|Chakraborty, Manas -|
|Kuriata, Agnieszka -|
|Henderson, J -|
|Wachter, Rebekka -|
|Levitus, Marcia -|
Submitted to: Biophysical Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 23, 2012
Publication Date: September 1, 2012
Citation: Chakraborty, M., Kuriata, A.M., Henderson, J.N., Salvucci, M.E., Wachter, R.M., Levitus, M. 2012. Protein oligomerization monitored by fluorescence fluctuation spectroscopy: Self-assembly of Rubisco activase. Biophysical Journal. 103:949-958. Interpretive Summary: In the process of photosynthesis, plants convert light into chemical energy. The energy produced by photosynthesis is then used to synthesize the carbon compounds that are harvested for food, fuel, fiber or other natural products. Heat stress inhibits photosynthesis, reducing the overall yield of the plant. Previous research from our group identified an enzyme called Rubisco activase as the component of photosynthesis that is most sensitive to inhibition by heat. Rubisco activase is a regulatory enzyme that controls the activity of Rubisco, the major carbon dioxide-fixing enzyme in plants. In this manuscript, we present important structural information about Rubisco activase, a protein that is composed of multiple subunits. Specifically, we use biophysical methods to characterize how the subunits of Rubisco activase assemble into an active enzyme. Knowledge of the structure of Rubisco activase is essential for understanding how Rubisco activase functions to control the activity of Rubisco. In addition, information about the assembly of its subunits will help elucidate the mechanism for heat inactivation of photosynthesis since subunit interactions are important for the stability of Rubisco activase and, if strengthened, would increase the temperature tolerance of the enzyme. This information eventually can be used to make changes that improve the activity and stability of Rubisco activase in order to improve the efficiency of photosynthesis under heat stress.
Technical Abstract: A methodology is presented to characterize complex protein assembly pathways by fluorescence correlation spectroscopy. We have derived the total autocorrelation function describing the behavior of mixtures of labeled and unlabeled protein under equilibrium conditions. Our modeling approach allows us to quantitatively consider the relevance of any proposed intermediate form, and Kd values can be estimated even when several oligomeric species coexist. We have tested this method on the AAA+ ATPase Rubisco activase (Rca). Rca self-association regulates the CO2 fixing activity of the enzyme Rubisco, directly affecting biomass accumulation in higher plants. However, the elucidation of its assembly pathway has remained challenging, precluding a detailed mechanistic investigation. Here, we present the first thermodynamic characterization of oligomeric states of cotton beta-Rca complexed with Mg•ADP. We find that the monomer is the dominating species below 0.5 micromolar. The most plausible model supports dissociation constants of about 4, 1 and 1 micromolar for the monomer-dimer, dimertetramer and tetramer-hexamer equilibria, in line with the coexistence of four different oligomeric forms under typical assay conditions. Large aggregates become dominant above 40 micromolar, with continued assembly at even higher concentrations. We propose that under some conditions, ADP-bound Rca self-associates by forming spiral arrangements that grow along the helical axis. Other models such as the stacking of closed hexameric rings are also discussed.