Matching Items (5)

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The Interaction of Rubisco and Rubisco Activase: A FRET-based Study

Description

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

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.

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2015-05

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Subunit exchange in spinach short-form Rubisco activase

Description

The primary carbon fixing enzyme Rubisco maintains its activity through release of trapped inhibitors by Rubisco activase (Rca). Very little is known about the interaction, but binding has been proposed to be weak and transient. Extensive effort was made to

The primary carbon fixing enzyme Rubisco maintains its activity through release of trapped inhibitors by Rubisco activase (Rca). Very little is known about the interaction, but binding has been proposed to be weak and transient. Extensive effort was made to develop Förster resonance energy transfer (FRET) based assays to understand the physical interaction between Rubisco and Rca, as well as understand subunit exchange in Rca.

Preparations of labeled Rubisco and Rca were utilized in a FRET-based binding assay. Although initial data looked promising, this approach was not fruitful, as no true FRET signal was observed. One possibility is that under the conditions tested, Rca is not able to undergo the structural reorganizations necessary to achieve binding-competent conformations. Rca may also be asymmetric, leading to less stable binding of an already weak interaction.

To better understand the structural adjustments of Rca, subunit exchange between different oligomeric species was examined. It was discovered that subunit exchange is nucleotide dependent, with ADP giving the fastest exchange, ATP giving slower exchange and ATPS inhibiting exchange. Manganese, like ADP, destabilizes subunit-subunit interactions for rapid and facile exchange between oligomers. Three different types of assemblies were deduced from the rates of subunit exchange: rigid types with extremely slow dissociation of individual protomers, tight assemblies with the physiological substrate ATP, and loose assemblies that provide fast exchange due to high ADP.

Information gained about Rca subunit exchange can be used to reexamine the physical interaction between Rubisco and Rca using the FRET-binding assay. These binding assays will provide insight into Rca states able to interact with Rubisco, as well as define conditions to generate bound states for structural analysis. In combination with assembly assays, subunit exchange assays and reactivation studies will provide critical information about the structure/function relationship of Rca in the presence of different nucleotides. Together, these FRET-based assays will help to characterize the Rca regulation mechanism and provide valuable insight into the Rubisco reactivation mechanism.

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Date Created
2017

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Understanding the Self-assembly Pathway of Higher Plant Rubisco Activase

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.

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.

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Date Created
2018

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Understanding Fluorescent Protein Photoconversion and Assembly of Spinach Rubisco Activase

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,

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.

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Date Created
2020

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Expression, Purification, and Electron Microscopic Analysis of the Rubisco-Rubisco Activase Complex

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

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.

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2022-05