Matching Items (94)
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Description
Structure is a critical component in drug development. This project supports antibody- facilitated structure determination for the following eleven membrane proteins: the human histamine and dopamine G protein-coupled receptors (HRH4 and DRD2) involved in a wide variety of pathologies such as allergies, inflammation, asthma, pain along with Parkinson's and schizophrenia

Structure is a critical component in drug development. This project supports antibody- facilitated structure determination for the following eleven membrane proteins: the human histamine and dopamine G protein-coupled receptors (HRH4 and DRD2) involved in a wide variety of pathologies such as allergies, inflammation, asthma, pain along with Parkinson's and schizophrenia respectively, the human cystic fibrosis transmembrane conductance regulator (CFTR), the human NaV1.8 voltage-gated sodium ion channel, the human TPC2 two-pore channel, the SARS virus proteins 3a, E and M, the MERS virus protein E and M, and the malarial chloroquine resistance transporter (PfCRT). Serum antibodies against these proteins were generated by genetic immunization, and both in vitro and in vivo expressed membrane proteins were created to characterize the serum antibodies. Plasmid clones were generated for genetic immunization, in vitro protein expression, and in vivo expression (HEK293T transfection). Serum antibodies were generated by genetic immunization of mice by gene gun. Genetic immunization promotes an immune response that allows for the generation of antibodies in the absence of purified protein. In vitro expression was accomplished through the novel technique: in vitro translation with hydrophobic magnetic beads (IVT-HMB). Transfections were performed using the HEK293T cell line to express the protein in vivo. The generated protein was then used in gel electrophoresis and silver stain and/or Western blot analyses to identify and visualize the proteins. These expressed proteins will allow for forthcoming characterization of the generated antibodies. The resulting antibodies will in turn enable structure determination of these important membrane proteins by co-crystallization.
ContributorsDrotar, Beniamin (Author) / Fromme, Petra (Thesis director) / Hansen, Debra T. (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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Description

This study aims to examine children’s fruit, vegetable, and added sugar consumption relative to the Dietary Guidelines for Americans and the American Heart Association’s recommendations, as well as to compare children’s reported consumption with parental perception of the child’s overall diet quality. Data were drawn from 2 independent, cross sectional

This study aims to examine children’s fruit, vegetable, and added sugar consumption relative to the Dietary Guidelines for Americans and the American Heart Association’s recommendations, as well as to compare children’s reported consumption with parental perception of the child’s overall diet quality. Data were drawn from 2 independent, cross sectional panels (2009–10 and 2014–15) of the New Jersey Child Health Study. The analytical sample included 2229 households located in five New Jersey cities. Daily consumption of fruit (cups), vegetables (cups), and added sugars (teaspoons) for all children (3–18 years old) were based on parent reports. Multivariate linear regression analyses estimated children’s adjusted fruit, vegetable, and added sugar consumption across parents’ perception categories (Disagree; Somewhat Agree; and Strongly Agree that their child eats healthy). Although only a small proportion of children meet recommendations, the majority of parents strongly agreed that their child ate healthy. Nonetheless, significant differences, in the expected direction, were observed in vegetable and fruit consumption (but not sugar) across parental perceptional categories for most age/sex groups. Dietary interventions tailored to parents should include specific quantity and serving-size information for fruit and vegetable recommendations, based on their child’s age/sex, and highlight sources of added sugar and their sugar content.

ContributorsEliason, Jessica (Author) / Acciai, Francesco (Author) / DeWeese, Robin (Author) / Vega-Lopez, Sonia (Author) / Ohri-Vachaspati, Punam (Author)
Created2020-08-03
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Description

Disparities in healthy food access are well documented in cross-sectional studies in communities across the United States. However, longitudinal studies examining changes in food environments within various neighborhood contexts are scarce. In a sample of 142 census tracts in four low-income, high-minority cities in New Jersey, United States, we examined

Disparities in healthy food access are well documented in cross-sectional studies in communities across the United States. However, longitudinal studies examining changes in food environments within various neighborhood contexts are scarce. In a sample of 142 census tracts in four low-income, high-minority cities in New Jersey, United States, we examined the availability of different types of food stores by census tract characteristics over time (2009–2017). Outlets were classified as supermarkets, small grocery stores, convenience stores, and pharmacies using multiple sources of data and a rigorous protocol. Census tracts were categorized by median household income and race/ethnicity of the population each year. Significant declines were observed in convenience store prevalence in lower- and medium-income and majority black tracts (p for trend: 0.004, 0.031, and 0.006 respectively), while a slight increase was observed in the prevalence of supermarkets in medium-income tracts (p for trend: 0.059). The decline in prevalence of convenience stores in lower-income and minority neighborhoods is likely attributable to declining incomes in these already poor communities. Compared to non-Hispanic neighborhoods, Hispanic communities had a higher prevalence of small groceries and convenience stores. This higher prevalence of smaller stores, coupled with shopping practices of Hispanic consumers, suggests that efforts to upgrade smaller stores in Hispanic communities may be more sustainable.

ContributorsOhri-Vachaspati, Punam (Author) / DeWeese, Robin (Author) / Acciai, Francesco (Author) / DeLia, Derek Michael, 1969- (Author) / Tulloch, David (Author) / Tong, Daoqin (Author) / Lorts, Cori (Author) / Yedidia, Michael J., 1946- (Author)
Created2019-07-03
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Description
First evolving in cyanobacteria, the light reactions of oxygenic photosynthesis are carried out by the membrane proteins, photosystem II and photosystem I, located in the thylakoid membrane. Both utilize light captured by their core antenna systems to catalyze a charge separation event at their respective reaction centers and energizes electrons

First evolving in cyanobacteria, the light reactions of oxygenic photosynthesis are carried out by the membrane proteins, photosystem II and photosystem I, located in the thylakoid membrane. Both utilize light captured by their core antenna systems to catalyze a charge separation event at their respective reaction centers and energizes electrons to be transferred energetically uphill, eventually to be stored as a high energy chemical bond. These protein complexes are highly conserved throughout different photosynthetic lineages and understanding the variations across species is vital for a complete understanding of how photosynthetic organisms can adapt to vastly different environmental conditions. Most knowledge about photosynthesis comes from only a handful of model organisms grown under laboratory conditions. Studying model organisms has facilitated major breakthroughs in understanding photosynthesis, however, due to the vast global diversity of environments where photosynthetic organisms are found, certain aspects of this process may be overlooked or missed by focusing on a select group of organisms optimized for studying in laboratory conditions. This dissertation describes the isolation of a new extremophile cyanobacteria, Cyanobacterium aponinum 0216, from the Arizona Sonoran Desert and its innate ability to grow in light intensities that exceed other model organisms. A structure guided approach was taken to investigate how the structure of photosystem I can influence the spectroscopic properties of chlorophylls, with a particular focus on long wavelength chlorophylls, in an attempt to uncover if photosystem I is responsible for high light tolerance in Cyanobacterium aponinum 0216. To accomplish this, the structure of photosystem I was solved by cryogenic electron microscopy to 2.7-anstrom resolution. By comparing the structure and protein sequences of Cyanobacterium aponinum to other model organisms, specific variations were identified and explored by constructing chimeric PSIs in the model organism Synechocystis sp. PCC 6803 to determine the effects that each specific variation causes. The results of this dissertation describe how the protein structure and composition affect the spectroscopic properties of chlorophyll molecules and the oligomeric structure of photosystem I, possibly providing an evolutionary advantage in the high light conditions observed in the Arizona Sonoran Desert.
ContributorsDobson, Zachary (Author) / Fromme, Petra (Thesis advisor) / Mazor, Yuval (Thesis advisor) / Redding, Kevin (Committee member) / Moore, Gary (Committee member) / Arizona State University (Publisher)
Created2022
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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

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

Oceanic life is facing the deleterious aftermath of coral bleaching. To reverse the damages introduced by anthropological means, it is imperative to study fundamental properties of corals. One way to do so is to understand the metabolic pathways and protein functions of corals that contribute to the resilience of coral

Oceanic life is facing the deleterious aftermath of coral bleaching. To reverse the damages introduced by anthropological means, it is imperative to study fundamental properties of corals. One way to do so is to understand the metabolic pathways and protein functions of corals that contribute to the resilience of coral reefs. Although genomic sequencing and structural modeling has yielded significant insights for well-studied organisms, more investigation must be conducted for corals. Better yet, quantifiable experiments are far more crucial to the understanding of corals. The objective is to clone, purify, and assess coral proteins from the cauliflower coral species known as Pocillopora damicornis. Presented here is the pipeline for how 3-D structural modeling can help support the experimental data from studying soluble proteins in corals. Using a multi-step selection approach, 25 coral genes were selected and retrieved from the genomic database. Using Escherischia coli and Homo sapiens homologues for sequence alignment, functional properties of each protein were predicted to aid in the production of structural models. Using D-SCRIPT, potential pairwise protein-protein interactions (PPI) were predicted amongst these 25 proteins, and further studied for identifying putative interfaces using the ClusPro server. 10 binding pockets were inferred for each pair of proteins. Standard cloning strategies were applied to express 4 coral proteins for purification and functional assays. 2 of the 4 proteins had visible bands on the Coomassie stained gel and were able to advance to the purification step. Both proteins exhibited a faint band at the expected migration distance for at least one of the elutions. Finally, PPI was carried out by mixing protein samples and running in a native gel, resulting in one potential pair of PPI.

ContributorsHuang, Joe (Author) / Klein-Seetharaman, Judith (Thesis director) / Fromme, Petra (Committee member) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
Description

Diisobutylene maleic acid, or DIBMA, offers a novel approach to integral membrane protein extraction without requiring the use of detergent. This copolymer extracts the protein along with the surrounding lipids, creating native nanodiscs. This method of solubilization is the preferred method, as traditional detergent solubilization can possibly alter the structural

Diisobutylene maleic acid, or DIBMA, offers a novel approach to integral membrane protein extraction without requiring the use of detergent. This copolymer extracts the protein along with the surrounding lipids, creating native nanodiscs. This method of solubilization is the preferred method, as traditional detergent solubilization can possibly alter the structural and functional integrity of the membrane protein. DIBMA solubilization, on the other hand, is able to create a more stable environment for the integral membrane protein, while allowing purification through commonly used chromatography methods similar to established detergent solubilization protocols. In this project, we study the ability of DIBMA to isolate the integral membrane protein, chloroplast ATP synthase, without the use of detergents.

ContributorsBalachandran, Kavya (Author) / Fromme, Petra (Thesis director) / Yang, Jay-How (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / College of Health Solutions (Contributor)
Created2023-05
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Description
This work comprises a cumulative effort to provide analysis of proteins relevant to understanding and treating human disease. This dissertation focuses on two main protein complexes: the structure of the Chimp adenovirus Y25 capsid assembly, as used in the SARS-CoV-2 vaccine, Vaxzveria, and the Dbl family RhoGEF (guanosine exchange factor)

This work comprises a cumulative effort to provide analysis of proteins relevant to understanding and treating human disease. This dissertation focuses on two main protein complexes: the structure of the Chimp adenovirus Y25 capsid assembly, as used in the SARS-CoV-2 vaccine, Vaxzveria, and the Dbl family RhoGEF (guanosine exchange factor) Syx and its associated small G protein, RhoA. The course of research was influenced heavily by the onset of the Covid-19 pandemic and associated lockdown, which pushed anyone with the means to do meaningful research to shift priorities towards addressing the greatest public health crisis since the 1918 flu pandemic. Analysis of the Syx-RhoA complex for the purposes of structurally guided drug design was initially the focus of heavy optimization efforts to overcome the numerous challenges associated with expression, purification, and handling of this protein. By analyzing E. Coli derived protein new important knowledge was gained about this protein’s biophysical characteristics which contribute to its behavior and may inform drug design efforts. Expression in SF9 insect cells resulted in promising conditions for production of homogeneous and monodispersed protein. Homology modeling and molecular dynamics simulation of this protein support hypotheses about its interactions with both RhoA as well as regions of the cytoplasmic leaflet of the cell membrane. Structural characterization of ChAdOx1, the adenoviral vector used in the AstraZeneca Covid-19 vaccine, Vaxzveria resulted in the highest resolution adenovirus structure ever solved (3.07Å). Subsequent biochemical analysis and computational simulations of PF4 with the ChAdOx1 capsid reveal interactions with important implications for vaccine induced thrombocytic throbocytopenia syndrome, a disorder observed in approximately 0.000024% of patients who receive Vaxzveria.
ContributorsBoyd, Ryan J (Author) / Fromme, Petra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2021
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Description
Particulate Guanylyl Cyclase Receptor A (pGC-A) is an atrial natriuretic peptide receptor, which plays a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain of pGC-A interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop a method

Particulate Guanylyl Cyclase Receptor A (pGC-A) is an atrial natriuretic peptide receptor, which plays a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain of pGC-A interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop a method that can regulate pGC-A, structural information regarding its intact form is necessary. Currently, only the extracellular domain structure of rat pGC-A has been determined. However, structural data regarding the transmembrane domain, as well as functional intracellular domain regions, need to be elucidated.This dissertation presents detailed information regarding pGC-A expression and optimization in the baculovirus expression vector system, along with the first purification method for purifying functional intact human pGC-A. The first in vitro evidence of a purified intact human pGC-A tetramer was detected in detergent micellar solution. Intact pGC-A is currently proposed to function as a homodimer. Upon analyzing my findings and acknowledging that dimer formation is required for pGC-A functionality, I proposed the first tetramer complex model composed of two functional subunits (homodimer). Forming tetramer complexes on the cell membrane increases pGC-A binding efficiency and ligand sensitivity. Currently, a two-step mechanism has been proposed for ATP-dependent pGC-A signal transduction. Based on cGMP functional assay results, it can be suggested that the binding ligand also moderately activates pGC-A, and that ATP is not crucial for the activation of guanylyl cyclase. Instead, three modulators can regulate different activation levels in intact pGC-A. Crystallization of purified intact pGC-A was performed to determine its structure. During the crystallization condition screening process, I successfully selected seven promising initial crystallization conditions for intact human pGC-A crystallization. One selected condition led to the formation of excellent needle-shaped crystals. During the serial crystallography diffraction experiment, five diffraction patterns were detected. The highest diffraction resolution spot reached 3 Å. This work will allow the determination of the intact human pGC-A structure while also providing structural information on the protein signal transduction mechanism. Further structural knowledge may potentially lead to improved drug design. More precise mutation experiments could help verify the current pGC-A signal transduction and activation mechanism.
ContributorsZhang, Shangji (Author) / Fromme, Petra (Thesis advisor) / Johnston, Stephen (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2021
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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

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