Browsing by Subject "structural biology"

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    Biophysical Characterization of Interactions Between Two Membrane Proteins: SERCA and Sarcolipin
    (2017-05) Dicke, Alysha
    Understanding the structures and interactions of proteins that interact with membranes has many implications. Membrane proteins play roles in the transfer of necessary materials and information between cells and their environments as well as within cells (e.g., between the cytosol and organelles). As such, they currently constitute more than half of all drug targets, and some peptides, such as antimicrobial peptides (AMPs), are also being investigated for their therapeutic use in treating bacterial infections for humans. However, studying the structures of membrane proteins has proven more challenging compared to soluble proteins. This is due to the necessity of including the membrane or a good membrane mimic to ensure the integrity of the membrane protein remains intact, as poor mimics or no membrane can detrimentally affect membrane protein structure and function. Some proteins, like the AMP chionodracine, are highly amenable to study with methods such as solution NMR spectroscopy (Chapter 2), but larger membrane proteins prove challenging or impossible to measure in solution due to the molecular weight limitations and frequently do not crystallize easily either. Solid-state NMR spectroscopy (ssNMR) has helped to overcome these obstacles and more methodology continues to be developed expanding the application of ssNMR. For example, Chapters 3 and 4 of this thesis describe new ssNMR methods using the sarco(endo)plasmic reticulum (SERCA) and sarcolipin (SLN), respectively. SERCA and SLN form a crucial complex in the membrane of the sarcoplasmic reticulum in skeletal muscles cells. Muscle relaxation is largely controlled by SERCA pumping calcium out of the cytosol using energy from ATP hydrolysis, and SLN inhibits SERCA as well as uncouples the ATP hydrolysis and calcium transport. SLN’s effect on SERCA leads to more heat production, which may be important to thermogenesis in mammals as well as an additional mechanism to control energy expenditure. Chapter 5 primarily uses ssNMR with the goal to better understand the mechanism by which SLN inhibits and uncouples SERCA. Overall, gaining a better understanding of how SERCA is regulated will aid in developing therapies for diseases resulting from improper calcium cycling.
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    Structure-Function Analysis of Recombinant Viral Proteins and Their Applications in Disease Diagnosis
    (2022-06) Di, Da
    Since the start of the COVID-19 pandemic in 2019, there have been hundreds of millions of reported cases of SARS-CoV-2 infection and millions of deaths associated with this virus-causing COVID-19 disease worldwide. Huge efforts have been devoted toward the development of antivirals, vaccines, and diagnostics against this virus that is constantly evolving genetically. Part of my doctoral thesis work involves the development of a highly sensitive SARS-CoV-2 nucleocapsid (N) protein-based enzyme-linked immunosorbent assay (ELISA), which could detect not only SARS-CoV-2 but also other SARS-CoV-like viruses. To do this, I devised a new method to express and purify the recombinant SARS-CoV-2 N protein and showed that it could retain its intended structure and biochemical function (as described in Chapter 2) and could be used toward the development of a highly sensitive ELISA to serologically screen human’s and companion animal’sbloods for evidence of SARS-CoV-2 exposures (as described in Chapter 3). A second major part of my doctoral thesis is focused on an attempt to express and purify recombinant proteins of another deadly human virus, called Lassa virus (LASV) that is responsible for up to 300,000 cases of infection and 5,000 deaths annually in several countries in West Africa. Toward this end, I successfully expressed and purified a couple of recombinant LASV proteins (L polymerase and nucleoprotein NP) and obtained preliminary electron microscopic three-dimensional (3D) structural reconstruction of these two essential viral proteins as an active viral polymerase holoenzyme (as described in Chapter 4). As part of this work, I de novo constructed a new PiggyBac transposase-transposon system and showed that it could be employed as a versatile and easy-to-use tool to generate mammalian stable-cell lines to express recombinant proteins (with almost unlimited cargo sizes) in a variety of human and animal cells, such as human kidney’s epithelial (293T), African green-monkey epithelial (Vero), baby hamster’s kidney epithelial (BHK21), and human cervical cancer (HeLa)v cells. Using this technical innovation, I generated a new HeLa cell line that could stably express the LASV Z (matrix) protein and showed that this new cell line could be induced by a chemical compound known as IPTG (Isopropyl β-D-1-thiogalactopyranoside) to express a consistently high level of the recombinant LASV Z protein and showed that its expression could effectively inhibit the cellular innate ability (i.e., innate immunity) to fight viral infection, and could therefore confer a replicative advantage for the virus in the infected cells (as described in Chapter 5). Taken all together, my doctoral dissertation work involves the development of several novel strategies to express and purify different viral proteins recombinantly for use successfully in not only diagnostic assay development for COVID-19 but also to investigate the structure and function of multiple SARS-CoV-2 and LASV proteins, all of which have provided novel insights into their essential biological functions and can serve as new targets for the development of novel preventative and/or therapeutic strategies against these two deadly human viral pathogens (as described in Chapters 2 and 5).