TY - JOUR
T1 - Protein oligomerization monitored by fluorescence fluctuation spectroscopy
T2 - Self-assembly of rubisco activase
AU - Chakraborty, Manas
AU - Kuriata, Agnieszka M.
AU - Nathan Henderson, J.
AU - Salvucci, Michael E.
AU - Wachter, Rebekka
AU - Levitus, Marcia
N1 - Funding Information:
This work was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through grants DE-FG02-09-ER16123 (to R.M.W and M.L.) and DE-FG02-08ER-20268 (to M.E.S.)
PY - 2012/9/5
Y1 - 2012/9/5
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84865777197&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84865777197&partnerID=8YFLogxK
U2 - 10.1016/j.bpj.2012.07.034
DO - 10.1016/j.bpj.2012.07.034
M3 - Article
C2 - 23009844
AN - SCOPUS:84865777197
SN - 0006-3495
VL - 103
SP - 949
EP - 958
JO - Biophysical journal
JF - Biophysical journal
IS - 5
ER -