Structural Characterization of Rubisco Activase

Rubisco activase is a chaperone-like AAA+ ATPase essential for maintaining photosynthetic activity by releasing inhibitory sugar phosphates from Rubisco’s active site. In higher plants, including spinach, Rca exists in α and β isoforms and is believed to function primarily as a nucleotide-dependent hexamer. Despite recent structural insights, questions remain about the full-length assembly of Rca and its interaction with Rubisco under physiological conditions. Here, we present a detailed biochemical and structural analysis of spinach β-Rca, including an improved purification protocol, oligomerization behavior under defined nucleotide conditions, and structural characterization using negative stain electron microscopy. Both SoβRca and Rubisco were purified to homogeneity, with SoβRca consistently forming hexamers in the presence of ATPγS. When mixed under activating conditions, SoβRca and Rubisco produced a reproducible early-eluting peak in SEC, distinct from either protein alone. Negative stain imaging of these fractions revealed large, asymmetric assemblies containing multiple Rubisco-like particles–raising the possibility of higher-order interactions. However, no strong conclusion can be drawn due to the low abundance of SoβRca in these fractions and the uncertainty surrounding the identity and stoichiometry of the observed complexes. Higher-resolution work, and further trials are required to resolve the structure and determine the relevance of these complexes.