ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
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- All Subjects: Biochemistry
Description
The green fluorescent protein (GFP)-like fluorescent proteins play an important role for the color of reef-building corals. Different colors of extant coral fluorescent proteins (FPs) have evolved from a green ancestral protein. Interestingly, green-to-red photoconversion FPs (Kaede-type Red FPs) are only found in clade D from Scleractinia (Faviina suborder). Therefore, I focus on the evolution of Kaede-type FPs from Faviina suborder ancestral FP. A total of 13 mutations have been identified previously that recapitulate the evolution of Kaede-type red FPs from the ancestral green FP. To examine the effect of each mutation, total ten reconstructed FPs were analyzed and six x-ray crystal structures were solved. These substitutions created a more hydrophilic environment around the carbonyl group of Phe61. Also, they increased the flexibility of the c-terminal chain, which keeps it from interacting with the entrance of the putative solvent channel. The photoconversion reaction shows a twophase kinetics. After the rapid initial phase, the overall reaction followed the firstorder kinetics. Based on the crystal structure analysis, I propose a new mechanism for Kaede-type FP photoconversion process, which a proton transfers via Gln38 to the carbonyl group of Phe61.
ContributorsKim, Hanseong (Author) / Wachter, Rebekka M. (Thesis advisor) / Fromme, Petra (Committee member) / Redding, Kevin E (Committee member) / Arizona State University (Publisher)
Created2012
Description
Rubisco activase (Rca) from higher plants is a stromal ATPase essential for reactivating Rubiscos rendered catalytically inactive by endogenous inhibitors. Rca’s functional state is thought to consist of ring-like hexameric assemblies, similar to other members of the AAA+ protein superfamily. However, unlike other members, it does not form obligate hexamers and is quite polydisperse in solution, making elucidation of its self-association pathway challenging. This polydispersity also makes interpretation of traditional biochemical approaches difficult, prompting use of a fluorescence-based technique (Fluorescence Correlation Spectroscopy) to investigate the relationship between quaternary structure and function. Like cotton β Rca, tobacco β Rca appears to assemble in a step-wise and nucleotide-dependent manner. Incubation in varying nucleotides appears to alter the equilibrium between varying oligomers, either promoting or minimizing the formation of larger oligomers. High concentrations of ADP seem to favor continuous assembly towards larger oligomers, while assembly in the presence of ATP-yS (an ATP analog) appears to halt continuous assembly in favor of hexameric species. In contrast, assembly in the “Active ATP Turnover” condition (a mixture of ATP and ADP) appears to favor an almost equal distribution of tetramer and hexamer, which when compared with ATPase activity, shows great alignment with maximum activity in the low µM range. Despite this alignment, the decrease in ATPase activity does not follow any particular oligomer, but rather decreases with increasing aggregation, suggesting that assembly dynamics may regulate ATPase activity, rather than the formation/disappearance of one specific oligomer. Work presented here also indicates that all oligomers larger than hexamers are catalytically inactive, thus providing support for the idea that they may serve as a storage mechanism to minimize wasteful hydrolysis. These findings are also supported by assembly work carried out on an Assembly Mutant (R294V), known for favoring formation of closed-ring hexamers. Similar assembly studies were carried out on spinach Rca, however, due to its aggregation propensity, FCS results were more difficult to interpret. Based on these findings, one could argue that assembly dynamics are essential for Rca function, both in ATPase and in regulation of Rubisco carboxylation activity, thus providing a rational for Rca’s high degree of polydispersity.
ContributorsSerban, Andrew J (Author) / Wachter, Rebekka M. (Thesis advisor) / Levitus, Marcia (Thesis advisor) / Redding, Kevin E (Committee member) / Van Horn, Wade D (Committee member) / Arizona State University (Publisher)
Created2018
Description
Biological systems have long been known to utilize two processes for energy conservation: substrate-level phosphorylation and electron transport phosphorylation. Recently, a new bioenergetic process was discovered that increases ATP yields: flavin-based electron bifurcation (FBEB). This process couples an energetically favorable reaction with an energetically unfavorable one to conserve energy in the organism. Currently, the mechanisms of enzymes that perform FBEB are unknown. In this work, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn), a FBEB enzyme, is used as a model system to study this phenomenon. Nfn is a heterodimeric enzyme that reversibly couples the exergonic reduction of NADP+ by reduced ferredoxin with the endergonic reduction of NADP+ by NADH. Protein film electrochemistry (PFE) has been utilized to characterize the catalytic properties of three ferredoxins, possible substrates for Nfn enzymes, from organisms that perform FBEB: Pyrococcus furiosus (PfFd), Thermotoga maritima (TmFd), and Caldicellulosiruptor bescii (CbFd). Additionally, PFE is utilized to characterize three Nfn enzymes from two different archaea in the family Thermococcaceae: two from P. furiosus (PfNfnI and PfXfn), and one from Thermococcus sibiricus (TsNfnABC). Key results are as follows. The reduction potentials of the [4Fe4S]2+/1+ couple for all three ferredoxins are pH independent and modestly temperature dependent, and the Marcus reorganization energies of PfFd and TmFd are relatively small, suggesting optimized electron transfer. Electrocatalytic experiments show that PfNfnI is tuned for NADP+ reduction by both fast rates and a low binding constant for NADP+. A PfNfnI variant engineered to have only cysteines as coordinating ligands for its [FeS] clusters has significantly altered rates of electrocatalysis, substrate binding, and FBEB activity. This suggests that the heteroligands in the primary coordination sphere of the [FeS] clusters play a role in controlling catalysis by Nfn. Furthermore, a variant of PfNfnI lacking its small subunit, designed to probe allosteric effects at the bifurcating site, has altered substrate binding at the NADP(H) binding site, i.e. the bifurcation site. PfXfn and TsNfnABC, representing different types of Nfn enzymes, have different electrocatalytic properties than PfNfnI, including slower rates of FBEB. This suggests that Nfn enzymes vary significantly over phylogenetically similar organisms despite relatively high primary sequence homology.
ContributorsJennings, David Peter (Author) / Jones, Anne K (Thesis advisor) / Redding, Kevin E (Committee member) / Torres, César I (Committee member) / Arizona State University (Publisher)
Created2018
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