|Chausse, Alison -|
|Wachter, Rebekka -|
Submitted to: Archives Of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 1, 2010
Publication Date: June 1, 2010
Citation: Barta, C., Chausse, A., Wachter, R.M., Salvucci, M.E. Structural Changes Associated with the Acute Thermal Instability of Rubisco Activase. Archives of Biochemistry and Biophysics (2010) 499:17-25. Interpretive Summary: In the process of photosynthesis, plants convert light into chemical energy. The energy produced by photosynthesis is then used to synthesize sugars and other foodstuffs. Heat stress inhibits photosynthesis, reducing the overall yield of the plant. Previous research from this research 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 the major carbon dioxide-fixing enzyme in plants. In this manuscript, we present new data that explains why the Rubisco activase enzyme is so sensitive to heat. Specifically, we use physical measurements to examine changes in the structure of the enzyme that occur when the enzyme is heated and describe conditions that make the enzyme more or less sensitive to heat. The results provide new insights into the heat inactivation of photosynthesis by showing that the properties of Rubisco activase make it inherently acutely sensitive. 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: The inhibition of photosynthesis at moderately high temperatures has been linked to a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation. This decrease is thought to be a consequence of the thermal instability of Rubisco’s chaperone, ribulose-1,5-bisphosphate carboxylase/oxygenase activase (Rubisco activase). To determine the structural basis for inactivation of Rubisco activase, the effects of ADP or ATP and/or Mg2+ on the thermal stability of the protein were determined and changes in protein structure were related to the loss of activity. Both ADP and ATP protected Rubisco activase from inactivation, whereas Mg2+ promoted inactivation. Prolonged or more intense heating caused aggregation characterized by formation of insoluble protein and disruption of the secondary structure content of the protein. In contrast, heating under more mild conditions or incubation at room temperature without ATP or ADP caused the active ~660 kD Rubisco activase to aggregate, forming a soluble, but inactive complex with an apparent molecular mass of greater than 2x106 D. Formation of this stable, high molecular mass aggregate correlated with an irreversible loss of Rubisco activase activity. Circular dichroism (CD) spectroscopy and intrinsic fluorescence established that the secondary structure of Rubisco activase was perturbed but not disrupted when subunits aggregated in the high molecular mass complex. Differences in the thermal stability between wild type Rubisco activase and a structurally-altered mutant were observed both for the recombinant proteins in vitro and when the proteins were expressed in transgenic Arabidopsis plants. That photosynthesis and Rubisco activation in plants with the mutant Rubisco activase were more sensitive to inhibition by moderate heat stress indicates that the inherent structural instability of Rubisco activase is the main determinant of the temperature-sensitivity of Rubisco activation.