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Biotechnology has the potential to deliver solutions to many global problems in medicine, materials science, nutrition, agriculture, natural resource preservation, and energy. Engineered cells and enzymes can perform chemical transformations that are rare or unknown in nature, and even catalyze reactions not accessible to traditional synthetic chemistry while also operating at gentler, more environmentally friendly conditions. Decreases in the price of DNA sequencing and synthesis has led to generation of vast databases that can be screened for any imaginable function. These sequence databases are even more powerful now due to the development of software to enable rapid generation of 3D protein structures, like AlphaFold2. However, tools to predict the function of these proteins or their performance in engineered cells are not yet robust, leading to long development times and limited successful applications to date. New tools and methods must be developed for the true potential of biotechnology to be unlocked.For my thesis, I explore metabolic pathway construction and screening, protein design, and protein sequence-structure-function relationships across broad contexts with the objective of tool and knowledge development for future efforts in biotechnology. My first chapter discusses the introduction of the triazine degradation pathway, of interest for remediation of contaminated sites, into E. coli and methods for characterizing pathway flux and identifying bottlenecks to guide engineering efforts and limit accumulation of metabolic intermediates. My second chapter focuses on methods for the design of supercharged proteins, which have many interesting potential applications, and parameters that increase the likelihood of successful design of these proteins. My third chapter regards the generation and characterization of a library of single point mutations in the enzyme B-glucosidase B and use of kinetic data to predict the effects of changes in sequence on enzyme function. These seemingly disparate topics all serve to improve tools for protein screening, production, functional prediction, and application, addressing several gaps toward improved development timelines and success rates for biocatalysts.

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.

Conservation genetics is a booming field focused on assessing the genetic structure and diversity among subpopulations of different species. However, the utilization of genetic analyses in management plans remains unclear because it is not known how often they are considered during creation of plans. Chapter one considers the question of how closely correlated the results from a genetic assessment on population structure are with the recommendations given at the conclusion of a study. Since conservation tends to have limited financial resources, it is imperative that the money spent on genetic studies is providing beneficial information for conservation. This analysis shows that genetic divergence is correlated with the recommendations, but different genetic markers (i.e. microsatellites) and divergence metrics (i.e. Fst) show different relationships between the recommendations and genetic divergence, possibly due to differences in the sample size associated with different markers.Small populations, such as those of conservation concern, that inbreed may lose alleles over time. This can be problematic if the alleles lost are beneficial, but making genetic assessments based on neutral genetic markers gives no information on the fitness of populations. Ideally, conservation programs should use quantitative genetics to assess population fitness. Chapter two looks at the change in additive genetic variance in populations following a dramatic decrease in population size (bottleneck). On average, populations experiencing a bottleneck showed an increase in additive genetic variance for populations that had levels of inbreeding equal to or less than that of sibling mating (0.25); above that a decrease in genetic variation was observed.Since genetic assessments using neutral markers can be costly, and the use of quantitative genetics may be impractical for informing conservation recommendations, chapter three looks at the use of statistical models to assess International Union for the Conservation of Nature Red list status using ecological characteristics. Here characteristics shared among species that have been assigned a threat status and those that have not been assigned a status, or those needing an update, are used in logistic regressions, regression tree analyses and discriminant function analyses to predict threat status and identify those species in most immediate need of attention. We found that logistic regression and discriminant function analysis are very good at predicting threat status. When resources are limited, using data on previously assessed species may be a good alternative to inform conservation recommendations.

RNA binding proteins (RBPs) impact cellular protein levels by regulating messenger RNA (mRNA) levels. Assignment of function to hundreds of emerging, uncharacterized RBPs is a critical bottleneck to a complete understanding of gene expression control. Here, large-scale tethering of nearly a thousand RBPs discovers 50 RBPs that affect reporter RNA turnover and translation. Enhanced UV crosslinking and immunoprecipitation (eCLIP) identify hundreds of endogenous mRNA targets affected by manipulating levels of more than a dozen candidate RBPs. Among these candidates, we characterize the ubiquitin-associated protein 2-like (UBAP2L) gene. Polysome profiling assays indicate that UBAP2L enhances translation of target mRNAs, likely due to ribosome interactions as supported by eCLIP data. UBAP2L can also be found in complex with fragile X mental retardation protein FMRP, and 52% of UBAP2L mRNA targets are also FMRP targets. UBAP2L depletion in a cortical neuronal model of Fragile X Syndrome (FXS) corrects molecular, cellular and electrophysiological defects relevant to autism spectrum disorder. Reduction of the Drosophila ortholog of UBAP2L in a FXS fly model rescues the neurodevelopmental defects due to loss of FMRP. Our efficient and scalable method identifies proteins involved in RNA metabolism and detailed studies of UBAP2L provides a new therapeutic strategy into human disease.

RNA binding proteins (RBPs) impact cellular protein levels by regulating messenger RNA (mRNA) levels. Assignment of function to hundreds of emerging, uncharacterized RBPs is a critical bottleneck to a complete understanding of gene expression control. Here, large-scale tethering of nearly a thousand RBPs discovers 50 RBPs that affect reporter RNA turnover and translation. Enhanced UV crosslinking and immunoprecipitation (eCLIP) identify hundreds of endogenous mRNA targets affected by manipulating levels of more than a dozen candidate RBPs. Among these candidates, we characterize the ubiquitin-associated protein 2-like (UBAP2L) gene. Polysome profiling assays indicate that UBAP2L enhances translation of target mRNAs, likely due to ribosome interactions as supported by eCLIP data. UBAP2L can also be found in complex with fragile X mental retardation protein FMRP, and 52% of UBAP2L mRNA targets are also FMRP targets. UBAP2L depletion in a cortical neuronal model of Fragile X Syndrome (FXS) corrects molecular, cellular and electrophysiological defects relevant to autism spectrum disorder. Reduction of the Drosophila ortholog of UBAP2L in a FXS fly model rescues the neurodevelopmental defects due to loss of FMRP. Our efficient and scalable method identifies proteins involved in RNA metabolism and detailed studies of UBAP2L provides a new therapeutic strategy into human disease.

Production of a positron emission tomography (PET) tracer involves three stages: production of the radioisotope, radio-labeling and purification of the tracer, and quality control (QC) testing of the final product (if the tracer is used in humans). Though the production of the positron-emitting radioisotope requires expensive and complex instrumentation (e.g. cyclotron or generator), in many cases, the radioisotope can simply be obtained from a commercial source. The radio-labeling and purification step is performed by machines called radiosynthesizers, which perform chemical synthesis and purification processes in a remote-controlled or automated manner within radiation-shielded “hot cells”. Quality control testing is performed to ensure each batch of radiopharmaceutical is safe prior to use in human subjects, and requires several analytical chemistry instruments. Though a couple of PET tracers are routinely available in final form from commercial sources, most other tracers need to be specially manufactured and quality tested at the researcher’s facility.Advances in radiosynthesizer technology such as automation, programmability, and reagent kits simplify the production of diverse tracers. Furthermore, recent efforts in miniaturized synthesizers based on microfluidics aim to reduce equipment cost, shielding and lab space needs, and quantities of expensive reagents, to make it more affordable to produce custom batches of PET tracers on demand.In the context of these emerging technologies, quality control testing still remains a bottleneck due to the high cost of the many expensive instruments, specially-trained staff, and documentation needed to determine the purity and ensure the levels of all potential contaminants are below acceptable limits. Several companies are developing automated systems for quality control testing to alleviate some of these issues. However, high cost and large size remain obstacles. We have therefore explored the use of microfluidic analytical chemistry techniques to address these remaining critical issues. In this dissertation, the feasibility of using capillary electrophoresis (CE) to analyze the chemical purity of PET tracers is investigated as a compact and inexpensive replacement for high performance liquid chromatography (HPLC) and other techniques that are normally used for this purpose. After establishing that CE has sufficient performance (comparable to HPLC) using several example chemical systems, the design, development, and characterization of a microscale chemical purity testing system are presented, along with a discussion of its capabilities and limitations. Overall, it appears that microchip CE could be used instead of HPLC for performing many of the required QC tests of PET tracers.

Many natural products, including fatty acids, polyketides, and non-ribosomal peptides have pharmacologically valuable properties. These products are biosynthesized using diverse pathways, but they employ very similar machineries and chemical transformations. Central to these pathways are carrier proteins, which covalently tether a growing intermediate. Small, dynamic carrier proteins facilitate transport of intermediates to subsequent reaction partners within the biosynthetic pathway, and protect them from unfavorable reactions in the cytosol. This dissertation focuses on these carrier proteins, characterizing them both structurally and biophysically. Solution state protein NMR methods are ideal for studying carrier proteins, given their small size and high flexibility.Several studies of the Escherichia coli fatty acid synthase (FAS) pathway yielded significant information about the structure and dynamics of the acyl carrier protein (ACP). Increasing the basic understanding of how modular synthases function, interact, and maintain product specificity has been a driving goal; such information is critical to engineering de novo modular pathways. Identification of the proteins in the pathways is essential, but understanding how they move and interact remains an intellectual bottleneck. Using mechanism-based crosslinking, the E. coli ACP was trapped in functional interaction with the cognate dehydratase protein, FabA. Structures were determined crystallographically in collaboration with the Tsai lab at the University of California, Irvine and by NMR in collaboration with the Opella lab at the University of California, San Diego. Residual Dipolar Couplings from NMR, together with Accelerated Molecular Dynamics studies in collaboration with the McCammon lab at the University of California, San Diego led to the identification of key residues necessary for this protein-protein interaction, and quantification of the ACP's dynamics both as a free protein and during interaction with a partner. Furthermore, studies of the actinorhodin polyketide synthase (PKS) pathway led to insights into the sequestration and cyclization of polyketide intermediates. To prevent unwanted side-reactions of polyketide intermediates, geometrically appropriate atom replacement mimetic probes were synthesized and loaded onto the actinorhodin ACP. Chemical shift perturbations of the protein amide resonances allowed monitoring of sequestration, as probes of longer length demonstrated larger perturbation of more residues than shorter polyketide probes.Additionally, studies of the prodigiosin and pyoluteorin pathways give new information about sequestration. Traditional NOE methods were employed to solve the solution NMR structure of the pyoluteorin carrier protein, PltL, in multiple cargo states. For the first time, sequestration of intermediates was observed in a non-ribosomal peptide synthetase (NRPS) system. Further studies are underway to determine if sequestration is observed in other NRPS systems. Finally, a follow-up to the ACP FabA project is in an early manuscript stage, detailing the E. coli ACP interaction with another partner, the ketosynthase FabB. NMR titration experiments, demonstrating key residues in the ACP FabB interaction are reported. In vivo complementation of the wt ACP with mutated ACPs showed that perturbing the ACP FabB interaction reduces E. coli's ability to produce unsaturated fatty acids at lower temperatures, connecting in vivo observations to in vitro effects.While challenges and unknowns remain, the work of this dissertation provides significant progress towards the possibilities of engineering novel pathways to produce pharmaceutically relevant or biochemically valuable products.