Theses and Dissertations
This collection includes both ASU Theses and Dissertations, submitted by graduate students, and the Barrett, Honors College theses submitted by undergraduate students.
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- Creators: Van Horn, Wade
Description
Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few TRP channels responsible for thermosensing. Despite sustained interest in the channel, the mechanisms underlying TRPM8 activation, modulation, and gating have proved challenging to study and remain poorly understood. In this thesis, I offer data collected on various expression, extraction, and purification conditions tested in E. Coli expression systems with the aim to optimize the generation of a structurally stable and functional human TRPM8 pore domain (S5 and S6) construct for application in structural biology studies. These studies, including the biophysical technique nuclear magnetic spectroscopy (NMR), among others, will be essential for elucidating the role of the TRPM8 pore domain in in regulating ligand binding, channel gating, ion selectively, and thermal sensitivity. Moreover, in the second half of this thesis, I discuss the ligation-independent megaprimer PCR of whole-plasmids (MEGAWHOP PCR) cloning technique, and how it was used to generate chimeras between TRPM8 and its nearest analog TRPM2. I review steps taken to optimize the efficiency of MEGAWHOP PCR and the implications and unique applications of this novel methodology for advancing recombinant DNA technology. I lastly present preliminary electrophysiological data on the chimeras, employed to isolate and study the functional contributions of each individual transmembrane helix (S1-S6) to TRPM8 menthol activation. These studies show the utility of the TRPM8\u2014TRPM2 chimeras for dissecting function of TRP channels. The average current traces analyzed thus far indicate that the S2 and S3 helices appear to play an important role in TRPM8 menthol modulation because the TRPM8[M2S2] and TRPM8[M2S3] chimeras significantly reduce channel conductance in the presence of menthol. The TRPM8[M2S4] chimera, oppositely, increases channel conductance, implying that the S4 helix in native TRPM8 may suppress menthol modulation. Overall, these findings show that there is promise in the techniques chosen to identify specific regions of TRPM8 crucial to menthol activation, though the methods chosen to study the TRPM8 pore independent from the whole channel may need to be reevaluated. Further experiments will be necessary to refine TRPM8 pore solubilization and purification before structural studies can proceed, and the electrophysiology traces observed for the chimeras will need to be further verified and evaluated for consistency and physiological significance.
ContributorsWaris, Maryam Siddika (Author) / Van Horn, Wade (Thesis director) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of English (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme (Rubisco) is responsible for the majority of carbon fixation and is also the least efficient enzyme on Earth. Rubisco assists 1,5-ribulose bisphosphate (RuBP) in binding CO2, however CO2 and oxygen have similar binding affinities to Rubisco, resulting in a low enzymatic efficiency. Rubisco activase (Rca) is an enzyme that removes inhibiting molecules from Rubisco’s active sites, promoting the Rubisco activity. The binding of Rubisco and Rca stimulates a high-rate of carbon fixation and lowers the overall CO2 concentration in the atmosphere. To study the interaction between the two complexes, Rubisco was extracted from baby spinach (Spinacia oleracea) and purified using anion-exchange chromatography and size-exclusion chromatography. Rca was designed to use a recombinant gene and overexpressed in Escherichia coli (E. coli). The purified proteins were verified using SDS-PAGE. The two proteins were assembled in vitro and the interaction of the protein complex was stabilized using glutaraldehyde cross-linking. The samples were then deposited on a carbon-coated electron microscopy (EM) grid, stained with uranyl formate, and observed under a transmission electron microscope (TEM). The ultimate goal is to image the specimen and reconstruct the structure of the protein complex at high resolution.
ContributorsHart, Hayden (Author) / Chiu, Po-Lin (Thesis director) / Redding, Kevin (Committee member) / Van Horn, Wade (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / Department of Military Science (Contributor)
Created2022-05