Protein oligomerization monitored by fluorescence fluctuation spectroscopy: Self-assembly of rubisco activase

Manas Chakraborty, Agnieszka M. Kuriata, J. Nathan Henderson, Michael E. Salvucci, Rebekka Wachter, Marcia Levitus

Research output: Contribution to journalArticle

31 Scopus citations

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, to our knowledge, thermodynamic characterization of oligomeric states of cotton β-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 ∼4, 1, and 1 micromolar for the monomer-dimer, dimer-tetramer, 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.

Original languageEnglish (US)
Pages (from-to)949-958
Number of pages10
JournalBiophysical journal
Volume103
Issue number5
DOIs
StatePublished - Sep 5 2012

ASJC Scopus subject areas

  • Biophysics

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