hexameric CI domain. Additionally, we obtained a relatively high resolution (3.2 Å) structure of a compressed form of KaiC where the CII domain breaks into a split washer with 2-fold symmetry, causing two of the CII domain subunits to interact more tightly with their respective CI domains. Using mutagenesis and various functional assays, I identified an allosteric conduit that connects the ATPase domains of the CI and CII domains of KaiC in both intra-protomer and inter-protomer contexts. Importantly, I trace these interactions back to cooperativity in KaiB association, with mutants along the pathway disrupting both KaiB affinity and cooperativity. Furthermore, I link KaiB cooperativity to ATP hydrolysis in the CI domain by identifying a key residue that senses CI nucleotide state. This residue is dispensable for both ATP hydrolysis as well as KaiB association, but is critical for both KaiB cooperativity and in vivo circadian rhythms. This, along with additional nucleotide dependence studies reported in Chapter 4, suggests that allosteric control of cooperativity through the CI active site is the structural basis for restriction of KaiB association to the nighttime KaiC phosphostates.Finally, in the latter half of Chapter 4 I summarize experiments that used the solvatocromatic dye Sypro Orange to detect changes in KaiC structure as a function of temperature and mutagenesis. I describe our preliminary efforts to build on these results by identifying additional solvatochromatic dyes that can leverage this effect to report continuously on the phase of biochemical oscillation by discriminate binding to different KaiC phosphostates at constant temperature.

Wildlife trafficking is a global issue with devastating ecological effects and synergistic relationships with other forms of international illicit networks. Furs and pelts are a major component of this trade and are more practical to transport. Proper identification of such items is important to be able to prosecute and punish poachers. Wildlife forensic investigators depend on reliable tools and methods to either include or exclude illegal versus legal species. However, due to the chemically and physically harsh production process, DNA-based methods often fail because the sample DNA is degraded. Morphological-based methods have poor species resolution, are time-consuming, and are dependent on a limited number of highly trained individuals. However, these challenges may be by-passed by focusing on the phylogenetic information in protein. DNA changes between closely related species are reflected at the protein level, allowing the phylogenetic information between species to lead to a species-level identification. Proteins are chemically more stable than DNA. Therefore, proteomic analysis of heavily processed items may provide an alternative, and robust, method of species identification. However, determining the amino acid sequences in mass spectrometry data requires the precise sequences to be present in a reference protein database. The hypothesis is that the protein sequences, or reference proteome, of a given species would match more than proteomes from other species and be more efficient at identifying peptide sequences in fur digests from the same species. This research focuses on the phylogenetic relationship between six cat species, lion (Panthera leo), leopard (Panthera pardus), tiger (Panthera tigris), cheetah (Acinonyx jubatus), Canada lynx (Lynx canadensis), and domestic cat (Felis catus); of which the first four had good-quality, morphologically-identified materials that were selected for analysis. The remaining two species, Canada lynx and domestic cat, were included in the phylogenetic comparison using their reference proteome databases. The selected fur or pelt reference material items were digested based on a previously-published method for protein extraction from human hair and analyzed by LC-MS/MS and PEAKS DB software. Species identification was achieved at three different levels: total peptide yield, protein coverage patterns, and presence of peptides containing species-specific markers. For each item sampled, the maximum number of total peptides identified using the PEAKS scoring algorithm was obtained using the corresponding theoretical database. For example, the raw peptides generated from the lion paw pelt yielded 13928 total identified peptides using the lion database. As evolutionary distance between the lion and related felid species increases, the total peptide yield decreases. Using the leopard, tiger, Canada lynx, domestic cat, and cheetah databases, the total peptide yields were less - 13766, 13758, 12604, 12307, and 11774, respectively. Similar results were obtained with the leopard pelt, tiger pelt, and cheetah coat sampled, demonstrating how species identification can be achieved through comparison of total peptide yields. Protein coverage patterns also indicated species of origin. Many keratin and keratin-associated proteins were identified from the hair and skin samples, including, but not limited to, Keratin 14, Keratin 16, Keratin 32, Keratin 35, Keratin 38, Keratin 75, Keratin 82, Keratin 85, Desmoplakin, and Corneodesmosin. Coverage patterns demonstrated the highest percent coverage when analyzing the raw peptides from a sample with its corresponding database. For example, the raw peptides generated from the lion sample that matched with Keratin 35 resulted in 84% coverage using the lion database, while the tiger, leopard, domestic cat, Canada lynx, and cheetah databases resulted in 77%, 75%, 77%, 75%, and 65%, respectively. Species-specific marker locations were identified by protein alignments between the sequences of all six species. Peptides containing these species-specific markers were then identified in the raw data and found to be present only in the samples that correlated with the species of origin. This paper demonstrates four individual examples of species-specific markers within Keratin 35 corresponding to the respective species of origin for all four sampled items: lion, leopard, tiger, and cheetah. In summary, the three approaches used in this project – the total peptide approach, the protein coverage pattern approach, and the species-specific peptide approach – individually demonstrated that species identification is plausible using phylogenetic proteomics. This data demonstrates the robustness and reliability of proteomic approaches to species identification from fur and lays the foundation for future research, such as the development of targeted proteomic assays, for broader application to wildlife forensic casework.

Pathogenic multidrug resistant bacteria cause a range of serious infections in humans. These bacteria have developed mechanisms to counteract the lethal effects of currently used antibiotics, creating a need for novel therapeutics. Gram-positive bacteria display a wide assortment of cell surface proteins that are important for bacterial survival and host-pathogen interactions. These key surface structures included pili, proteinaceous fibers that assist in microbial survival by mediating adhesion to host tissues and aid in the formation of biofilm. A large number of gram-positive bacterial species assemble pili and append surface proteins to the cell wall using sortase cysteine transpeptidase enzymes. These enzymes link the components of the pilus together via covalent lysine isopeptide bonds which confer enormous tensile strength. This dissertation describes my investigation of the assembly mechanism of the archetypal SpaA-pilus from Corynebacterium diphtheriae. It also describes my contributions to develop small molecule sortase inhibitors that could function as anti-infective agents and my work towards exploiting the activity of sortase enzymes as a protein engineering tool. This thesis focuses on sortase enzymes and can be divided into two major sections: studies to determine how sortases construct pili (Chapters 2-4) and work designed to discover a small molecule inhibitor for the Staphylococcus aureus Sortase A enzyme (Chapter 5). All of the studies have been published in peer reviewed papers, with the exception of work detailed in Chapter 4. Chapter 2 describes the NMR solution structure of the lysine isopeptide bond interface that connects the pilin components of the pilus. This structural information combined with biophysical and cellular analyses led to the formulation of the “latch” mechanism of pilus assembly. Chapter 3 describes an enzyme kinetic study of the sortase enzyme from C. diphtheriae (CdSrtA) which catalyzes the formation of lysine isopeptide bonds between components of the SpaA pilus. In this study, the rate-limiting step of catalysis was determined and variants of CdSrtA with improved activity were discovered. Chapter 4 describes research that employed biochemical and structural approaches to investigate how the incorporation of the SpaB pilin subunit terminates pilus assembly. Key differences were observed between the reaction that terminates assembly and the process of polymerization that builds the shaft of the pilus. Chapter 5 describes efforts to discover a small molecule inhibitor of the Staphylococcus aureus Sortase A enzyme that has the potential to be a therapeutically useful anti-infective agent. The first half of Chapter 5 describes the work done to improve the activity of previously discovered pyridazinone-based molecules using computational and synthetic chemistry methods. The second half describes the implementation of a novel cell-based screen to identify sortase inhibitors. This work leveraged the unique sortase-dependent growth phenotype of Actinomyces oris to screen for sortase inhibitors. Over 200,000 small molecules were screened for their ability to impair A.oris’s growth, which led to the identification of three molecular scaffolds that inhibit sortase activity in vitro. In totality, my thesis research has shed light on how sortase enzymes assemble pili in gram-positive bacteria, led to improved variants of CdSrtA sortase that can be used in protein engineering, and helped identify new small molecule sortase inhibitors with potential therapeutic applications.

HP1(Heterochromatin Protein 1) is a major component of heterochromatin, which is a highly condensed form of DNA playing an important role in multiple cellular activities including gene silencing. New research proposes that HP1 proteins could compartmentalize DNA into compacted chromatin by phase separation, which could be promoted by diverse HP1-binding proteins. STAT is a promising candidate. Besides its canonical role in JAK-STAT signaling pathway, previous research in our lab indicated that STAT contains HP1-binding PXVXI motif. Unphosphorylated STAT could bind to HP1 and maintain the stability of heterochromatin while phosphorylated STAT disperses from HP1, resulting in heterochromatin disruption. Thus, I hypothesized that phosphorylation induced conformational change on STAT, switching it from an HP1-binding state to a DNA-binding state. In this paper, I constructed computational models among HP1α, STAT3, and DNA to examine the influence of phosphorylation on STAT3’s binding affinity to both HP1α and DNA. During the preparation stage, I modified and constructed biomolecular structures for protein docking by Pymol and SWISS-MODEL. I imported the prepared biomolecular structures into HADDOCK and the web server provided potential binding complexes as output. I used the PRODIGY program to measure the Gibbs free energy and equilibrium constant of binding among unphosphorylated STAT3, HP1α and DNA as well as phosphorylated STAT3, HP1α and DNA. Compared to phosphorylated STAT3 homodimers, unphosphorylated STATA3 homodimers have higher binding affinity to HP1α and lower binding affinity to DNA. Although computational model has limitations and needs confirmation by further experiments in vitro, my results support the conclusion that phosphorylation drives STAT from HP1-binding to DNA-binding.

For much of human history, microbial infections have been a problem. The discovery of antibiotics has led to the prevention of many deaths and allowed for surgeries that would be too risky otherwise to be performed. In the post-antibiotic era antibiotic resistance has become an ever-growing crisis. Overuse and misuse of antibiotics have allowed for the development of resistance to antibiotics by many strains of bacteria. There have also been very few new antibiotics that have been developed in the past three decades. Antibiotics act on single biological pathways of bacteria and can be deactivated with relative ease, particularly when bacteria are exposed to a subinhibitory concentration. Pseudomonas aeruginosa (PA) is a gram-negative bacterium that has been able to develop resistance to many antibiotics. Nanoparticulate matter has properties that differ from their bulk counterparts. Many metallic nanoparticles have been examined as antimicrobial agents. These nanoparticles tend to harm bacterial growth by many different pathways simultaneously, making the development of resistance by bacteria much more difficult. Silver has been used as an antimicrobial agent for much of human history. Metallic silver nanoparticles have been explored as antimicrobial agents, but systematic evaluation of other forms of silver nanoparticles has been rare. Silver halides are photoreactive materials, most used in paper photography. In this work, I systematically evaluate the antimicrobial activity of silver halide nanoparticles against PA. The preliminary results indicate that silver halide nanoparticles exhibit higher antimicrobial activity and better silver resistance profiles when compared with the metal silver nanoparticles.

The greater part of this work describes the biochemical and morphological changes accompanying grain development in cereals. Attention is focussed on those aspects most likely to be involved in the regulation of grain yield and composition. Thus, since starch is the major component of grain yield much of the work is concerned with the developmental enzymology of carbohydrate synthesis. The composition, properties and structure of the developing starch granules and amyloplasts have also been described. Grain development is associated with a range of catabolic processes and a number of enzymes active in carbohydrate degradation has been identified. Although oxygen levels in the developing endosperm are unknown, recent work suggests that the mitochondria present are fully func- tional. It is possible that oxygen required for grain respiration is derived from pericarp photosynthesis. At the same time this tissue may fix carbon dioxide derived from endosperm respiratory processes. Thus, the pericarp may be involved in the overall control of endosperm metabolic activity. Studies of endosperm cell division indicate that this continues for more than half the developmental period, later divisions producing mainly aleurone cells. Hence, stimulation of endosperm cell division early in development will increase the yield of carbohydrate whereas at later stages it will increase the yield of protein. Further studies describe the pattern of mineral ion accumulation, the growth and nutrition of the develop - ing embryo and the morphological changes in the crease region through which nutrients move from the vascular tissue of the pericarp to the endosperm. A second group of papers is concerned with the control of a- amylase and polyphenol oxidase activity during cereal grain germination. Finally, there is a small group of papers describing research on the properties of photosynthesising tissues including marine algae.

Leishmaniasis is a neglected tropical disease which affects millions of people every year across 6 continents. Current antileishmanial drugs have become less effective due to resistance, have a high toxicity, and are too expensive for many lower socioeconomic countries. New drug candidates can be explored and synthesized by natural drug synthesis and high throughput screening (HTS). This paper aimed to explore both methods in search of novel antileishmanial compounds. For the natural product approach, the ABC ring of berberine will be optimized to improve its antileishmanial properties. For the HTS approach, a piperazine based small molecule has been synthesized and will be evaluated for its antileishmanial properties.

Chapter 1 presents the development of an efficient method for the expression and purification of aggregation-prone amyloid-β (Aβ) peptides, including Aβ(M1-42), 15N-labeled Aβ(M1-42), and Aβ(M1-42) familial mutants. Aβ peptides are central to the pathogenesis of Alzheimer’s disease. Aβ peptides are highly aggregation-prone, making them challenging to prepare and purify. Advances in amyloid research rely on improved access to Aβ peptides. Chemical synthesis of Aβ can lead to impurities, such as amino acid deletion products, that are difficult to eliminate during purification. Expression of Aβ(1-42) peptide requires the generation of fusion protein and cleavage by protease to remove the N-terminal methionine group that originates from the translational start codon, which can make the preparation of Aβ costly and time-intensive. In this chapter, I collaborated with fellow graduate student Stan Yoo to express Aβ(M1-42), a widely used form of Aβ with properties comparable to those of the native Aβ(1−42) peptide. Expression of Aβ(M1−42) is simple to execute and avoids the expensive and difficult enzymatic cleavage step associated with expression and isolation of Aβ(1−42). We then developed an efficient method to afford Aβ(M1-42) and 15N-labeled Aβ(M1-42) at around 19 mg per liter of bacterial culture with high purity using simple and inexpensive steps in three days. This method relies on the combination of protein biology tools such as bacterial expression and inclusion body solubilization, as well as peptide chemistry tools such as preparative HPLC purification of the solubilized inclusion bodies. This chapter also describes a simple method for the construction of recombinant plasmids and the preparation of Aβ peptides containing familial mutations. These methods may enable experiments that would otherwise be hindered by insufficient access to Aβ, such as NMR experiments with 15N-labeled Aβ. We anticipate that this method can be adjusted for the expression and purification of other amyloidogenic proteins.Chapter 2 presents the preparation of Aβ peptide with an N‑terminal cysteine [Aβ(C1–42)], the development of tailored chemical reaction conditions for the conjugation of aggregation-prone Aβ(C1–42) peptide with fluorophores or biotin, and the biophysical studies of labeled Aβ peptides.N-terminally functionalized Aβ peptides are important in amyloid and Alzheimer’s disease research. Site-specific labeling on the N-terminus of Aβ minimizes perturbation in the structure and function of the peptide, as the central and C-terminal regions of Aβ are more involved in fibril and oligomer formation. Although synthetic N-terminally functionalized Aβ peptides are commercially available, these peptides are expensive and limited to biotin and a few common fluorophores. Expressed Aβ peptides offer advantages over synthetic Aβ because they are free from amino acid deletions and epimeric contaminants, and they have been found to be more biological relevant because they aggregate more quickly and are more neurotoxic than synthetic Aβ. The method of preparing Aβ(C1–42) relies on the hitherto unrecognized observation that the expression of the Aβ(MC1-42) gene yields the Aβ(C1–42) peptide, because the N-terminal methionine is endogenously excised by E. coli. This observation is significant, because it allows the preparation of a useful Aβ(C1–42) peptide without the additional N-terminal methionine that originates from the translational start codon. The Aβ(C1–42) peptide represents a minimal modification of native Aβ(1–42), and the addition of a single cysteine residue at the N-terminus enables labeling of the expressed Aβ with complete site specificity using widely available maleimide-based reagents.The Aβ peptide is challenging to handle and label because of its strong propensity to aggregate. This chapter details the development of tailored chemical reaction conditions for handling and labeling of this aggregation-prone peptide. The labeling chemistry was optimized to be performed at pH 9 in HPLC fractions Aβ(C1–42), where Aβ remains mostly monomeric. Biophysical studies show that the labeled Aβ peptides behave like unlabeled Aβ and suggest that labeling of the N-terminus does not substantially alter the properties of the Aβ. This chapter also goes on to demonstrate the utility of the labeled Aβ peptides through fluorescence microscopy to visualize their interactions with mammalian cells and bacteria. Ready access to labeled Aβ bearing fluorophores will advance amyloid and Alzheimer’s disease research by enabling experiments for investigating the pathogenic mechanism, transport, and clearance of Aβ, as well as for screening anti-Aβ antibodies.Chapter 3 presents the introduction of intramolecular disulfide bonds to Aβ peptides that stabilizes Aβ into oligomeric state and the discovery of a disulfide-stapled peptide, AβC18C33, that forms homogeneous dimers and does not fibrilize. Aβ aggregates rapidly and exists as three different forms in equilibria: monomers, oligomers, and fibrils. Although Aβ fibrils are the most commonly found species in the brain tissue of patients with Alzheimer’s disease, soluble Aβ oligomers are the toxic contributors to neurodegeneration. Aβ oligomers are heterogenous and metastable: they can rapidly aggregate into more thermodynamically stable fibrils, which makes it challenging to isolate them and study their structures and biological properties. No high-resolution structures of Aβ oligomers have been reported thus far. Filling this gap is necessary for an enhanced understanding of the molecular basis of Alzheimer’s disease.In this chapter, we aimed to generate stable, non-covalent Aβ oligomers by introducing intramolecular disulfide linkages to Aβ. The intramolecular linkages were designed to enforce a β-hairpin conformation, the key conformation that favors Aβ oligomer formation and disfavors fibrilization. We have designed, prepared, and studied a variety of mutant Aβ peptides containing intramolecular linkages. Among these mutant peptides, we discovered AβC18C33, an Aβ peptide containing a disulfide bond between positions 18 and 33, that forms stable, homogeneous dimers and does not fibrilize, as evidenced by the results of a series of biophysical studies. The Aβ dimer has been proposed to be the basic building block of many larger Aβ oligomers and is thought to be one of the most neurotoxic and pathologically relevant species in Alzheimer’s disease. We thereby anticipate that the AβC18C33 peptide to serve as a stable, non-fibrilizing, and noncovalent Aβ dimer model for the exploration of the structure and pathogenesis of Aβ dimers, the generation of Aβ-specific antibodies, and the screening of Aβ-targeted drugs. Our laboratory is exploring ways to obtain high-resolution structures of this dimeric Aβ model peptide, using techniques including cryo-EM, NMR, and X-ray crystallography. Chapter 4 presents the structure-based design of a cyclic peptide inhibitor towards SARS-CoV-2 using free molecular modeling and docking software and publicly available X-ray crystallographic structures. When the COVID-19 pandemic forced the temporary closure of our laboratory in March 2020, we began working on the structure-based drug design of inhibitors against the SARS-CoV-2 virus main protease. These efforts resulted in the design of a cyclic peptide inhibitor, UCI-1. Working with other group members, I published a tutorial paper based to describe the structure-based drug design process and teach others how to do it using the publicly available software UCSF Chimera and AutoDock Vina.SARS-CoV-2 is a highly infectious virus that causes COVID-19, a serious respiratory infection that has caused over 178 million infections and over 3.8 million deaths worldwide as of June 2021. Main protease is a crucial enzyme that SARS-CoV-2 utilizes for site-specifically cleaving the polyprotein that is translated from viral mRNA and generating mature proteins that are necessary for replication and infection. The essential role of main protease, as well as the success of HIV protease inhibitors in the treatment of AIDS, make main protease an attractive therapeutic target in the treatment of COVID-19.This chapter begins with the analysis of the X-ray crystallographic structure of the main protease of the SARS coronavirus (SARS-CoV) bound to a peptide substrate. This chapter then describes the structural modification of the peptide substrate using the UCSF Chimera molecular modeling software to create a cyclic peptide inhibitor. Finally, this chapter presents the use of molecular docking software AutoDock Vina to show the interaction of the cyclic peptide inhibitor with both SARS-CoV main protease and the highly homologous SARS-CoV-2 main protease.The supporting information section of this chapter provides an illustrated step-by-step protocol showing the inhibitor design process. These and other molecular modeling studies helped our laboratory decide to pursue the synthesis of the cyclic peptide and experimentally evaluate its promise as an inhibitor of SARS-CoV-2 main protease. The molecular modeling studies presented in this chapter may help students and scientists design their own drug candidates for COVID-19 and the coronaviruses that may cause future pandemics.

The field of Combinatorial chemistry was first introduced in the early 1980s. In 1984 Geysen et al1 reported the use of a 96-well plate multi pin system for the synthesis and selection of peptides. This method was later modified by synthesizing the peptides onto cellulose paper, allowing the production of “spot libraries”2 . These methods3–7 , among others played a crucial role in the development of the field of combinatorial chemistry. Their contributions, the development of high-throughput screening methods and the advancements made in analytical characterization3 , revolutionized the pharmaceutical industry, and paved the way for the development of diverse and unique methodology4,8–13. One such method is the One-Bead OneCompound (OBOC) combinatorial method developed by Kit Lam in 199114. OBOC uses Fmoc-chemistry coupled with solid-phase peptide synthesis (SPPS) and “split-mix synthesis” to efficiently generate large peptide libraries with extensive synthetic diversity contained on millions of beads. Each of which contains a single unique peptide sequence that can be analyzed using Edman microsequencing or mass spectrometry. Figure A-1 shows the stepwise procedure used for OBOC libraries. Upon completion of the final step each library is globally deprotected using 4-methylpiperdine and trifluoroacetic acid (TFA) before being prepared for screenings. Using this method with 19 reaction vessels to synthesize a pentapeptide library we can produce up to 2,476,099 (195 ) individual peptides14 . The diversity of these libraries is further increased through the utilization of unnatural amino acids and small molecules allowing us to synthesize libraries containing billions of possibilities. This is one of the traits that makes iii OBOC a unique and versatile method that has been used to identify novel ligands for targets against live tumor cells15, protein inhibition16,17, and molecular imaging16,18–21 . While this method does have it disadvantages the first two chapters will be used to discuss projects in which using this method has been advantageous. The final chapter will go into more detail about the limitations and methods that can be used to overcome these limitations. In chapter 1 we discuss the use of OBOC technology to discover short peptide dye pairs to be used as molecular probes. These genetically encoded small illuminants (GESI) can be fused to integral membrane proteins (IMPs) and utilized to monitor changes to those proteins’ environment, such as structural or post-translational modifications (PTMs). GESI are smaller iv (~2kDa) than frequently used fluorescent probes like green fluorescent protein (GFP), which allows their grafting into IMPs without interfering with their physiological properties. These peptides, discovered from screening several cyclic OBOC libraries with confocal microscopy22 using molecular rotor dye(MRD) such as bromocresol purple as probes. This novel GESI technology, once fully developed, will greatly expand the capabilities of the molecular imaging toolbox. Here we report the discovery of several GESI peptides that were successfully transfected between GFP domain and the transmembrane domain of platelet-derived growth factor receptor (PDGFR) using HEK293T cells. Using Bromocresol Purple (BCP) we were able to demonstrate the utility of the proposed GESI imaging system in living cells. In Chapter 2 we focus on developing peptide binders to an allosteric binding pocket adjacent to the RGD motif binding site on integrin αvβ3. Integrins are a family of transmembrane proteins viewed as potential therapeutic targets because of their integral function in various physiological and pathological pathways. One of the prototypical binding sites associated with some of these integrins is known as the RGD binding site, named for its high affinity for RGD tripeptide motif. Recent studies on the binding mechanism of αvβ3 with its ligand Fractalkine in the absence of its receptor, CX3CR1, has supported the hypothesis that there is an allosteric binding pocket located on the β subunit. This site is adjacent to the classical RGD binding site and contrary to this site, is most accessible when the integrin is in its low affinity conformation. This theory is shown to apply to other proteins and not just Fractalkine when solubilized αvβ3, in its inactive state, was tested against human secreted phospholipase A2 type IIA (sPLA2- IIA), which is known to bind this integrin as well. By targeting an allosteric binding site, we v hope to develop peptides that are more specific and have reduced overall off-target effects. In this study we designed and prepared 6 cyclic and linear OBOC peptide libraries. These libraries were then screened using cell and protein binding assays with K562(+αvβ3) cells and a GST fusion protein of Site 2 peptide (QPNDGQSHVGSDNHYSASTTM, residues 267–287of β3, C273 is changed to S). Positive hits from both screenings were validated using both previous methods and with soluble integrin αvβ3 integrin, yielding 5 lead peptides. Of these only one peptide, Peptide 10, showed binding to K562(+αvβ3) when analyzed using flow cytometry. Lastly, we access various methods that can be used to reduce the non-specific binding found in OBOC libraries. This non-specific binding is inherent in many library screening methods and can be addressed in a variety of ways. In the OBOC method, the following three approaches are often used either individually or in combination to increase the screening stringency so that true positive ligands with high affinity can be identified: (i) including soluble competing ligands in the screening buffer, (ii) decreasing the concentration of screening probes, and (iii) down substituting the peptide displayed on the resin surface. Additionally, compounds that show binding and read as positive hits on resin can fail to translate well in solution, yielding peptides that must undergo significant optimization to be viable. It is important to develop methods to minimize non-specific binding. Here we use bivalirudin, an FDA-approved anticoagulant, as a model ligand against thrombin, and systematically assess the contribution of stealth peptides, linkers, peptide substitution, and resins to non-specific binding of proteins to peptide-beads, which could be readily determined by pull-down assay followed by gel electrophoresis. vi We found that replacing 50% of the peptide ligand on the bead surface with zwitterionic stealth hexapeptide EKEKEK, or simply (EK)3 could greatly lower the undesirable nonspecific binding. This new information was incorporated into the design of a focused OBOC library, which was then screened against thrombin.

Mutual Information (MI) based methods are used to predict contact residues within proteins and between interacting proteins. There have been many high impact papers citing the successful use of MI for determining contact residues in a particular protein of interest, or in certain types of proteins, such as homotrimers. In this dissertation we have carried out a systematic study to assess if this popularly employed contact prediction tool is useful on a global scale. After testing original MI and leading MI based methods on large, cross-species datasets we found that in general the performance of these methods for predicting contact residues both within (intra-protein) and between proteins (inter-protein) is weak. We observe that all MI variants have a bias towards surface residues, and therefore predict surface residues instead of contact residues. This finding is in contrast to the relatively good performance of i-Patch (Hamer et al. [2010]), a statistical scoring tool for inter-protein contact prediction. i-Patch uses as input surface residues only, groups amino acids by physiochemical properties, and assumes the existence of patches of contact residues on interacting proteins. We examine whether using these ideas would improve the performance of MI. Since inter-protein contact residues are only on the surface of each protein, to disentangle surface from contact prediction we filtered out the confounding buried residues. We observed that considering surface residues only does indeed improve the interprotein contact prediction ability of all tested MI methods. We examined a specific "successful" case study in the literature and demonstrated that here, even when considering surface residues only, the most accurate MI based inter-protein contact predictor,MIc, performs no better than random. We have developed two novel MI variants; the first groups amino acids by their physiochemical properties, and the second considers patches of residues on the interacting proteins. In our analyses these new variants highlight the delicate trade-off between signal and noise that must be achieved when using MI for inter-protein contact prediction. The input for all tested MI methods is a multiple sequence alignment of homologous proteins. In a further attempt to understand why the MI methods perform poorly, we have investigated the influence of gaps in the alignment on intra-protein contact prediction. Our results suggest that depending on the evaluation criteria and the alignment construction algorithm employed, a gap cutoff of around 10% would maximise the performance of MI methods, whereas the popularly employed 0% gap cutoff may lead to predictions that are no better than random guesses. Based on the insight we have gained through our analyses, we end this dissertation by identifying a number of ways in which the contact residue prediction ability of MI variants may be improved, including direct coupling analysis.

Oxidative stress is a physiological event caused by an overaccumulation of reactive oxygen species (ROS) within the body. While ROS are a natural by-product of oxygen metabolism, too many can lead to cell and tissue damage and contribute to many etiologies. Bisphenol A (BPA), a component of many plastic products, has been shown to induce oxidative stress. While the industrial usage of BPA usage has lessened, the safety of its replacements is unknown. This paper will primarily discuss ROS and mechanisms of oxidative stress, the usage of BPA and its analogues, etiologies associated with oxidative stress resulting from exposure to BPA and analogues, and a proposed experimental design to determine oxidative stress from exposure to BPA and its analogues.

The shape and charge of a protein play significant roles in protein dynamics in the biological system of humans and animals. Characterizing and quantifying the shape and charge of a protein at the single-molecule level remains a challenge. Solid-state nanopores made of silicon nitride (SiNx) have emerged as novel platforms for biosensing such as diagnostics for single-molecule detection and DNA sequencing. SSN detection is based on measuring the variations in ionic conductance as charged biomolecules translocate through nanometer-sized channels driven by an external voltage applied across the membrane. In this paper, we observe the translocation of asymmetric cylindrical structure CRISPR-Cas9 protein and symmetric cylindrical structure Avidin protein driven by an electric field through the solid-state nanopore. We also observe how glycerol impacts on the time durations and current blockage amplitudes produced by the translocation of two proteins in nanopore by using different glycerol concentrations.

Coenzyme Q (ubiquinone or Qn) is a benzoquinone lipid of varying isoprenoid tail length ‘n’, with essential functions within the respiratory electron transport chain and in cellular antioxidant defense. Several Coq polypeptides coordinate to drive the biosynthesis of Qn at the inner mitochondrial membrane, and are organized into a high-molecular weight complex known as the ‘CoQ synthome’, Absence of individual Coq enzymes results in decreased content of Qn and other Coq proteins, in addition to destabilization of the CoQ synthome. Patients with partial defects in Q10 biosynthesis suffer from a variety of debilitating diseases. Therefore, the studies outlined here seek to produce a more comprehensive understanding of the Qn biosynthetic pathway, allowing for more targeted therapeutic strategies. This work employs Saccharomyces cerevisiae as a model organism due to the high level of COQ gene functional conservation between yeast and humans. In S. cerevisiae, Coq11 was recently identified to associate with members of the CoQ synthome, and was required for efficient de novo Q6 biosynthesis. The function of Coq11 remains uncharacterized. Chapter 1 provides an overview of the coenzyme Q biosynthetic pathway in both humans and yeast, while highlighting the identification of Coq11 as a novel member of the CoQ synthome. Chapter 2 explores the functional relationship between Coq11 and Coq10, which have evolved as protein fusions in several fungal genomes. Data collected with help from Dr. Roland Stocker’s laboratory, Dr. Mario Barros, and Jenny Ngo from Dr. Orian Shirai’s laboratory, has demonstrated that the coq10Δ mutant respiratory deficiency, sensitivity to lipid peroxidation, and low de novo Q6 biosynthesis is rescued by deletion of COQ11. Further, yeast lacking COQ11 have increased expression of several Coq proteins and a stabilized CoQ synthome. These results indicate that Coq11 may serve as a modulator of Q6 biosynthesis. Chapter 3 outlines additional experimental characterization towards understanding the role of Coq11 within the Q6 biosynthetic pathway. With assistance from Dr. Lukas Susac from the laboratory of Professor Juli Feigon, various strategies for Coq11 purification have been documented, with some showing promise for future purification attempts. We have also revealed that Coq11 deletion in combination with deletion of two phenylacrylic acid decarboxylases, Pad1 and Fdc1, fails to effect Q6 biosynthesis. In collaboration with Dr. Mario Barros, several Coq11 overexpression vectors have been constructed and evaluated for their ability to restore Q6 biosynthesis in the coq11∆ mutant. The data reveal that yeast does not tolerate Coq11 overexpression from the majority of constructs, suggesting that Coq11 is a negative regulator of Q6 biosynthesis. In Chapter 4, two novel phosphatidylethanolamine methyltransferase deletion mutants, cho2 and opi3, were identified to have significantly higher Q6 compared to wild type. Dr. Anita Ayer from the Stocker laboratory conducted a preliminary large-scale screen of the S. cerevisiae diploid homozygous knockout library for mutants displaying altered Q6 content. With help from Dr. Lucía Fernández-del-Río, we confirmed that the cho2∆ and opi3∆ mutants have increased Q6 as well as a stabilized CoQ synthome, despite retaining wild-type amounts of Coq proteins. Finally, Appendices I-VIII contain previous publications that detail various aspects of coenzyme Q characterization in several organisms, in addition to a methods paper regarding the detection of protein-protein interaction networks. Together, this work provides novel insights regarding the biosynthesis, cellular functions, and regulation of coenzyme Q.

This paper reviews functions of Amyloid-β (Aβ) in healthy individuals compared to the consequences of aberrant Aβ in Alzheimer’s disease (AD). As extraneuronal Aβ accumulation and plaque formation are characteristics of AD, it is reasonable to infer a pivotal role for Aβ in AD pathogenesis. Establishing progress of the disease as well as the mechanism of neurodegeneration from AD have proven difficult (Selkoe, 1994). This thesis provides evidence suggesting the pathogenesis of AD is due to dysfunctional neuronal processes involving Aβ’s synaptic malfunction, abnormal interaction with tau, and disruption of neuronal homeostasis. Significant evidence demonstrates that AD symptoms are partially due to aberrant Aβ, and further experimental research may focus on repairing or preventing the noxious effects of Aβ.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides the highest mass resolving power and mass measurement accuracy for confidant identification of biomolecules such as proteins and peptides. Chapter 1 briefly provides introductory information on FT-ICR MS, description of the 14.5 T and 9.4 T FT-ICR MS instruments in our laboratory and basics of liquid chromatographic separation, electrospray ionization (ESI), hydrogen/deuterium exchange (HDX) technique. The last part of this chapter gives an overview of the bottom-up and top-down approaches for protein identification and characterization and finally, tandem mass spectrometry methods used for dissociation of biomolecules. HDX monitored by MS is an important non-perturbing tool to study protein structure and protein-protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast HPLC and supercritical fluid chromatography have been shown to decrease back-exchange by our laboratory. Chapter 2 shows that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent. This work has been published in J. Am. Soc. Mass Spectrom., 2012, 23, 699-707. ESI produces multiply charged ions, thereby lowering the mass-to-charge ratio for peptides and small proteins to a range readily accessed by trapped-ion (quadrupole, orbitrap, and ion cyclotron resonance (ICR)) mass analyzers (m/z = 400-2,000). For Fourier transform mass analyzers (orbitrap and ICR), higher charge also improves signal-to-noise ratio, mass resolution, and mass accuracy. Addition of m-nitrobenzyl alcohol (m-NBA) or sulfolane has previously been shown to increase the charge states of proteins. Moreover, polar aprotic DMF improves chromatographic separation of proteolytic peptides for mass analysis of solution-phase protein HDX for improved (78-96%) sequence coverage. In chapter 3, we show that addition of each of various modifiers (DMF, thiodiglycol, dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone) can significantly increase the charge states of proteins up to 78 kDa. Moreover, incorporation of the same modifiers into reversed-phase liquid chromatography solvents improves sensitivity, charging, and chromatographic resolution for intact proteins. This work has been published in Anal. Chem., 2010, 78, 7515 - 7519. In chapter 4, we investigate the extent of charging of different peptides and polyethylene glycols electrosprayed from different solutions with various modifiers to gain more insight into the supercharging mechanism. Small peptides without significant secondary structure exhibits higher sensitivity and shifts to higher charge states with DMSO. Also, DMSO increases charge and S/N for PEG derivatives as well. Furthermore, we discuss the preliminary results of ESI droplet size measurement with phase Doppler particle analyzer (PDPA) and also review all possible reasons that could potentially contribute to supercharging. Chapter 5 talks about the top-down electron capture dissociation (ECD) characteristics of multiply charged molecular ions of various proteins formed by the incorporation of supercharging reagents. Here, we show that addition of modifier and ECD of higher charge states can significantly increase the number of structurally informative backbone cleavages and diagnostic product ions. Moreover, though ECD of higher charge states improves protein sequencing, the sequence coverage for cytochrome c gradually decreases beyond a particular charge state. Also, there is no significant difference in the fragmentation of same charge state with various modifiers. ECD of supercharged ions can be anticipated to improve top-down sequencing and characterization especially for high molecular weight protein complexes. Previously, the highest-mass protein for which FT-ICR unit mass resolution had been obtained was 115 kDa at 7 T. In chapter 6, we present baseline resolution for an intact 147.7 kDa monoclonal antibody (mAb), by prior dissociation of non-covalent adducts, optimization of detected total ion number, and optimization of ICR cell parameters to minimize space charge shifts, peak coalescence, and destructive ion cloud Coulombic interactions. The resultant long ICR transient lifetime (as high as 20 s) results in magnitude-mode mass resolving power of ~420,000 at m/z 2,593 for the 57+ charge state (the highest mass for which baseline unit mass resolution has been achieved), auguring for future characterization of even larger intact proteins and protein complexes by FT-ICR MS. We also demonstrate up to 80% higher resolving power by phase correction to yield an absorption-mode mass spectrum. This work has been published in Anal. Chem., 2011, 83, 8391-8395. The dissociation efficiency of intact proteins decreases as the molecular size and structural complexity increases due to the non-covalent interactions that stabilize the higher order structures of intact protein. Chapter 7 is a short review that describes the challenges and improvements in the direct top-down dissociation of large intact proteins (>60 kDa). Various innovative methods of collisional activated dissociation (CAD) such as prefolding dissociation, octopole-CAD, multipole-storage-assisted dissociation (MSAD) etc and electron driven dissociation techniques coupled with mostly ultra-high resolution FT instruments are covered. The appendixes include the manuscripts I have published during my Ph.D. Appendix A is the paper that covers the method development and optimization to decrease back-exchange in HDX by incorporation of aprotic modifiers. Appendix B is a paper we published in Analytical Chemistry, covering enhanced charging and improved chromatographic separation of intact proteins by addition of new organic modifiers. Appendix C is a publication that describes record unit mass baseline resolution for an intact 148 kDa monoclonal antibody by FT-ICR MS in collaboration with Pfizer, Inc.

Structural biology is a field studying the relationship between structure and function of macro biomolecules, including proteins. Proteins with numerous different functions are synthesized by recruiting amino acids into a linear polypeptide chain. The linear protein sequence, determined by a gene, can fold into higher level structures. For a given protein, correct structure/conformation is important to maintain its proper biological function. Several biophysical tools such as X-ray crystallography, NMR and cryo-EM can be used to investigate protein structure. However, each method has its limitations and usually complementary methods are required to answer a complicated biological question. Hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS) has become a informative tool to study solution phase protein conformation. High-resolution mass analysis (such as Fourier transform ion cyclotron resonance mass spectrometer) is particularly advantageous for the HDX MS method, by accurately assigning the peptide fragments after protease digestion and in resolving extensive overlap of peptide isotopic distributions, both before and after HDX MS. Chapter 1 is started by introducing the theory of the FT-ICR MS and the actual instrumentation set-up of the Hybrid LTQ 14.5 T FT-ICR Mass Spectrometer. An introduction of several biophysical methods to study structural biology is also covered, followed by the examples of the usage of mass spectrometry in this field. The last portion of this chapter gives an overview of the HDX MS method, its principle, experimental strategy and the historical applications.In Chapter 2, a new automation system is described as part of the method development effort for HDX-MS in my PhD career. The new system permits flexible modification of all HDX parameters, e.g., number of HDX reactions, reaction volume, injection volume, HDX duration, quench duration, digestion duration, and chromatography duration. Additional steps such as pre-HDX handling of protein sample, additional replicates, or post-digestion treatments can be easily added and automatically interlaced. Chapter also describes the software developments for HDX-MS data analysis, including several add-ons developed in Python: 'Peak list based data evaluation.', 'Compare HDX data for multiple protein states.' and 'HDX heat-map and modeling HDX results on 3D structure.' Chapter 3 describes the HDX-MS application on the epitope mapping problems. Three projects are described consequently. The first project is a 'proof of principle' study trying to establish the HDX-MS method on a known antigen-antibody system. The second and third projects are the applications of HDX-MS method on detecting unknown epitopes. The result of the first project is published in Analytical Chemistry (Zhang, Q. et al. Anal. Chem. 2011, 83, 7129-7136.). The manuscripts of the latter two projects are being submitted soon. Chapter 4 describes a collaborative project combining cryo-EM and HDX-MS to solve the structure of COPII cage. COPII vesicles are involved in transporting proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. A 12 Å resolution structure of the COPII cage, from which the tertiary structure of Sec13 and Sec31 is clearly identifiable, is generated by cryo-EM data. This structure and a homology model of the Sec13-31 are coupled with flexible fitting methods to create a reliable pseudo-atomic model of the COPII cage. Data from hydrogen/deuterium exchange mass spectrometry analysis is combined with this model to characterize four distinct contact regions at the vertices of the COPII cage. These results are published in Nature Structure Molecular Biology recently. Chapter 5 discusses the use of solution-phase HDX experiments to probe the allosteric effect of ATP on molecular chaperone GroEL. The ~800 kDa tetradecameric GroEL plays an essential role in the proper folding of many cellular proteins via an ATP-driven cycle of conformational changes. We capture the preceding yet pivotal step in its functional cycle by use of a non-hydrolysable ATP analog, ATPγS, to mimic the ATP-bound GroEL intermediate conformational state. Comparison of HDX-MS results for apo GroEL and GroEL-ATPγS enables the characterization of the nucleotide-regulated conformational changes throughout the entire protein with high sequence resolution. GroEL is by far the largest protein entity yet accessed by HDX-MS, and the results achieved here establish the groundwork for further HDX-MS characterization of such large complexes in solution. This work has been recently accepted by Scientific Reports. The appendices include the papers I have published during my graduate study. Appendix A is a publication that covers the collaboration work with Dr. Kenneth Roux (FSU department of biology) on the epitope mapping of food allergens. Appendix B is the paper we published in Nature Structure Molecular Biology recently, covering the COPII cage structure project.

G protein coupled receptors play important roles in cellular signaling and are stimulated by a multitude of extracellular signals. These proteins initiate signal transduction cascades that elicit a variety of cellular responses. Once activated, G protein coupled receptors activate G proteins which further stimulate target proteins to elicit cellular responses such as enzyme activity, translation and ion channels60. In this study, we set out to solve the crystal structure of a constitutively active form of the alpha subunit of the retinal G protein, transducin. This activated G protein, designated as αT* SFD QLRC, elicits the ability to stimulate its effector molecule, PDE, to levels comparable to activated native retinal alpha. Crystals of αT* SFD QLRC were grown and x-ray diffraction data sets were observed at low resolution. Following insights from a recent paper by Hu et al, 2018, a C210S mutant was created to help improve diffraction. Data sets of αT* SFD QLRC C210S were collected to 2.8 Å and compared to previously solved GDP-bound (1TAG) and GTPγS-bound (1TND) native αT subunits. The crystal structure of αT* SFD QLRC C210S reveals that despite being fully active in vitro, GDP, and not GTP, is bound in the structure. As a result, Switch II and Switch III appear to be in a transition state between the inactive GDP-bound state and the active GTP-bound state and are un-modelled due to low electron density. Future work involves improving crystallization of αT* SFD QLRC C210S to obtain a GTP-bound structure so that the structure can be used to elucidate the mechanism by which the transducin α subunit, especially the phenylalanine residue of the SFD mutation, confers maximum PDE activity.

The cpTat system transports fully folded proteins into the thylakoid using the proton motive force and, together, cpTatC, Tha4, and Hcf106 transport precursors across the bilayer membrane. Tha4 is predicted to form the translocation pore; however, Hcf106 may also have a role because of the large size of some cpTat substrates and its homology to Tha4. In this study, direct binding between Hcf106 and a cpTat substrate, the Oxygen Evolving Complex precursor (pOE17), was investigated. Prior work showed direct binding to Tha4 for four variants of the substrate (pOE17-D68C, -S84C, -K99C, and -T115C) using cysteine crosslinking. The current study showed little to no direct binding between Hcf106 and the last variant of the substrate (pOE17-T115C); although, that variant could interact with Tha4. Together with precursor-Tha4 interaction maps, we concluded that both cpTat components bind directly to the substrate and the strongest interactions occur at the C-tail and the C-terminal end of the amphipathic helix (APH) region. This study also investigates whether cpTat substrates stay in contact with Hcf106 throughout transport. Protease treatments were performed on crosslinked Hcf106 and pOE17 at the C-tail and N-terminal residues of Hcf106. Unexpectedly, membrane-protected and -unprotected crosslinked products were detected in the N-terminus of Hcf106. The paper presents limitations with the study and directions for future research.

Acetyl-CoA, an essential metabolite at the junction of various anabolic and catabolic pathways, is generated both from the breakdown of carbohydrates, lipids, and amino acids, and from the activation of acetate. AMP-forming acetyl-CoA synthetase, a key enzyme for acetate activation into acetyl-CoA in all domains of life, is a member of the adenylate-forming enzyme superfamily. Although members in the superfamily have different biological functions, they all share the property of forming an acyl-adenylate intermediate. Here I describe my research on characterization of a medium-chain acyl-CoA synthetase from Methanosarcina acetivorans (MacsMa) and identification of key residues in acyl substrate and CoA binding and catalysis. Additionally, this dissertation reports the characterization of Acs4 from Methanosaeta concilii (Acs4Msc) and further investigation of the role of a highly conserved Trp residue in Acs. MacsMa was biochemically characterized to catalyze the acyl-CoA synthesis with preference for 2-methylbutyrate. However, when propionate was used as the acyl substrate, propionyl-CoA was not produced. Propionyl-AMP and PPi were released in the absence of CoA, whereas in the presence of CoA, the propionyl-AMP intermediate was converted to AMP and propionate and released along with PPi. These findings suggested that although acyl-CoA synthetases may have the ability to utilize a broad range of substrate for the acyl-adenylate forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step, in which case the enzyme has devised a mechanism for the release of the propionyl-adenylate intermediate. In order to investigate the molecular basis of enzymatic mechanism of MacsMa, several key amino acid residues were identified through inspecting the enzyme structure and sequence alignment. Kinetic characterization of these residues suggested that MacsMa has highly conserved acyl and CoA binding pockets. Six residues, Lys256, Cys298, Gly351, Trp259, Trp237, and Trp254 were identified to be essential in acyl substrate binding and catalysis and also Lys256 had an important role in structure stability; Seven residues, Lys519, Lys461, Gly459, Tyr460, Arg490, Tyr525, and Tyr527 were found to be important in CoA binding and catalysis without significantly affecting the first half adenylate-forming reaction. Another interesting finding was that 3'-phosphate of CoA was proved to be critical in catalysis of thioester-forming step of the reaction in MacsMa. However, Acs is able to utilize 3'-dephospho CoA well though not as efficient as with CoA. Characterization of CoA binding and catalysis also suggested that MacsMa had a similar CoA binding pocket to CBAL. Investigation of the MacsMa and active site(s) help gain a better understanding of biochemistry and two-step reaction mechanism in Acs and the evolution of the active site and substrate binding pocket in short- and medium-chain acyl-CoA synthetase (Sacs/Macs) family. In order to investigate the biochemistry and kinetics of the Methanosaeta Acs's, Acs4Msc was biochemically characterized to show little to no acyl-adenylate synthetase/acyl-CoA synthetase activity. The Trp416 in Methanothermobacter thermautotrophicus Acs1 (Acs1Mt) has been shown to be highly conserved in other Acs's and very important in determining acyl substrate utilization but replaced by Phe in Acs4Msc. To further investigate the role of this highly conserved Trp active site in Acs, the Phe528Trp Acs4Msc variant was characterized to still display little to no acyl-adenylate/acyl-CoA synthetase activity as observed with wild-type enzyme. Furthermore, the Acs1Mt Trp416Phe variant showed similar kinetic parameters for either acetate or propionate for acyl-CoA synthetase; however, this variant released propionyl-adenylate as a free product, suggesting that Acs1Mt have different interactions with acetate and propionate and the benzoyl ring in Phe may reduce the ability of the enzyme to retain the propionyl-adenylate for the second-half thioester-forming step. The appendix section includes a recently published paper focusing on the structure of MacsMa. I contributed to the enzymatic activity characterization of MacsMa and am an author on this paper.

We frequently find proteins exist in oligomeric forms in nature. The abundance of dimers, trimers and tetramers with cyclic or dihedral symmetries in the Protein Data Bank is a good testimony. Even more, it is not rare to find proteins form highly ordered, symmetric, large complexes. These oligomeric forms are usually essential for their functions. Ferritin forms an octahedral cage with 24 subunits to store iron; some virus capsid proteins assemble into icosahedral cages; vaults, which are large dihedral particles widely conserved in eukaryotes, have biological functions yet to be discovered. These fascinating structures inspire three types of questions: How do individual subunits interact form such symmetric complexes? How can we reproduce such complexes with protein engineering? How do we put engineered symmetric protein complexes to application? My thesis work consists of projects addressing all three questions.My first project, described in Chapter 1, concerns bacterial microcompartments (MCP), which are large proteinaceous organelles enclosed by an icosahedral or pseudo-icosahedral shell. MCPs usually enclose special metabolic pathways that are inefficient or toxic in the cytosol. To do so, MCPs must form a sealed barrier with its shell proteins. It was hypothesized that at least one type of the proteins forming the shell of MCPs has to be pentameric instead of hexameric. Indeed, we proved that the BMV proteins, a family of protein highly conserved in MCP operons, formed pentamers in solution. Together with other crystallographic evidence, we conclude BMV proteins form pentamers to cap and seal the MCP shell. In addition to MCPs, I worked on another natural oligomeric protein, bactofilin. Bactofilins are fiber-forming proteins that are widely conserved among bacteria. These proteins have roles in diverse biological functions including but not limited to cell motility, cell wall synthesis and modification. Chapter 2 describe my preliminary biochemical and structural work on bactofilins.Next, I moved on to symmetry-based engineering protein complexes. In Chapter 3, I included a recent review paper on the theory and successes in symmetry-based protein engineering that I participated in preparing. Designed complexes need to be validated at high resolution with X-ray crystallography, but for a long time, the low yield and solubility of the designs complicated their validation. In Chapter 4, we show that mutating solvent-exposed side chains to charged amino acids improved the solubility of a previously low yield tetrahedral design and enabled validation by crystallography. Next, I advanced to a bigger challenge in designing symmetric nanoparticles—icosahedral particles. Icosahedral particles are made up of 60 asymmetric units, as compared to 12 in tetrahedral particles, making them much more difficult to design with accuracy. I was able to validate three different icosahedral design with crystallography, making them the largest designed protein assemblies ever crystallized to date. This work is described in Chapter 5. Additionally, I have made other independent design efforts, one to combine DNA and protein as building materials to design tetrahedral complexes, another to design protein sheets with layer group symmetry. These efforts are documented in Chapter 6.IIn the last chapter, I utilized the validated tetrahedral designs as a scaffold in cryo-electron microscope (cryo-EM) for small targets. Despite recent advancements in cryo-EM techniques, small targets remain difficult. By arranging small targets around tetrahedral particles, we can overcome the size limit and provide multiple views to alleviate the commonly seen orientation preference. My project used a type of versatile adaptor protein, designed ankyrin repeat proteins (DARPins), to connect the tetrahedral particles to the imaging targets. We show that the resulting construct is amenable to structural analysis by single particle cryo-EM, allowing us to identify and solve the structure of the attached DARPin at near-atomic detail. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other proteins, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell.In conclusion, my thesis work contributes to the understanding of natural oligomeric complexes, expands our capacity in designing symmetric assemblies, and puts forward an example of a useful application of the designed assemblies.

Technology guides the practice of scientific inquiry. In the biological sciences, DNA sequencing has encouraged the commingling of traditional experimental biology and computer science. In this thesis, I describe computational and biochemical methods to advance nanopore DNA sequencing technology. Nanopore DNA sequencing is an single-molecule technology that shows promise in the area of read lengths, instrument portability, and, as shown in this work, chemical modification detection. A nanopore sequencing device contains a nanometer-sized pore that separates two electrolyte buffer reservoirs. A voltage potential is applied across the nanopore and the device records the ionic current through pore. As DNA polymers translocate through the pore they modulate the ionic current by partially obstructing the pore in a sequence-dependent manner. Commercial sequencing devices contain arrays of pores that are individually controlled.The first chapter of this work provides a technical background and practical application of the technology. In the following chapters I describe new algorithms that improve the utility of the technology. In Chapter 2 I describe a hidden Markov model for the nanopore ionic current. I describe how multiple model topologies were implemented, one of which included modeling the time domain. I describe how a hierarchical Dirichlet process can be used to model new non-canonical (modified) bases affording a HMM-HDP model. In chapter \ref{chap:detecting_modifications} I show how the model can be trained to detect multiple chemical modifications on synthetic DNA and genomic DNA samples. In the final section of this chapter I show how the model was incorporated into a cloud-based pipeline that allows for horizontal scaling of the datasets.The last section of the paper describes a biochemical method to re-read DNA sequences using a nanopore and a helicase enzyme. Multiple biochemical techniques have been used to increase the accuracy of nanopore sequencing; re-reading the same strand allows for stochastic errors to be differentially placed in multiple passes.

INTRODUCTION: Healthcare practices are rapidly evolving, shifting to multidisciplinary initiatives, and prompting a reevaluation of the current structure in the preparation of medical students. The response of medical schools is to adopt newly modeled curricula that use a flipped classroom structure to implement an integrated curriculum encouraging the practice of multidisciplinary inquiry within the basic sciences to develop physicians capable of thoughtful clinical reasoning skills. This pedagogical shift in medical education and the prevailing reaction of medical schools to fundamentally reform curriculum has lead to the emergence of a demand for innovative educational technology capable of effective distribution of pre-class material. OBJECTIVE: Assess student experience of SoftChalk online biochemistry modules as a pre-class learning tool to determine effectiveness in fostering student learning and engagement. In evaluating perceptions on improvement, future modules can be knowledgeably revised to maximize educational gains and elucidate effective/ineffective implementation practices. Data was stratified by previous biochemistry experience to determine if students who have taken the same traditionally instructed graduate biochemistry course (BI751) would have differing thematic opinions of the flipped model’s pre-learning environment. METHODS: Participants were Boston University’s first year medical school students (n=165) class of 2019’ in an integrated curriculum containing basic science modules. After completion of the modules a mixed-methods anonymous survey with a thematic approach to assess experience and improvement of SoftChalk biochemistry modules were emailed, with reminders. The survey contained demographic data, qualitative free response questions, and likert scale assessment questions with no incentive for completion. The data was analyzed independently by researchers to assess common themes and stratified by previous biochemistry experience. RESULTS: Three main themes emerged in assessment of data: SoftChalk as a quality learning tool, lack of integration/consistency, and formatting concerns with an overall positive perception of the pre-learning tool. Respondents commented on quality of SoftChalk as a pre-learning tool; the majority deeming it helpful, interactive, and having beneficial activities. Integration and consistency concerns surfaced in both experience, with commentary on pre-class modules being too dense, and improvement by consolidating information into one resource. The major formatting concern was the ability to maintain module value in paper form. Themes were furthered by the quantitative data with students perceiving SoftChalk as effective, providing a foundation for material in-class, and questions having the correct difficulty. When data was stratified BI751 disagreed that SoftChalk helped students stay on track with course material despite the class on average agreeing. CONCLUSIONS: While SoftChalk is an effective pre-class learning tool, the challenge is in reversing students’ perceptions that basic material should be instructed and that a comprehensive syllabus is necessary. Future SoftChalk modules can enhance success if pre-class modules are condensed, have a stronger transition from pre-class information to in-class activities and maintain consistency among instructor expectations.

Chemoselective reactions are powerful tools for the progression of basic science and biochemical technologies. Bond-forming reactions are routinely used for monitoring the status of cellular activity and in the production of precision therapeutics and diagnostics. However, the development of such reactions is a challenging feat, as one must account for the broad swathes of interfering chemical entities present within the biological milieu. One way to address this complicating factor is through the use of “bioorthogonal” chemistry. These reactions involve two reactive groups with xenochemical properties, i.e., reactivity preferences that are inert to biological moieties yet exquisitely reactive with one another. By necessity, bioorthogonal reactions are forged from deep chemical insight. A complementary strategy to achieve reaction selectivity in biological settings seeks to use proximity enhancement. Positioning two groups in close proximity to one another raises the higher effective local concentration of the desired partners, increasing the rate by which they react to afford highly selective conjugation, even amidst complex systems. In this dissertation, I explore several applications of this concept to develop a variety of tools for basic bioscience and clinical diagnostics. In Chapter 1, I provide a brief overview of several landmark papers that employ proximity-enhanced reactions in chemical biotechnology.In Chapter 2, I describe the use of proximity-enhanced reactivity to direct ligation and detection of protein-specific glycosylation on live cells. In Chapter 3, I describe the application of proximity enhancement to selectively accelerate the ligation of DNA strands that have been chemically attached to different antibodies. In this particular project, we used paired antibodies that bind either to biotin or a protein of interest, driving the ligation and formation of a DNA amplicon in the presence of a biotinylated protein. In combination with chemistries that selectively attach biotin to glycans, this technology allows the use of qPCR to detect very low levels of glycosylated proteins. In Chapter 4, I describe the development of a new technology, termed Antibody Detection by Agglutination-PCR (ADAP), which uses PCR to detect antibodies with a high degree of multiplexing and sensitivity.In Chapter 5, I describe a method for re-purposing cyclooctynes to selectively ligate to thiols present on serum albumin as a strategy to improve the serum-half lives of therapeutic molecules.In Chapter 6, I describe the computational modeling and synthesis of dendrimer-based glycan mimetics.

The Iron-dependent Regulator (IdeR) is a 230-amino acid transcriptional repressor that regulates iron homeostasis, oxidative stress response and virulence in Mycobacterium tuberculosis. IdeR binds to DNA as a dimer of dimers to repress gene expression and as four dimers to upregulate gene expression (upregulation is only for iron storage genes). IdeR contains two metal binding sites both of which have always been assumed to bind Fe(II) in vivo and it is the binding of metal by IdeR which induces the dimerization. IdeR is able to be activated for DNA binding in vitro by various divalent transition metals including: Mn(II), Fe(II), Co(II), Ni(II), Zn(II) and Cd(II). The model for activation of IdeR has always been investigated using divalent metals other than Fe(II) because of the rapid oxidation of Fe(II) to Fe(III). The objective of our work was to use Fe(II) to investigate the metal activation and DNA binding properties of IdeR. We also wanted to characterize a hyperactive mutant of IdeR (IdeR(D177K)) to better understand the cause of the hyperactive phenotype. To investigate the Fe(II) binding of IdeR we directly measured the Fe(II) bound using equilibrium dialysis and we investigated DNA binding using a fluorescence anisotropy assay. We determined that IdeR has the ability to bind Fe(II) into both metal binding sites, but that both sites bind the Fe(II) with affinities that differ by a factor of 25. We also determined that IdeR with only one Fe(II) bound per monomer was inactive and not able to bind to DNA. Through the use of IdeR metal binding site mutants we were able to determine that the first equivalent of Fe(II) bound to the ancillary metal site. This result was in contrast to all other papers published on IdeR, or its closest homologue (DtxR), that have shown that mutations to the primary metal binding site completely knock out repressor activity and mutations to the ancillary metal binding site only abrogate repressor activity. We determined that IdeR activation for promoter binding is enhanced when either Mn(II) or Zn(II) is present in solution before titrating in Fe(II), with Zn(II) having a much greater enhancement compared to Mn(II). We also determined that Zn(II) is able to bind at both metal binding sites in IdeR, but with KD values that differ by a factor of 70 and that the first equivalent of Fe(II) and the first equivalent of Zn(II) bind to different metal binding sites and the first equivalent of Zn(II) binding is ~36 picomolar. We determined that having Zn(II) present in solution before titrating in Fe(II) reduced the amount of Fe(II) required to activate promoter binding by 30 fold. These results suggest that IdeR is a mixed-metal repressor, where Zn(II) acts as a structural metal and Fe(II) acts to trigger the physiologically relevant promoter binding. We also set out to characterize the hyperactive repressor (IdeR(D177K)). We determined that this hyperactive mutant binds Fe(II) with a lower affinity that wild-type. The Fe(II) dependent promoter binding affinity is lower for IdeR(D177K) than wild-type IdeR. When Zn(II) is present we see that IdeR(D177K) also requires 30 fold less Fe(II) to activate promoter binding, but that IdeR(D177K) still requires a higher concentration of Fe(II) than wild-type IdeR. We also looked at the promoter binding affinity while keeping the metal concentrations constant and we saw that regardless of the metal species present (Fe(II), Zn(II), or Fe(II)-Zn(II)) IdeR(D177K) and wild-type IdeR had similar affinities for the promoter. IdeR(D177K) does not have a decrease sensitivity to changes in the free metal concentration once bound to the promoter as evidenced by our EDTA titration assay. The hyperactivity of IdeR(D177K) is due to it being bound to the promoter for a longer period of time compared to wild-type IdeR when both Zn(II) and Fe(II) are present.

Because of petroleum's expansive boiling point and carbon number range, two analytical methods are primarily used to unlock composition and structure: 2-dimensional gas chromatography mass spectrometry (GCxGC/MS) and liquid infused Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). The strength of one method is literally the weakness of the other: GCxGC/MS, despite being able to resolve isomers, cannot analyze polars and molecules larger than ~C35. Alternatively FT-ICR MS can analyze polars and molecules up to C100 but cannot resolve isomers. Fortunately the continuous boiling point and carbon number nature of petroleum allows the techniques to compliment one other and resolve potential ambiguities. Environmental oxidative transformation after an oil spill tends to increase polarity, decreases separation efficiency of GCxGC and increases instrumental resolving power demands. This shifts the onus onto FT-ICR MS for analysis and also demands advances in high resolution isolation (excitation) for structural determination and dynamic range enhancement. Here, we present a detailed compositional analysis of the Deepwater Horizon oil spill contamination transformation products by FT-ICR including the main mechanism of heavy end transport through permeable beach sand. We also show advances in stored waveform inverse Fourier transform (SWIFT) isolation FT-ICR dynamic range enhancement, IRMPD dissociation and the isolation resolving power fundamental limit (coulombic shielding). Finally we show an enhancement in positive ion electrospray sensitivity of weathered crude oil by optimization of the spray, a development that allowed us to observe one of the more atypical transformative signatures of the Deepwater Horizon spill. High resolution isolation (excitation) by stored waveform inverse Fourier transform (SWIFT) is fundamentally limited by long period of excitation time at low voltage (similar to detection being long data acquisition period at low ion number). Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry in general relies upon linearity between the ion cyclotron excitation and the observed response. However, nonlinearities result from non-ideal applied electric and magnetic fields and Coulombic interactions. Here, we report nonlinear response at low excitation electric field magnitude due to Coulombic shielding. The measured ICR signal magnitude exhibits an excitation voltage threshhold that increases monotonically with the number of shielding ions (i.e., nonresonant ions). If shielding ions are not present, ICR signal magnitude versus excitation voltage is linear (e.g., for quadrupole-isolated ions of nearly a single m/z). Finally, we show that shielding results in a reduced cyclotron radius at low excitation voltage, resulting in an increased rate of transient decay; thereby exacerbating response nonlinearity and excitation threshold for long data acquisition period. Of the estimated 4.4 million barrels of crude oil released into the Gulf of Mexico from the Deepwater Horizon (DWH) oil spill, much washed ashore onto sandy beaches from Louisiana to the Florida panhandle. Here comprehensive two dimensional gas chromatography (GCxGC) confirms the source (Deepwater Horizon (DWH), Macondo, Mississippi Canyon Block 252 crude oil), and determines the extent of weathering and detailed molecular level composition of oil spill contamination that reached Pensacola Beach, Florida. Samples collected from an intertidal zone of Pensacola Beach at various sediment depths were analyzed by positive and negative ion electrospray (ESI) ionization Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The results display a greater than two-fold increase in number of elemental compositions, particularly oxygen compounds (CcHhN1Ox and CcHhOx classes that serve as molecular tracers of oxidation) relative to the original Macondo wellhead oil. The increase in number of oxygen-containing species is consistent with the five-fold increase in O:C ratio by bulk elemental analysis, and reveals extensive oxidative weathering from biotic and abiotic modification in the marine environment. Time-of-flight (TOF) mass analysis of model compounds strongly suggests that oxidative incorporation of ketones in the parent crude oil is responsible for the increased diversity of oxygen-containing species detected by (+) ESI (from C20 to C80). Carboxylic acids are the dominant oxygen functionality detected by (-) ESI FT-ICR MS analysis (C10 - C50). Tandem mass spectrometry by infrared multiphoton dissociation (IRMPD) pinpoints the location of oxidative modifications to alkyl side chains, confirmed by their labile nature (oxygen loss) in tandem MS fragmentation to generate purely hydrocarbon fragments. Ion exchange separates ketones from carboxylic acids. Subsequent GCxGC mass spectrometry analysis unequivocally verifies the presence of ketones (specifically 2-ones, C6 - C26) in the sediment extracts. Finally, a decrease in carbon number and double bond equivalent (DBE) of petrogenic species with increased depth in the sediment extracts is consistent with the preferential downward migration of compact, aromatic species of higher water solubility. More than 1000 km of Gulf of Mexico (GOM) beaches were oiled after the Deepwater Horizon (DWH) explosion of April 2010. Little is known about transport of the petroleum in the permeable beach sand, particularly the high boiling point, petroleum "heavy ends" (polar compounds and/or those above C35) inaccessible by gas chromatography. Here, we present electrospray (ESI) and atmospheric pressure photoionization (APPI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) of oil extracted from binned sand samples. The sand samples were obtained from vertical sections of sand in the intertidal zone of Pensacola Beach (PB), Florida in the summer and fall of 2010. The native petroleum constituents (petrogenic) show a shift to lower carbon number and double bond equivalent (DBE) at depths above and below the oil "stripe" (the most heavily oiled binned sand depth). The shift to smaller, less aromatic compounds is explained by both the behavior of increasingly weathered crude oils and also the known water solubility of smaller, less aromatic compounds that would accompany vertical migration from both tidal burial and washing. The oxygenated environmental transformation products (CxHyOz) have relative abundance distributions consistent with water solubility and ionization of the proposed functional groups and and highlight chemical separation of the crude oil within the sand. Elemental composition assignment confidence in mass spectrometry is typically assessed by monoisotopic mass accuracy. For a given mass accuracy, resolution and detection of other isotopologues can further narrow the number of possible elemental compositions. However, such measurements require ultrahigh resolving power and high dynamic range, particularly for compounds containing low numbers of nitrogen and oxygen (both 15N and 18O occur at less than 0.4% natural abundance). Here, we demonstrate validation of molecular formula assignment from isotopic fine structure, based on ultrahigh resolution broadband Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Dynamic range is enhanced by external quadrupole and internal stored waveform inverse Fourier transform (SWIFT) isolation to facilitate detection of low abundance heavy atom isotopologues. Broad ionization of the largest possible number of compounds is critical to most Fourier transform ion cyclotron resonance (FT-ICR) crude oil or complex mixture analyses. Here, we show an up-to-three-fold improvement in sensitivity by optimization of acid/analyte concentration in the positive ion electrospray mixture. The results show an increased detection of both oxygen and hydrocarbon classes relative to nitrogen simply by bulk analyte dilution at sufficiently high acid concentration. A similar relative increase in oxygen detection is observed by positive ion electrospray time-of-flight (TOF) analysis of model compounds, particularly with compounds containing ketone functional groups. The model compound work demonstrates that at sufficiently low concentration of analyte and sufficiently high acid concentration, a steady-state is observed in the ratio of detection of nitrogen and oxygen. Here, we report isolation of a largely single stored waveform inverse Fourier transform (SWIFT) isolated crude oil Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) m/z and subsequent fragmentation by infrared multiphoton dissociation (IRMPD) of largely a single crude oil m/z. A sample preparation method that employs higher than typical analyte to acid ratio (2 mg/ml analyte, 0.05% formic acid in spray mixture) was performed to preferentially ionize nitrogen (N1 heteroclass) and simplify m/z species in the FT-ICR cell, ergo an artificially simplified spectra. External quadrupole and internal SWIFT isolation were used to isolate the peak before fragmentation (isolation resolving power of ~11,000). IRMPD was used to fragment the peak at a series of irradiation times between 0.5 seconds and 2.0 seconds with the number of N1 fragment ions between 114 and 200. 13C fragments at irradiation times greater than 0.75 seconds show the presence of a smaller impurity in the FT-ICR cell post-isolation. Recent studies have shown extensive oxidative modification to the Macondo wellhead oil upon release into the Northern Gulf of Mexico as a result of biotic and abiotic environmental processes. The transformations include a ketone signature that is masked in positive ion electrospray by the native petroleum N1 class at high analyte concentration relative to acid modifier in the spray solution. Here, we compare Deepwater Horizon oil spill ketone oxidative transformations to those from the Cosco Bosan oil spill in San Fransisco Bay. In contrast to the Gulf of Mexico samples, the San Francisco Bay oil spill do not contain the magnitude or diversity of positive ion electrospray ketone oxidative products found in the Deepwater Horizon contamination. The data presented lead the authors of this paper to posit that the ketone oxidative transformations are a Gulf of Mexico specific event.

Chaperonins are ubiquitous molecular chaperones that are found in all animal kingdoms. They all share a common structure and function to assist the folding of other proteins. All chaperonins consist of two stacked rings, which come together to form a central cavity where folding can take place. The eukaryotic cytosolic chaperonin containing TCP1 (CCT) is a member of the group II chaperonins, which are defined as having octameric or nonameric rings composed of more than one type of subunit.;CCT is an essential part of eukaryotic cell function, and it has been estimated that it folds up to 10% of newly synthesized cytosolic proteins (3). Some of the proteins that have been shown to depend on CCT for their biogenesis include cytoskeletal proteins (alpha-, and beta-actin, alpha-, beta-, and gamma-tubulin), histone deacetylases (HDAC3, Set3p, and Hos2p), cell cycle regulators (Cdc20p, and Cdc55p), and proteins possessing the seven-bladed beta-propeller WD40 domain, including the beta-subunit of the heterotrimeric guanine nucleotide-binding protein (G-protein) complex (4, 5). Heterotrimeric G proteins are a highly conserved group of molecules involved in a great number of signaling processes. They are composed of three subunits (alpha-, beta-, and gamma), which are bound together in the inactive form. In this heterotrimeric state, guanosine di-phosphate (GDP) is bound to the G alpha-subunit. Upon activation, the alpha-subunit exchanges GDP for guanosine tri-phosphate (GTP). This exchange triggers Gbetagamma to dissociate from Galpha and both Galpha-GTP and Gbetagamma are then free to activate downstream effectors. This form of signal transduction is so common that G-proteins regulate or modulate nearly every cellular and physiological process. Because it is so critical for normal cellular function, dysregulation of G-protein signaling is associated with many human diseases (6) and more than half of the current pharmaceutical therapies target a component of the G-protein pathway (7). While much is known about the signal transduction, far less is known about the biogenesis and assembly of G-proteins.;The overall objective of my research has been to elucidate the mechanisms of the chaperonin containing Tailless Complex Polypeptide 1 (CCT) with respect to heterotrimeric guanine nucleotide-binding protein (G protein) signaling. The CCT co-factor, phosducin-like protein (PhLP) is required for Gbeta biosynthesis, and therefore its mechanism is critical for our understanding of this process. In the first chapter of my thesis, I take a structure-based approach to investigate the interaction between PhLP and Gbeta. Currently there are no available structures of this complex, nor is there a single published structure of PhLP. Therefore, I used a homology modeling approach to generate the first known 3D structure of PhLP. Then in order to gain insight into this interaction, I docked PhLP to the Gbetagamma and compared it to the crystal structure of phosducin (Pdc), a closely related protein that also forms a complex with Gbetagamma. The second chapter is centered on a truncated splice isoform of PhLP, referred to as PhLPSHORT (PhLPs), which has been shown to be a potent inhibitor of CCT. A recent paper published by my lab showed that PhLPs expression led to a decrease in alpha-, beta-, and gamma-subunits of the G-protein transducin at the protein level. Later experiments showed that this decrease occurred at the mRNA level as well. While the decrease in Gbeta and Ggamma wasn't surprising, the decrease in Galpha was unexpected since it is not a known substrate of CCT. In order to find a connection between CCT and Galpha, I looked for any perturbations of gene expression that occurred in transgenic mice constitutively expressing PhLPs using microarray results. Furthermore, because these mice express PhLPs at an early age I repeated these experiments in inducible transgenic mouse lines that we developed to express PhLPs in adult mice. Taken together, my research provides additional data to refine the mechanism of Gbeta biosynthesis and suggests that there is an additional level of regulatory control over G-proteins that connects the expression of all three subunits. (Abstract shortened by UMI.).

Lignin is one of the most abundant biopolymers in plants and plays an important role in plant structure and stress defense. Lignin is also considered to be a hallmark of vascular plants because of its crucial role in plant terrestrialization. However, lignin is an undesired component in the pulp and paper industry, bioethanol production, and forage digestibility. Thus, understanding the functions and the evolution of lignin biosynthesis genes can not only advance our knowledge of the evolution of land-adaptation for vascular plants but also help guide the effort to exploit the potential for genetic manipulation of lignin for desirable traits in economically important crops. In this dissertation I studied a cinnamyl alcohol dehydrogenase (CAD) gene family in an important basal angiosperm species Liriodendron tulipifera L. A total of seven LtuCAD homologs were identified from a comprehensive Liriodendron EST database. The comparison of amino acid sequences, phylogeny, expression analysis, and complementation in the Arabidopsis cad4 cad5 double mutant indicate that LtuCAD1 is involved in lignin biosynthesis in Liriodendron. This finding provides the opportunity to manipulate the expression of LtuCAD1 for lignin modification in Liriodendron and broadens our knowledge of lignin biosynthesis in basal angiosperms. I also investigated the impact of expressing a parsley tyrosine-rich peptide (TYR) in poplar. With an aim to facilitate lignin removal during the utilization of woody biomass as a biofuel feedstock, transgenic poplars carrying the TYR sequence were previously generated, and a number of transgenic lines released more polysaccharides following protease digestion and were more flexible than wild type plants. In the current study, it was found that expression of the parsley tyrosine-rich peptide did not compromise the plant stem growth, susceptibility to pathogen, nor cause significant wood chemistry alternation in the transgenic poplars. Only five transcripts were found differentially expressed in the transgenic plants, all with at least 4-fold decrease of transcript abundance relative to the wildtype. These five transcripts encode a sulfotransferase domain protein, a NB-ARC domain-containing disease resistance protein, and one putative protein, respectively. The results suggest that it is possible to increase cell wall digestibility and flexibility without compromising growth and pathogen resistance of the poplar plants expressing a tyrosine-rich peptide encoding sequence.

Life is made possible by an astounding array of interactions between highly-evolved biomolecules that serve a variety of specific functions. Their functions are dictated by structure and flexibility or “dynamics,” both of which can change upon interaction with a partner molecule. Understanding these changes and the pathways that link them have revealed key insights into both disease and the foundations of life. However, because these interactions are often “allosteric,” wherein changes are propagated far from the site of contact, the subsequent changes are difficult to predict and sometimes counter-intuitive to understand, thus motivating directed, basic research.Here, we aim to understand allostery by characterizing the activation mechanism of the paradigmatic, allosteric, ligand-responsive protein trp RNA-binding Attenuation Protein (TRAP), which regulates genes under control of the trp operon in Bacilli. The ring-shaped 11-mer TRAP is activated to recognize a specific trp-mRNA target upon binding up to 11 tryptophan (Trp) molecules; in contrast to the Trp-bound “holo” state of TRAP, the Trp-free “apo” state is inactive. In study 1, to understand how the apo and holo states of TRAP differ with respect to structure and dynamics, we used state of the art nuclear magnetic resonance (NMR) experiments. We learn that apo TRAP is flexible in the μs-ms time window, and that Trp binding yields local changes in structure, and a global reduction in μs-ms flexibility. In study 2, to understand the time-resolved pathway of the apo to holo activation process, we monitored Trp fluorescence upon rapid-mixing of Trp and apo TRAP in solution. We demonstrate that Trp encounters its binding site on TRAP a remarkable 2,000 times before entry is permitted, that activation occurs within one sec via step-wise increases in bondedness, order and compression, and we characterize two Trp-bound states reflecting holo TRAP flexibility on the sec timescale, putatively responsible for Trp release. In study 3, to characterize partially-bound TRAP donuts (i.e., between apo and holo states), we titrated Trp into TRAP and monitored structure via NMR and binding energetics via isothermal titration calorimetry (ITC). NMR data reveal that TRAP samples conformations in the supra-ms time window that are neither the apo nor holo states, and that the structure and dynamics of a given protomer can be altered even if Trp is not bound at that site. The ITC data reveal allostery in Trp binding that, if described via nearest-neighbor interactions between TRAP protomers, must invoke negative binding cooperativity (i.e., binding a second Trp molecule is less favorable than binding the first). Finally, because this research relies heavily on NMR experiments, we wrote a comprehensive methodological review paper, and developed an innovative analytical software package, “GUARDD.”

The human health threat from marine algal toxins is increasing with expanding human population, increased seaside populations, and the concomitant increase in aquaculture operations and demand for seafood. As humans increase demand for seafood, they create waste and activity that may increase the likelihood of harmful algal blooms (HABs) and phycotoxin production by some of these blooms, notably the production of the causative toxins for ciguatera fish poisoning (CFP), paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP) and diarrhretic shellfish poisoning (DSP). The mainstay for regulatory detection of these toxins has long been the mouse bioassay (MBA), with intraperitoneal (i.p.) injection of suspect extracts and subsequent monitoring for symptoms and time of death. The general sentiment in the research community is that there is a need to eliminate, or at least reduce, the use of the mouse bioassay in testing for algal toxins, due to technical limitations of the procedure and its ethical drawbacks. A number of functional and analytical methods have been developed to this end. This paper reviews the rise of harmful algal blooms, toxin syndromes, historical use of and the subsequent need to find an alternative to the mouse bioassay in detection of algal toxins and obstacles to the development of these alternative methods. The role of large importing countries in this process is then considered; particular attention is paid to the United States, as there is little discussion of their efforts in the literature.

The [beta]-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the existence of two different thiolase isoforms, Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9 AACT) and Thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16 OACT). Glyoxysomal AACT is the last enzyme in the p-oxidation of fatty acids in plant glyoxysomes. Glyoxysomal AACT has been successfully cloned, expressed and purified from sunflower cotyledons (Dyer et al., 2006). In this paper we investigate the optimal conditions for both expression and purification. These include the choice of vector, the growth conditions and the purification parameters for the maximum possible yield of AACT protein.

Premature birth is currently the leading cause of infant morbidity and mortality in the United States. One common aspect of premature birth leading to morbidity and mortality is lung immaturity, which often entails a lack of proper lung morphogenesis and pulmonary surfactant production. Presently, a thorough understanding of the molecular regulation, transport, and modification of lipids required to produce functional pulmonary surfactant is lacking, which hinders progress toward improved care for patients with immature lungs. Understanding the molecular mechanisms regulating lipid trafficking will allow the development of more effective preventative and treatment strategies for patients with immature lung pathologies like Respiratory Distress Syndrome (RDS). The following document describes the rationale for the study of Fatty acid binding protein 5 (FABP5) in alveolar type II cells. Previous gene deletion and over-expression data implicate Fabp5 as being a potential component of lipid regulation during perinatal lung maturation. Specifically, Fabp5 down-regulation strongly correlates with decreased lipogenesis and improper production of pulmonary surfactant. In addition, Fabp5 expression has been shown to be regulated by proteins known to control lipogenesis in vivo. As a result, this paper describes the previous and bioinformatic data supporting the hypothesis that Fabp5 regulates lung lipid homeostasis.

Ultrathin films consisting of an alternating sequence of positively and negatively charged polyelectrolytes have been prepared by means of the electrostatic layer-by-layer sequential assembly technique. To augment their typical applications in the water treatment, personal care as well as the pulp and paper industry, the structure and the design of these polyelectrolytes were tailored synthetically to satisfy the requirements of different types of applications. Some were used for surface modifications, hydrophobic and hydrophilic coatings, corrosion protection, conducting and biocompatible surfaces. Others were found to be very efficient for membrane and chromatographic applications. The ease with which these multilayer coatings can be constructed, their robustness and stability make them very good candidates for industrial applications. The dissertation focuses mainly on the structure-property relationships of these polyelectrolytes and their corresponding thin films. Various polyelectrolytes were synthesized or modified in a strategic approach and gave novel and promising properties. Some of them exhibited permeabilities that were higher than any membranes reported in the literature. Also, some are potentially very useful for designing drug delivery systems such as tablets or encapsulations since they were shown to control the permeability of sample drugs and vitamins very efficiently based on their sensitivity to pH changes. Other synthesized polyelectrolytes proved to be very effective in preventing protein adsorption or promoting cell growth and differentiation. Some systems were very useful as robust stationary phases for simple chiral separations in capillary electrochromatography. Along with modifications and improvements, the approach might one day be applied commercially for chiral separations using high performance liquid chromatography and replace currently used stationary phases. Last but not least, the potential for these polyelectrolytes and their corresponding films is immense. Slight variations in their structural properties could transform into significant improvements in their physical properties and behavior. Hydrophobicity, permeability, swelling, thickness and stability are very important properties of these films and could be tuned to achieve new and unprecedented properties.

Recent evidence has suggested that oncogenic transformation of mammalian tissue culture cells results in altered expression of several surface components. One of these changes is the reduced ability of transformed cells to synthesise complex gangliosides. This modification has been correlated with the other phenotypic alterations of transformed cells and with their tumorigenic potential. We have adopted a novel approach to elucidate the relationship between the impaired synthesis of complex gangliosides in transformed cells and other aspects of the transformed phenotype. Mutants of Balb/c 3T3 mouse fibroblasts, which normally contain substantial levels of the gangliosides GM3. GM2. GM1 and GD1a. have been selected for failure to express ganglioside GM1 at the cell surface. Other phenotypic properties of these mutants have been investigated, to determine whether the expression of any of the characteristics normally associated with transformed cells are dependent on the loss of ability to synthesise GM1 or the more complex gangliosides, GD1a and GTla. Cholera toxin, which is known to bind specifically to ganglioside GM1 with high affinity, has been used in the selection of these mutant cell lines. A method involving antibody-dependent complement-mediated lysis of cells able to bind cholera toxin was used as the one of choice. Previously, there has been some doubt as to whether any other cell surface receptors for cholera toxin exist in Balb/c 3T3 fibroblasts. Evidence is presented in this thesis, and in an accompanying paper, that the major receptor for cholera toxin in these cells is a ganglioside with similar characteristics to GM1. We have further demonstrated that a substantial proportion of this lipid is insoluble in neutral detergent and remains with Triton X-100 extracted cytoskeletons of Balb/c 3T3 cells. The possibility that GMlis indirectly associated with the cytoskeleton is discussed.

A particulate enzyme preparation made from 1-week-old dark-grown pea (Pisum sativum) seedlings has been shown to incorporate glucuronic acid from UDP-D-[U-14C]-glucuronic acid into a hemicellulosic product. Optimum conditions for the incorporation include the presence of Mn2+ ions between 4 and 10 mmol dm-3 and a pH of approximately 7.5. UDP-D-xylose at 1 mmol dm-3 allows incorporation to continue for at least 8 h. In its absence, the reaction stops within 30 min. Analysis of the product by partial and total acid hydrolysis, followed by paper chromatography or electrophoresis, indicates that the polysaccharide produced is a glucuronoxylan.

This thesis describes a project which had a simple central objective: to take an existing preparation of dissociated cells from the immature cerebellum and develop conditions in which these cells could survive and differentiate in monolayer culture. The aim was to develop a system in which brain development could be investigated, particularly at the biochemical level in a simplified environment. The study of cellular differentiation in vitro has two advantages: 1. Aspects of a cell's development which are intrinsically programmed in the cell at that stage can be distinguished from aspects which are dependent on some extrinsic influence, for instance from another cell type. 2. Cultures have a more restricted cellular composition than the brain and therefore may go some way to overcoming the cellular heterogeneity of brain tissue which renders interpretation of most conventional biochemical measurements difficult. To realise the first it is necessary to compare a cell's differentiation in vitro with its normal course of development in vivo. To realise the second, cultures must be of a simplified and defined composition in which biochemical measurements can be attributed to particular cell types. Both requirements imply the use of cultures which contain in quantified proportions a few cell types which can be specifically identified by reliable criteria, rather than in terms of such general categories as 'neurons'. The use of a single area of the brain, the cerebellum, was an important first step in restricting the cellular composition of the cultures. The cerebellum contains large numbers of relatively few neuronal types which differ greatly in perikaryal size and therefore were amenable to a size-based separation of cell types by unit gravity sedimentation. Culture conditions were established for the survival and differentiation of the mixed cerebellar cell suspension and some of the separated cell fractions. This led to cultures containing a high proportion of granule neurons. However a fraction enriched in Purkinje cells did not survive well in culture. Finally, the composition of the cultures was further defined in a study involving cell type-specific immunological markers and the different uptake of the inhibitory neurotransmitter, GABA. The description of this work is organised in seven more or less self-contained chapters, and can be informally divided into two parts. Chapter 1 is a general introduction to the problems of studying development, to the advantages of the cerebellum, in particular the detailed knowledge of some mechanisms of cerebellar development gained from work with mutants and X-irradiation and to the current knowledge, or lack of it, concerning the molecular basis of cellular interactions. Chapter 2 is a description of the cell isolation method used here, and of some improvements to the method effected during the course of this work. Chapter 3 completes the first part of the thesis and deals briefly with the history of brain cell separation techniques before detailing the separation of cerebellar cells by a unit gravity sedimentation technique. This was a joint project with James Cohen and Gary Dutton at The Open University and Graham Wilkin and Robert Balazs at MRC, Carshalton. Chapter 3 concentrates mainly on the part of the project which was the primary work of this author, that is the size analysis of cells using a Coulter counter and points out those parts of the work, in particular electron microscopy, which were performed by others. The second part begins with Chapter 4, which is a review of nervous system culture work with the aim of establishing whether there are reasons why the contribution of cell cultures to our biochemical knowledge has been quite limited, although they have been in use for many years and would appear an ideal controlled system for biochemical studies. Chapter 5 is a description of the search for culture conditions in which one week postnatal cerebellar cells would survive and grow, and of their pattern of development in culture. Chapter 6 details the use of markers to define culture composition more closely; cerebellar cultures were found to be over 80% in a single neuronal type, the granule cell. Cultures of embryonic cerebellum were found to contain a distinct population of neurons which are absent from cultures of older animals, Chapter 7 briefly summarises some future directions for this work and suggests that defined cell culture systems in alliance with modern immunological techniques, may offer the best route to find the elusive cell surface recognition molecules which are generally believed to mediate the cellular interactions that control development. Some of this work, together with related aspects which are primarily the work of others, has been published elsewhere. Copies of these papers are attached.

Yeast 3-phosphoglycerate kinase (PGK), a glycolytic enzyme, was isolated, purified and characterised, and subjected to cyanogen bromide cleavage. Three of the four cyanogen bromide fragments were purified by gel filtration, and enzymically digested with trypsin, chymotrypsin, pepsin, thermolysin and staph. protease. The resulting peptides were purified by high voltage electrophoresis on paper, and were sequenced by the dansyl-Edman technique. Of the 243 amino acids constituting these three cyanogen bromide fragments, 221 were placed in order by direct sequencing, composition, and comparison with the horse muscle enzyme sequence, with which yeast PGK exhibits an estimated 65% identity. By fitting the sequence to the 2.5 R-resolution electron density map of yeast PGK, the positions of the reactive alutamyl residue (Glu-403) and of the reactive tyrosyl residue (Tyr-195) have been located, at sites distant from that of bound ATP. The remote location of the glutamyl residue supports the suggestion that PGK must undergo a substrate-induced conformational change in order to bring Glu-403 into closer proximity with the ~-phosphoryl of ATP. The glycine lining of the ATP-binding pocket of the horse muscle enzyme is conserved in the yeast enzyme, as are the glycines near the t-phosphoryl of bound ATP. However, the location of the hinge region of the molecule, tentatively identified in the horse muscle enzyme sequence, is not conserved in the yeast PGK sequence. The structure of PGK has been compared with that of other kinases, and similarities with hexokinase and adenylate kinase noted.

The aim of the study was to determine the efficiency with which the carbon skeletons of essential amino acids are utilised by the pig when they are limiting in its diet. This requires the oxidation rate of an amino acid to be measured accurately using a tracer labelled with an isotope of carbon or hydrogen. The use of carbon labelled tracers requires the animal to be enclosed in a sealed chamber for the collection of CO2; to avoid this the use of [3H]-labelled tracers was investigated. The oxidation rates of [14C]-labelled tracers when the tracee was limiting and the animal's nitrogen intake was 2 g/kg0.75.d, showed that leucine was used with an efficiency of 0.88; for phenylalanine and histidine the efficiency was over 0.99. When the animal's nitrogen intake was reduced to 0.23 gN/kg0.75.d, and the limiting amino acid was removed entirely from the diet, the oxidation rates did not change significantly. The oxidation rates of [3H]amino acids were very variable compared to the [14C]amino acids and the techniques used were critically analysed. Three reasons were identified for the differences between the [14C]- and [3H]tracers. These were the measurement of tritiated water, the purity of the infused tracers and the position of the isotope in the labelled amino acid. The first two were solved by the adaption of techniques to the high accuracy required. The third reason was a problem of isotope becoming incorporated into synthetic pathways via exchange reactions. No amino acids were available labelled with a [3H] atom which would not be incorporated; therefore it was necessary to synthesise one. L-[33H]valine was chosen because a suitable precursor for its synthesis, L-[2,3-3H]valine was available and because L-[1-14C]valine was also available for comparison The L-[2,3-3H]valine was acetylated to produce N-acetyl-D, L-[3-3H]valine which was then hydrolysed by the stereospecific enzyme acylase I to yield N-acetyl-D-[3-3H]valine and L-[3-3H]valine. The products were separated using a cation exchange column and the L-[3-3H]valine was purified by paper chromatography. Contamination with D-[3-3H]valine was measured using D-amino acid oxidase and L-[2,3-3H]valine using L-amino acid oxidase. The oxidation rates of L-[1-14D]valine and L-[3-3H]valine were compared in the rat and the oxidation rate of L-[3-3H]valine was significantly higher. The experiment was repeated using L-[3-3H]valine synthesised by Amersham International plc. using the same method; the structure was established by NMR. In this case both tracers were oxidised at the same rate. The difference in the first experiment was therefore probably due to slight contamination of L-[3-3H]valine with L-[2,3-3H]valine, the tritium in position 2 being removed by reversible transamination of valine, i.e., not only during its oxidation. Finally, the two tracers were compared in the pig. It was found that the valine flux rates calculated from the two tracers were in closer agreement when a correction was made for infused D-labelled valine. Using the techniques developed throughout this work, L-[1-14C]valine and L-[3-3H]valine measured the same rate of valine oxidation. L-3-3H]valine was a more precise tracer of valine oxidation.