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Rubisco is a very important protein which catalyzes the addition of CO2 to ribulose-1,5-bisphosphate (RuBP) to form two molecules of 3-phosphoglycerate in photosynthesis. Rubisco activase is the protein which functions to uninhibit Rubisco, however proof of a physical interaction has never been shown. A possible method for determining

Rubisco is a very important protein which catalyzes the addition of CO2 to ribulose-1,5-bisphosphate (RuBP) to form two molecules of 3-phosphoglycerate in photosynthesis. Rubisco activase is the protein which functions to uninhibit Rubisco, however proof of a physical interaction has never been shown. A possible method for determining the interaction of the two proteins is by Förster Resonance Energy Transfer (FRET) based analysis of the two proteins. Attempts to get a FRET signal from these two proteins have been unsuccessful. To get better results, Ficoll 70, a crowding agent, was used. Analysis suggests that Ficoll 70 does not affect the fluorescence of Alexa-fluor 488 and Alexa-fluor 647 used to label the two proteins. Further analysis also suggests that while the Alexa label on Rubisco activase does not affect the ATPase activity of the protein, the protein also does not have a high rate of ATP turnover.
ContributorsTichacek, Laura Renee (Author) / Wachter, Rebekka (Thesis director) / Levitus, Marcia (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
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Description
Proteins function as molecular machines which perform a diverse set of essential jobs. To use these proteins as tools and manipulate them with directed engineering, a deeper understanding of their function and regulation is needed. In the studies presented here, the chemical mechanism of a fluorescent protein and the assembly

Proteins function as molecular machines which perform a diverse set of essential jobs. To use these proteins as tools and manipulate them with directed engineering, a deeper understanding of their function and regulation is needed. In the studies presented here, the chemical mechanism of a fluorescent protein and the assembly behavior of a chemo-mechanical protein were explored to better understand their operation. In the first study a photoconvertible fluorescent protein (pcFP) was examined which undergoes a photochemical transformation upon irradiation with blue light resulting in an emission wavelength change from green to red. Photo-transformable proteins have been used in high resolution, subcellular biological imaging techniques, and desires to engineer them have prompted investigations into the mechanism of catalysis in pcFPs. Here, a Kinetic Isotope Effect was measured to determine the rate-limiting step of green-to-red photoconversion in the reconstructed Least Evolved Ancestor (LEA) protein. The results provide insight on the process of photoconversion and evidence for the formation of a long-lived intermediate. The second study presented here focuses on the AAA+ protein Rubisco activase (Rca), which plays a critical role in the removal of inhibitors from the carbon-dioxide fixing enzyme Rubisco. Efforts to engineer Rubisco and Rca can be guided by a deeper understanding of their structure and interactions. The structure of higher plant Rca from spinach, and its interactions with its cognate Rubisco, were investigated through negative-stain electron microscopy (EM) and cryo-EM experiments. Multiple types of higher-order oligomers of plant Rca were imaged which have never been structurally characterized, and the AAA+ core of plant Rca was shown to bind Rubisco side-on, similar to bacterial Rca’s. Higher resolution structures of these aggregates and complexes are needed to make definitive observations on protein-protein interactions. However, the results presented here provide evidence for the formation of regulatory structures and an experimental foundation for future exploration of plant Rca through cryo-EM.
ContributorsBreen, Isabella (Author) / Wachter, Rebekka (Thesis advisor) / Chiu, Po-Lin (Thesis advisor) / Levitus, Marcia (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2020