Skip to main content
ARS Home » Pacific West Area » Maricopa, Arizona » U.S. Arid Land Agricultural Research Center » Plant Physiology and Genetics Research » Research » Publications at this Location » Publication #286680
Title: Biophysical characterization of higher plant Rubisco activase

Author
item HENDERSON, NATHAN - Arizona State University
item HAZRA, SURATNA - Arizona State University
item DUNKLE, ALISON - Arizona State University
item Salvucci, Michael
item WACHTER, REBEKKA - Arizona State University

Submitted to: Biochimica et Biophysica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2012
Publication Date: 1/1/2013
Citation: Henderson, N.J., Hazra, S., Dunkle, A.M., Salvucci, M.E., Wachter, R.M. 2013. Biophysical characterization of higher plant Rubisco activase. Biochimica et Biophysica Acta. 1834(1):87-97.

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 various biophysical methods to characterize subunit interactions of Rubisco activase that affect its enzymatic activity. Knowledge of these interactions is essential for understanding how Rubisco activase functions to control the activity of Rubisco. In addition, we examine the physical stability of Rubisco activase at high temperature and characterize how various low molecular weight compounds affect thermal stability. 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: Rubisco activase (Rca) is a chaperone-like protein of the AAA+ family, which uses mechanochemical energy derived from ATP hydrolysis to release tightly bound inhibitors from the active site of the primary carbon fixing enzyme ribulose 1,5-bisphosphate oxygenase/carboxylase (Rubisco). Mechanistic and structural investigations of Rca have been hampered by its exceptional thermolability, high degree of size polydispersity and propensity towards subunit aggregation. In this work, we have characterized the thermal stability and self-association behavior of recombinant Rca preparations, and have developed ligand screening methods. Thermal denaturation profiles generated by circular dichroism indicate that creosote and tobacco short-form Rcas are the most stable proteins examined, with an estimated mid-point temperature of 45 - 47 deg C for protein denaturation. We demonstrate that ADP provides a higher degree of stabilization than ATP, that magnesium ions have a small stabilizing effect on ATP-bound, but a significant destabilizing effect on ADP-bound Rca, and that phosphate provides weak stabilization of the ADP-bound form of the protein. A dimeric species was identified by size-exclusion chromatography, suggesting that the two-subunit module may comprise the basic building block for larger assemblies. Evidence is provided that chromatographic procedures reflect nonequilibrium multimeric states. Dynamic light scattering experiments performed on nucleotide-bearing Rca support the notion that several larger, highly polydisperse assembly states coexist over a broad concentration range. No significant changes in aggregation are observed upon replacement of ADP with ATP. However, in the absence of nucleotides, the major protein population appears to consist of a monodisperse oligomer smaller than a hexamer.