Almost all therapeutic proteins contain disulfide bonds to stabilize their native structure. Recombinant DNA technology enables many therapeutic proteins to be produced in bacteria, but the expression of native proteins is not always efficient due to the limited ability of bacteria to form disulfide bonds in vivo. It is often necessary to employ in vitro oxidative folding process to form the native disulfide bonds to obtain the native structure of disulfide-containing proteins. Aromatic disulfides are small molecules designed to match some of the physical properties of the active site of protein disulfide isomerase (PDI), which catalyzes the folding process of disulfide-containing proteins in eukaryotes. Three aromatic thiols with varying charges, PA, SA and QAS thiol, were used to fold reduced BPTI in vitro. Bovine pancreatic trypsin inhibitor (BPTI) is positively charged (pI = 10.5) at pH 7.3, and we hypothesized that mixed disulfide intermediates formed between BPTI and negatively charged small molecule thiols were more likely to precipitate due to their minimized net charge. Protein precipitation was observed during folding with negatively charged thiols, PA and SA, but not positively charged thiol QAS. At the folding pH of 7.3, almost 90% of native BPTI was produced in 2 h with the conditions of 0.25 mM QAS disulfide and 10 mM QAS thiol. Only 25% of native BPTI was produced in 2 h with the best conditions for glutathione and glutathione disulfide. Aromatic thiols with an elongated alkyl group on the aromatic ring, butyl, hexyl and octyl thiol, were hypothesized to increased interactions with the hydrophobic core of disulfide-containing proteins during folding, allowing more facile access to buried disulfide bonds. However, the longer the hydrocarbon chain, the more likely protein precipitation was to occur. About 90% native BPTI was formed in 1 h with 0.25 mM hexyl disulfide and 10 mM hexyl thiol. A method using capillary electrophoresis (CE) to analysis the oxidative folding process of reduced BPTI with small molecule thiols and disulfides was also developed. Folding of reduced BPTI with QAS disulfide was analyzed using CE in a shorter run time. The consumption of protein samples and solvent solutions was minimized.

The conformational dynamics of RNA are crucial to its role in numerous essential biological functions, requiring a comprehensive view of masses of individual molecular motions in order to fully understand these processes. Obtaining such a unified view, however, presents many challenges. Even the simplest RNA structures undergo rearrangements within an intricate three-dimensional network of secondary and tertiary interactions, resulting in motions that span a broad range of timescales. This complexity gives rise to a large number of experimental techniques sampling different aspects of the folding process, leaving a rather fragmented picture of RNA folding overall. In order to address the divergence and limitations of existing ensemble and single-molecule methods, this work describes the application of carbon nanotube (CNT)-based single-molecule field-effect transistors (smFET) as a platform for studying the folding and unfolding dynamics of RNA. smFET is capable of measuring individual biomolecular dynamics for long durations, while at a sufficiently high time resolution to capture the relevant timescales for RNA folding. This technique, moreover, avoids potential sources of interference or damage to the molecule as found in other available methods, and capitalizes on the detailed information that can be garnered from studying a single molecule as opposed to an ensemble. By taking advantage of the highly sensitive electronic properties and nanoscale dimensions of CNTs, and tethering a comparably sized and charged single biomolecule like RNA, it is possible to monitor the folding and unfolding of the molecule and characterize the kinetics associated with these motions. This thesis describes the optimization and application of such smFET technology to RNA stem-loops, which are extremely prevalent and thermodynamically stable elements of RNA secondary structure. Chapter 1 introduces the biological context of these molecules as well as the mechanisms of CNT-based smFET sensing. In Chapter 2, the methods used to fabricate smFET devices are described along with the experiments conducted to optimize single-molecule tethering. These methods and protocols were then applied to the studies detailed in Chapter 3, which examines the implications of the thermodynamic and kinetic analyses of RNA stem-loop folding and unfolding as investigated with smFET. Chapter 4 concludes with a brief overview of what has been accomplished and potential future directions for this platform. The work expressed here thus presents a cohesive view of RNA stem-loop folding, integrating the past results of both experimental and computational studies. With this improved smFET methodology, this technique can be applied to many other essential and increasingly complex biological systems to achieve a fuller and richer understanding of the processes that govern life.

The accumulation of misfolded proteins within intracellular aggregates is a distinctive feature observed within multiple neurodegenerative diseases (NDDs). However, the genes and pathways that regulate protein misfolding, aggregation, and subsequent cellular toxicity remain poorly understood. Here I describe a high-throughput discovery platform that enables the simultaneous screening of dozens of neurodegenerative disease models to rapidly uncover genetic modifiers that alter the solubility and toxicity of a wide variety of aggregation-prone proteins. From these studies, I identify the human HSP40 chaperone, DNAJB6 as a potent rescuer of the misfolding and proteotoxicity of multiple RNA-binding proteins implicated in Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) including FUS, TDP-43, and hnRNPA1. I, with collaborator help, further demonstrate that DNAJB6 has an intrinsic ability to phase separate under physiologic conditions and can alter the properties of FUS containing condensates by maintaining them in a gel-like state over long periods, preventing FUS aggregation. By conducting domain mapping studies and a deep mutational scan on DNAJB6, I am able to gain detailed insight into its mechanism of action while also uncovering a series of novel variants with enhanced activity. During the development of this multiplexed screening approach for neurodegenerative disease models, research was interrupted by a global pandemic caused by SARS-CoV-2. I realized that the themes of studying proteotoxicity of multiple related, yet distinct models could be applied towards drug development to identify inhibitors of the essential 3CL proteases encoded by multiple coronaviruses, which cause proteotoxicity when expressed in cells. As such, I develop and describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This essay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, I identify compounds with broad 3CLpro inhibitory activity. I also adapt the assay for use in compound screening and in doing so uncover additional SARS-CoV-2 3CLpro inhibitors. I observe strong concordance between data emerging from this assay and those obtained from live virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. I identify two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested including 3CLpro enzymes from understudied zoonotic coronaviruses.

Proteins are composed of a unique sequence of amino acids, whose order guides a protein to adopt its particular fold and perform a specific function. It has been shown that a protein's 3-dimensional structure is embedded within its primary sequence. The problem that remains elusive to biochemists is how a protein's primary sequence directs the folding to adopt such a specific conformation. In an attempt to gain a better understanding of protein folding, my research tests a novel model of protein packing using protein design. The model defines the knob-socket construct as the fundamental unit of packing within protein structure. The knob-socket model characterizes packing specificity in terms of amino acid preferences for sockets in different environments: sockets filled with a knob are involved in inter-helical interactions and free sockets are involved in intra-helical interactions. Equipped with this knowledge, I sought to design a unique protein, Ksα1.1, completely de novo. The sequence was selected to induce helix formation with a predefined tertiary packing interface. Circular dichroism showed that Ksα1.1 formed α-helical secondary structure as intended. The nuclear magnetic resonance studies demonstrated formation of a high order oligomer with increased protein concentration. These results and analysis prove that the knob-socket model is a predictive model for all α-helical protein packing. More importantly, the knob-socket model introduces a new protein design method that can potentially hold a solution to the folding problem.

Spider silk is a natural high-performance biopolymer with superior mechanical propetiies. Although these fibers out perfmm several man-made and natural biomaterials, there are cha llenges to be circumvented before commercialization. One of the silkproducing glands warranting further study is the pyrifonn gland, which produces gluelike threads functioning to cement dragline silk to substrates. We focused on the molecular properties of PySp 1, the major component of pyrifonn silk from Latrodectus hersperus, and its putative Oiiholog, PySp2, from Nephi/a clavipes. To date, there are no reports describing the secondary structure of PySp internal block repeats. Moreover, because the PySp C-terminus amino acid residues are distinct from MaSp C-terminus and the morphology of these glands is different, we hypothesized that PySp C-terminal domains form distinct secondary structures. The MaSp C-terminus has been shown to regulate the silk assembly process and whether the PySp C-terminus performs a similar function is unknown. In order to test this supposition, we used the following experimental approaches: I) we developed a series of PySp prokaryotic expression constructs carrying various block repeat modules representative of the internal iterations found within the protein chain; 2) we constructed prokaryotic expression vectors coding for the PySp C-terminal domains; 3) we expressed and purified the PySp C-terminal domains from bacteria; 4) we performed structural analyses of the purified PySp C-terminal domains using cd spectroscopy and atomic force microscopy. After expression and purification of the PySp C-tennini proteins, our studies support that this domain displays a predominantly ~-sheet structure, distinctive from the NMR-determined ahelical nature of MaSp C-tennini. The difference in secondary structure implies the MA and pyriform glands use different biochemical mechanisms during fiber extrusion to control protein folding and assembly. By investigating protein folding and fiber formation for different spider silk types, its characteristics can be customized for spinning different materials for industrial applications.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18918号 / 薬科博第32号 / 新制||薬||4(附属図書館) / 31869 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 松﨑 勝巳, 教授 加藤 博章, 教授 石濱 泰 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20008号 / 農博第2192号 / 新制||農||1045(附属図書館) / 学位論文||H28||N5017(農学部図書室) / 33104 / 京都大学大学院農学研究科農学専攻 / (主査)教授 裏出 令子, 教授 松村 康生, 教授 三上 文三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20431号 / 農博第2216号 / 新制||農||1048(附属図書館) / 学位論文||H29||N5052(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 裏出 令子, 教授 松村 康生, 教授 三上 文三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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