No description available.

Rubisco activase operates as the chaperone responsible for maintaining the catalytic competency of Ribulose 1,5-bisphophate carboxylase oxygenase (Rubisco) in plants. Rubisco is notoriously inefficient, rapidly self-inactivating under physiological conditions. Rubisco activase uses the power released from the hydrolysis of ATP to power a conformational change in Rubisco, reactivating it. Rubisco activase has been previously shown to form a large range of species in solution; however, little has been done to relate the size of oligomeric species and physiological activity. In this thesis data is presented from a range of biophysical techniques including analytical ultracentrifugation, static light scattering, and small angle X-ray scattering combined with activity assays to show a strong relationship between oligomeric state and activity. The results suggest that small oligomers comprising 2-4 subunits are sufficient to attain full specific activity, a highly unusual property for enzymes from the AAA+ family. Studies utilising a number of Rubisco activase variants enabled the determination of how Rubisco and Rubisco activase may interact within a plant cell. A detailed characterisation of the α-, β-, and a mixture of isoforms further broadened our knowledge on the oligomerisation of Rubisco activase. Of particular importance was the discovery of a thermally stable hexameric Rubisco activase variant. It is hoped that these findings may contribute to development of more heat tolerant Rubisco activase and lead research into more drought resilient crop plants.

abstract: 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. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2017

abstract: 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. / Dissertation/Thesis / Masters Thesis Biochemistry 2020

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiii, 149 p.; also includes graphics. Includes abstract and vita. Advisor: F. Robert Tabita, Dept. of Microbiology. Includes bibliographical references (p. 144-149).

In this diploma work influence of needle age at Rubisco activity and content in Norway spruce (Picea abies) was studied. The plants were cultivated in conditions with ambient (A) CO2 concentration (350 µmol CO2/mol) and elevated (E) CO2 concentration (700 µmol CO2/mol). Sampling was done two times during the growing season (in the middle of June and in the end of September) were taken. Initial and total Rubisco activities were measured spectrophotometrically. Rubisco content was determined by SDS–PAGE method. Rubisco activity in 18-months-old needles was in E higher than in A. Rubisco contents in current-year needles and one-year-old needles were in A higher than in E in September. These differences were statistically significant that demonstrates the down-regulation of Rubisco content in conditions of elevated CO2 concentration. It seems the course of activities and content depending on age of the needles are antiparallel, that means that decrease of content is followed by increase of activity.

La nécessité de coordonner l’expression des gènes provenant de génomes différents chez les plantes a conduit à l’émergence de mécanismes imposant un contrôle nucléaire sur l’expression génétique de l’organelle. Des signaux antérogrades, exercés par des protéines reconnaissant des séquences spécifiques, existent en parallèle avec un contrôle des synthèses chloroplastiques dépendant de l’assemblage (CES). Ensemble, ils coordonnent la formation stoichiométrique des complexes photosynthétiques.La Ribulose bisphosphate carboxylase/oxygénase (Rubisco) est une enzyme localisée dans le chloroplaste qui contient deux sous-unités. La grande sous-unité (LSU) et la petite sous-unité (SSU) sont codées par les génomes chloroplastique et nucléaire respectivement. Elles s’assemblent dans le stroma du chloroplaste pour former une holoenzyme hexadécamérique (LSU8SSU8). Pendant mon travail au laboratoire, j’ai tenté de décrire les étapes régulatrices majeures de la synthèse de la Rubisco chez Chlamydomonas reinhardtii en me focalisant sur la régulation post-transcriptionelle de la LSU.J’ai montré que la protéine PPR – MRL1 est un facteur limitant pour l’accumulation de l’ARN messager de rbcL. Bien qu’il ait été décrit précédemment comme un facteur stabilisateur du transcrit susnommé, MRL1 s’est révélé avoir un rôle dans la traduction.J’ai par ailleurs démontré que chez Chlamydomonas, l’expression de la Rubisco est contrôlée par la présence de la SSU. En son absence, la traduction de rbcL est inhibée par son propre produit – la grande sous-unité non assemblée. J’ai pu montrer qu’un intermédiaire d’assemblage, constitué de LSU en complexe avec sa chaperonne RAF1, est nécessaire pour cette régulation, ce qui prouve que ce processus dépend de l’état d’oligomérisation de la LSU. Parallèlement, j’ai caractérisé le devenir de la LSU non assemblée quand la régulation CES est perturbée, et grâce à cela ait contribué à améliorer la connaissance de son processus de repliement et d’assemblage. / The necessity to coordinate the expression of genes originating from different genomes within the plant cell resulted in the appearance of mechanisms imposing nuclear control over organelle gene expression. Anterograde signaling through sequence-specific trans-acting proteins (OTAFs) coexists in the chloroplast with an assembly dependent control of chloroplast synthesis (CES process) that coordinates the stoichiometric formation of photosynthetic complexes.Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is a chloroplast-located carbon fixing enzyme constituted of two subunits. Large subunit (LSU) and small subunit (SSU) are encoded in the chloroplast and nuclear genomes respectively. In the stroma they assemble to form a hexadecameric holoenzyme (LSU8SSU8). In this study I tried to highlight major regulatory points of its synthesis in Chlamydomonas reinhardtii focusing on the posttranscriptional regulation of LSU.I showed that the MRL1 PPR protein is a limiting factor for rbcL mRNA accumulation. Whereas it has been previously designated as a stabilization factor for the abovementioned transcript, MRL1 appeared also to have a function in rbcL translation.Most notably, I have demonstrated that in Chlamydomonas reinhardtii Rubisco expression is controlled by the small subunit (SSU) presence. In its absence rbcL undergoes an inhibition of translation through its own product – the unassembled Rubisco large subunit. This process depends on LSU-oligomerization state as I was able to show that the presence of a high order LSU assembly intermediate bound to the RAF1 assembly chaperone is essential for the regulation to occur. In parallel I shed light on the fate of unassembled LSU in a deregulated CES context, thereby improving our understanding of the process of its folding and assembly.

No description available.

Diurnal changes of initial and total activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) under conditions of ambient (350 µmol mol-1) and elevated (E = 700 µmol mol 1) concentration CO2 were measured in needles of Norway spruce. Needles of Norway spruce were taken on 22th July in two-hours from 3:30 a. m. till 10:30 p. m. The Rubisco activity was determined by spectrophotometry, the Rubisco content was detected by SDS-PAGE. The Rubisco activity was in most samples statistically significantly lower in needles cutlivated under ambient carbon dioxide then in needles cultivated under elevated carbon dioxide. Diagrams of the Rubisco activity shows absence of night inhibitor CA1P. The Rubisco activity was directly proportional to the intensity of photosynthetically active radiation in time from 7:00 a. m. till 7:30 p. m. The Rubisco content was statistically significantly higher in needles of Norway spruce cultivated under ambient carbon dioxide than in needles cultivated under elevated CO2, which shows acclimation of the Rubisco content during long-term effect of elevated CO2 concentration.

No description available.