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- All Subjects: Rubisco
- Creators: Wachter, Rebekka M.
- Creators: Redding, Kevin E
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
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.
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.
ContributorsForbrook, Dayna S (Author) / Wachter, Rebekka M. (Thesis advisor) / Allen, James (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2017