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- All Subjects: Biochemistry
- Genre: Doctoral Dissertation
- Creators: Chiu, Po-Lin
- Status: Published
Description
Cryogenic Electron Microscopy (Cryo-EM) is a method that can be used for studying the structure of biological systems. Biological samples are frozen to cryogenic temperatures and embedded in a vitreous ice when they are imaged by electrons. Due to its ability to preserve biological specimens in near-native conditions, cryo-EM has a significant contribution to the field of structural biology.Single-particle cryo-EM technique was utilized to investigate the dynamical characteristics of various protein complexes such as the Nogo receptor complex, polymerase ζ (Polζ) in yeast and human integrin ⍺vβ8-pro-TGFβ1-GARP complex. Furthermore, I proposed a new method that can potentially improve the sample preparation for cryo-EM. The Nogo receptor complex was expressed using baculovirus expression system in sf9 insect cells and isolated for structural studies. Nogo receptor complex was found to have various stoichiometries and interactions between individual proteins. A structural investigation of the yeast apo polymerase ζ holoenzyme was also carried out. The apo Polζ displays a concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. Furthermore, a lysine residue that obstructs the DNA-binding channel in apo Polζ was found and suggested a gating mechanism. In addition, cryo-EM studies of the human integrin ⍺vβ8-pro-TGFβ1-GARP complex was conducted to assess its dynamic interactions. The 2D classifications showed the ⍺vβ8-pro-TGFβ1-GARP complex is highly flexible and required several sample preparation techniques such as crosslinking and graphene oxide coating to improve protein homogeneity on the EM grid. To overcome challenges within the cryo-EM technique such as particle adsorption on air-water interface, I have documented a collaborative work on the development and application of lipid monolayer sandwich on cryo-EM grid. Cryogenic electron tomography (cryo-ET) along with cryo-EM were used to study the characteristics of lipid monolayer sandwich as a potential protective layer for EM grid. The cryo-ET results demonstrated that the thickness of lipid monolayer is adequate for single-particle cryo-EM processing. Furthermore, there was no appearance of preferred orientations in cryo-EM and cryo-ET images. To establish that this method is actually beneficial, more data must be collected, and high-resolution structures of protein samples must be obtained using this methodology.
ContributorsTruong, Chloe Du (Author) / Chiu, Po-Lin (Thesis advisor) / Liu, Wei (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2021
Description
Structural-based drug discovery is becoming the essential tool for drug development withlower cost and higher efficiency compared to the conventional method. Knowledge of the
three-dimensional structure of protein targets has the potential to accelerate the process
for screening drug candidates. X-ray crystallography has proven to be the most used and
indispensable technology in structural-based drug discovery. The provided
comprehensive structural information about the interaction between the disease-related
protein target and ligand can guide the chemical modification on the ligand to improve
potency and selectivity. X-ray crystallography has been upgraded from traditional
synchrotron to the third generation, which enabled the surge of the structural
determination of macromolecular. The introduction of X-ray free electron laser further
alleviated the uncertain and time-consuming crystal size optimization process and
extenuated the radiation damage by “diffraction before destruction”. EV-D68 2A
protease was proved to be an important pharmaceutical target for acute flaccid myelitis.
This thesis reports the first atomic structure of the EV-D68 2A protease and the structuresof its two mutants, revealing it adopting N-terminal four-stranded sheets and C-terminal
six-stranded ß-barrels structure, with a tightly bound zinc atom. These structures will
guide the chemical modification on its inhibitor, Telaprevir. Integrin ⍺Mβ2 is an integrin
with the α I-domain, related to many immunological functions including cell
extravasation, phagocytosis, and immune synapse formation, so studying the molecular
ligand-binding mechanism and activation mechanism of ⍺Mβ2 is of importance. This
thesis uncovers the preliminary crystallization condition of ⍺Mβ2-I domain in complex
with its ligand Pleiotrophin and the initial structural model. The structural model shows consistency with the previous hypothesis that the primary binding sites are metal iondependent
adhesion sites on ⍺Mβ2-I domain and the thrombospondin type-1 repeat (TSR)
domains of Pleiotrophin. Drug molecules with high potency and selectivity can be
designed based on the reported structures of the EV-D68 2A protease and ⍺Mβ2-I domain
in the future.
ContributorsLiu, Chang (Author) / Liu, Wei (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Purification, Characterization, and Structural Determination of Proteins Vital to Infectious Disease
Description
The work in this dissertation progressed the research of structural discovery for two targets critical in the fight of infectious disease. Francisella lipoprotein 3 (Flpp3) is a virulent determinant of tularemia and was the first protein of study. The proteins soluble domain was studied using a hybrid modeling theory that used small angle X-ray scattering (SAXS) in combination with computation analysis to generate a SAXS-refined structure. The SAXS-refined structure closely resembled the NMR structure (PDB: 2MU4) which contains a hydrophobic cavity inside the protein that could be used for drug discovery purposes. The full-length domain of Flpp3 purified from the outer membrane of E. coli was also studied with a combination of biophysical characterization methods. Mass spectrometry and western blot analysis confirmed Flpp3 being translocated to the outer membrane, while SDS-PAGE confirmed the purity of Flpp3 in the monomeric form after size exclusion chromatography. Using Circular Dichroism (CD) the monomeric form of Flpp3 was shown to be almost fully refolded into having a primarily β-stranded secondary structure. This information advances the progress of both tularemia research and outer membrane protein research as no natively folded outer membrane protein structures have been solved for F. tularensis.The second protein worked on in this dissertation is the nonstructural protein 15 from SARS-CoV-2, also called NendoU. Nsp15 is an endoribonuclease associated with aiding the virus responsible for the current COVID-19 pandemic in evasion of the immune system. An inactive mutant of Nsp15 was studied with both negative stain electron microscopy and cryogenic electron microscopy (Cryo-EM) in the presence of RNA or without RNA present. The initial findings of negative stain electron microscopy of Nsp15 with and without RNA showed a difference in appearance. Negative stain analysis of Nsp15 is in the presence of a 5nt RNA sequence in low salt conditions shows a conformational change when compared to Nsp15 without RNA present. As well the presence of RNA appeared to shift the electron density in Cryo-EM studies of Nsp15. This work advances the research in how Nsp15 may bind and cleave RNA and aid in the evasion of the host cell immune system.
ContributorsGoode, Matthew (Author) / Fromme, Petra (Thesis advisor) / Guo, Jia (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2022
Description
Infectious diseases are the third leading cause of death in the United States and the second leading cause of death in the world. This work aims to advance structural studies of vital proteins involved in the infection process of both a bacterial and a viral infectious disease in hopes of reducing infection, and consequently, fatality rates. The first protein of interest is OspA, a major outer surface protein in Borrelia burgdorferi – the causative bacterium of Lyme disease. Previous functional studies of OspA allude to both a role in colonization of B. burgdorferi in the tick vector and in evasion of the human immune system. This work describes the first ever structural studies of OspA as it is seen by the immune system: in the outer membrane. OspA was expressed in and purified from the outer membrane of Escherichia coli prior to characterization via circular dichroism (CD), native polyacrylamide gel electrophoresis, and electron microscopy. Characterization studies of OspA provide the first evidence of multimeric formation of OspA when translocated to the outer membrane, which presents a new perspective from which to build upon for the design of vaccinations against Lyme disease. The second protein of interest is nonstructural protein 15 (Nsp15), a protein responsible for facilitating immune system evasion of SARS-CoV-2 – the virus responsible for the COVID-19 pandemic. Nsp15 functions to enzymatically cleave negative sense viral RNA to avoid recognition by the human immune system. The work described in this dissertation is dedicated to the electron microscopy work utilized to reveal structural information on an inactive variant of Nsp15 bound to RNA sequences. Negative stain electron microscopy was used to verify Nsp15 structural integrity, as well as reveal a low-resolution image of structural deviation when RNA is bound to Nsp15. Cryo-electron microscopy was performed to solve structural density of Nsp15 without RNA to a resolution of 3.11 Å and Nsp15 bound to 5-nucleotides of RNA to a resolution of 3.99 Å. With further refinement, this structure will show the first structural data of Nsp15 bound to a visible RNA sequence, revealing information on the binding and enzymatic activity of Nsp15.
ContributorsKaschner, Emily (Author) / Fromme, Petra (Thesis advisor) / Hansen, Debra T (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2022
Description
Macromolecular structural biology advances the understanding of protein function through the structure-function relationship for applications to scientific challenges like energy and medicine. The proteins described in these studies have applications to medicine as targets for therapeutic drug design. By understanding the mechanisms and dynamics of these proteins, therapeutics can be designed and optimized based on their unique structural characteristics. This can create new, focused therapeutics for the treatment of diseases with increased specificity — which translates to greater efficacy and fewer off-target effects. Many of the structures generated for this purpose are “static” in nature, meaning the protein is observed like a still-frame photograph; however, the use of time-resolved techniques is allowing for greater understanding of the dynamic and flexible nature of proteins. This work advances understanding the dynamics of the medically relevant proteins NendoU and Taspase1 using serial crystallography to establish conditions for time-resolved, mix-and-inject crystallographic studies.
ContributorsJernigan, Rebecca Jeanne (Author) / Fromme, Petra (Thesis advisor) / Hansen, Debra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Hogue, Brenda (Committee member) / Arizona State University (Publisher)
Created2022
Description
Receiving signals and responding to the environment is crucial for survival for every living organism. One of those signals is being able to detect environmental and visceral temperatures. Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential melastatin 8 (TRPM8) are ion channels within cells that allow higher organisms to detect hot and cold temperatures, respectively. These TRP channels are also implicated in diverse physiological roles including pain, obesity, and cancer. As a result, these channels have garnered interest as potential targets for therapeutic interventions. However, the entanglement of TRPV1 and TRPM8 polymodal activation where it responds to a variety of different stimuli has caused adverse side effects of body thermal dysregulation and misregulation when antagonizing these channels as drug targets. This dissertation will dissect the molecular mechanism and regulation of TRPV1 and TRPM8. An in-depth look into the complex and conflicting results in trying to find the key area for thermosensation as well as looking into disentangling the polymodal activation modes in TRPV1. The regulatory mechanism between TRPM8 with phosphoinositide interacting regulator of TRPs (PIRT) and calmodulin will be examined using nuclear magnetic resonance (NMR). A computational, experimental, and methodical approach into ancestral TRPM8 orthologs using whole-cell patch-clamp electrophysiology, calcium mobilization assay, and cellular thermal shift assay (CETSA) to determine whether these modes of activation can be decoupled. Lastly, smaller studies are covered like developing a way to delivery full-length and truncated protein using amphipols to artificial and live cells without the biological regulatory processes and the purification of the TRPM8 transmembrane domain (TMD). In the end, two successful methods were developed to study the polymodal activation of proteins.
ContributorsLuu, Dustin Dean (Author) / Van Horn, Wade D (Thesis advisor) / Redding, Kevin E (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2023
Description
The understanding of protein functions in vivo is very important since the protein is the building block of a cell. Cryogenic electron microscopy (cryo-EM) is capable of visualizing protein samples in their near-native states in high-resolution details. Cryo-EM enables the visualization of biomolecular structures at multiscale ranging from a cellular structure to an atomic structure of protein subunit.Neurodegenerative diseases, like Alzheimer’s disease and frontotemporal dementia, have multiple dysregulated signaling pathways. In my doctoral studies, I investigated two protein complexes relevant to these disorders: one is the proNGF- p75 neurotrophin receptor (p75NTR)- sortilin neurotrophin complex and the other is the p97R155H mutant complex. The neurotrophins are a family of soluble basic growth factors involved in the development, maintenance, and proliferation of neurons in the central nervous system (CNS) and peripheral nervous system (PNS). The ligand for the neuronal receptors dictates the fate of the neuronal cells. My studies focused on understanding the binding interfaces between the proteins in the proNGF-p75NTR-sortilin neuronal apoptotic complex. I have performed the biochemical characterization of the complex to understand how the complex formation occurs.
Single amino-acid mutation of R155H on the N-domain of p97 is known to be the prevalent mutation in 40% patients suffering from neurodegenerative disease. The p97R155H mutant exhibits abnormal ATPase activity and cofactor dysregulation. I pursued biochemical characterization in combination with single-particle cryo-EM to explore the interaction of p97R155H mutant with its cofactor p47 and determined the full-length structures of the p97R155H-p47 assemblies for the first time. About 40% p97R155H organizes into higher order dodecamers, which lacks nucleotide binding, does not bind to p47, and closely resembles the structure of p97 bound with an adenosine triphosphate (ATP)-competitive inhibitor, CB-5083, suggesting an inactive state of the p97R155H mutant. The structures also revealed conformational changes of the arginine fingers which might contribute to the elevated p97R155H ATPase activity. Because the D1-D2 domain communication is important in regulating the ATPase function, I further studied the functions of the conserved L464 residue on the D1-D2 linker using mutagenesis and single-particle cryo-EM. The biochemical and structural results suggested the torsional constraint of the D1-D2 linker likely modulates the D2 ATPase activity. Our studies thus contributed to develop deeper knowledge of the intricate cellular mechanisms and the proteins affected in disease pathways.
ContributorsNandi, Purbasha (Author) / Chiu, Po-Lin (Thesis advisor) / Mazor, Yuval (Committee member) / Hansen, Debra T (Committee member) / Arizona State University (Publisher)
Created2022
Description
Borrelia burgdorferi (Bb), the causative agent of Lyme disease, is a unique pathogen, with a complex genome and unique immune evasion tactics. It lacks genes encoding proteins involved in nutrient synthesis and typical metabolic pathways, and therefore relies on the host for nutrients. The Bb genome encodes both an unusually high number of predicted outer surface lipoproteins of unknown function but with multiple complex roles in pathogenesis, and an unusually low number of predicted outer membrane proteins, given the necessity of bringing in the required nutrients for pathogen survival. Cellular processing of bacterial membrane proteins is complex, and structures of proteins from Bb have all been solved without the N-terminal signal sequence that directs the protein to proper folding and placement in the membrane. This dissertation presents the first membrane-directed expression in E. coli of several Bb proteins involved in the pathogenesis of Lyme disease. For the first time, I present evidence that the predicted lipoprotein, BBA57, forms a large alpha-helical homo-multimeric complex in the OM, is soluble in several detergents, and purifiable. The purified BBA57 complex forms homogeneous, 10 nm-diameter particles, visible by negative stain electron microscopy. Two-dimensional class averages from negative stain images reveal the first low-resolution particle views, comprised of a ring of subunits with a plug on top, possibly forming a porin or channel. These results provide the first evidence to support our theories that some of the predicted lipoproteins in Bb form integral-complexes in the outer membrane, and require proper membrane integration to form functional proteins.
ContributorsRobertson, Karie (Author) / Hansen, Debra T. (Thesis advisor) / Fromme, Petra (Thesis advisor) / Van Horn, Wade (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020
Description
G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data. Additionally, differential and specific cholesterol binding in the CCK receptor subfamily was observed while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. Mutation of this conserved CRAC sequence of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase, however, as most techniques, it has limitations. Using an optimized SFX experimental setup in a helium atmosphere we determined the room temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution and compared it with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, we demonstrated the capability of utilizing high XFEL beam transmissions, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete data set.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
ContributorsGeiger, James (Author) / Liu, Wei (Thesis advisor) / Mills, Jeremy (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020
Description
Since its conception over a century ago, X-ray crystallography (XRC) has become the most successful method used to elucidate the structures and functions of biological molecules at atomic resolution. The extensive use of XRC has led to meaningful discoveries across many scientific fields, notably its contributions to rational drug design. Traditional drug discovery relies on the use of trial-and-error based approaches in cellular and animal models of disease to identify chemical probes that elicit desirable therapeutic effects based off changes in phenotype. However, this approach lacks critical information in regards to the biological target in which the compound interacts with. In contrast, the use of rational drug design presents the opportunity to identify chemical probes that target specific protein targets of known medical importance and study their interactions using three dimensional structures that can be used to suggest new drug candidates. The main focus of my research presented in this dissertation aims to utilize XRC to discover novel therapeutics. In this work, I begin by describing the use of structure-based drug discovery for the rational design of hydrocarbon-stapled peptides that block Focal Adhesion Kinase (FAK) scaffolding in cancer (Chapter 2). FAK is an intracellular tyrosine kinase that has been linked to many cancers through its interaction with Paxillin LD motifs as it relates to tumor growth, invasion, metastasis, and suppression of apoptosis. The results of this study demonstrate the effectiveness hydrocarbon-stapling has on the native Paxillin LD2 sequence with ~50 fold greater binding affinity by surface plasmon resonance (SPR) that can be explained by the unique structural interactions observed by XRC. Next, I present a series of methods which lays the foundations for the discovery of novel anti-bacterial drugs that target 3-Deoxy-D-manno-octulosonate-8-phosphate (KDO8P) Synthase, a critical enzyme in the biosynthesis of gram-negative lipopolysaccharides (Chapter 3).
ContributorsThifault, Darren G. (Author) / Fromme, Petra (Thesis advisor) / Mills, Jeremy (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021