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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The FOF1 ATP synthase is responsible for generating the majority of adenosine triphosphate (ATP) in almost all organisms on Earth. A major unresolved question is the mechanism of the FO motor that converts the transmembrane flow of protons into rotation that drives ATP synthesis. Using single-molecule gold nanorod experiments, rotation of individual FOF1 were observed to measure transient dwells (TDs). TDs occur when the FO momentarily halts the ATP hydrolysis rotation by the F1-ATPase. The work presented here showed increasing TDs with decreasing pH, with calculated pKa values of 5.6 and 7.5 for wild-type (WT) Escherichia coli (E. coli) subunit-a proton input and output half-channels, respectively. This is consistent with the conclusion that the periplasmic proton half-channel is more easily protonated than the cytoplasmic half-channel. Mutation in one proton half-channel affected the pKa values of both half-channels, suggesting that protons flow through the FO motor via the Grotthuss mechanism. The data revealed that 36° stepping of the E. coli FO subunit-c ring during ATP synthesis consists of an 11° step caused by proton translocations between subunit-a and the c-ring, and a 25° step caused by the electrostatic interaction between the unprotonated c-subunit and the aR210 residue in subunit-a. The occurrence of TDs fit to the sum of three Gaussian curves, which suggested that the asymmetry between the FO and F1 motors play a role in the mechanism behind the FOF1 rotation. Replacing the inner (N-terminal) helix of E. coli c10-ring with sequences derived from c8 to c17-ring sequences showed expression and full assembly of FOF1. Decrease in anticipated c-ring size resulted in increased ATP synthesis activity, while increase in c-ring size resulted in decreased ATP synthesis activity, loss of Δψ-dependence to synthesize ATP, decreased ATP hydrolysis activity, and decreased ACMA quenching activity. Low levels of ATP synthesis by the c12 and c15-ring chimeras are consistent with the role of the asymmetry between the FO and F1 motors that affects ATP synthesis rotation. Lack of a major trend in succinate-dependent growth rates of the chimeric E. coli suggest cellular mechanisms that compensates for the c-ring modification.
ContributorsYanagisawa, Seiga (Author) / Frasch, Wayne D (Thesis advisor) / Misra, Rajeev (Committee member) / Redding, Kevin (Committee member) / Singharoy, Abhishek (Committee member) / Wideman, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023
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