Alum Speaker

Using FRET to Examine the Physical Interaction Between Rubisco and Rca

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Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme on Earth and represents the primary entry point of CO2 into the biosphere. Rubisco’s extremely slow carboxylation rate of ribulose-1,5-bisphosphate and its propensity toward inhibition make it a notoriously inefficient enzyme, however, Rubisco activase (Rca) is essential in maintaining Rubsico activity by catalyzing the rapid release of trapped inhibitors to reactivate Rubisco for CO2 fixation. Rca uses ATP hydrolysis as the driving force behind a conformational motion that restores activity to inhibited Rubisco active sites. Adding to the complexity of this system is the high size polydispersity of Rca in solution, as well as the species dependent variations in reactivation: in some cases, Rca reactivates Rubisco from a different species more effectively than Rubisco from its own species. A bound complex of Rubisco-Rca has not yet been isolated, but the interaction has been proposed to be weak and transient. Information about the physical interaction of Rca and Rubsico will assist in deconstructing the Rubisco reactivation mechanism and may lead to a more efficient enzyme and therefore, increase the efficiency of carbon fixation in plants. Based on our fluorescence correlation spectroscopy (FCS) assembly studies of Rca showing a primarily hexameric species in solution at 30μM, a fluorescence based,steady state Rubisco-Rca binding assay is being developed to study their interaction. Chlamydomonas reinhardtii Rubisco mutants have been designed and isolated to target site specific labeling of introduced cysteine residues. Both Rca and Rubisco have been successfully labeled with specific fluorophores and utilized in a Förster resonance energy transfer (FRET) based binding assay to monitor their proximity and calculate equilibrium binding constants. As of yet, an indisputable FRET response has not been seen, but many parameters, such as using different combinations of species (i.e. cotton Rubisco+cotton Rca or Chlamy Rubisco+spinach Rca) and varying nucleotide concentrations, are still being altered. These FRET assays, along with FCS, reactivation and kinetic assays, and crystallographic studies, will provide critical information about how Rca interacts and reactivates Rubisco. Through the work in the Wachter lab, we hope to gain a full understanding of the mechanism of Rubisco remodeling from a structural and mechanistic point of view.