科研之友
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摘要
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 K-d 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 beta-Rca complexed with Mg center dot ADP. We find that the monomer is the dominating species be low 0.5 micromolar. The most plausible model supports dissociation constants of similar to 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.
- 出版日期2012-9-5
