The dependency of slow CLC-ec1 dimerization kinetics on lipid solvent volume.

Abstract

Although protein association equilibrium has been vastly studied in water, questions remain about how membrane proteins assemble and operate within the oily solvent environment of the membrane. However, recent developments in the field provide the potential for understanding the molecular basis of these reactions. In particular, our lab has developed approaches for quantifying the equilibrium affinities of membrane protein oligomers in membranes, a physical model for protein association and folding. Using single-molecule photobleaching microscopy, the lab has demonstrated CLC-ec1 participates in a fully reversible, equilibrium dimerization reaction in membranes. Our studies reveal the kinetics of dimerization are slower than most biological reactions taking days to equilibrate at 37 ºC in 2:1 POPE/POPG lipid bilayers. Here, we investigate the molecular basis for the slow kinetics by measuring subunit exchange kinetics in membranes. We hypothesize the dimerization reaction involves a high-energy transition state that requires the formation of a vacuum cavity in between the two subunits inaccessible to the surrounding solvent. We expect measuring the reaction in mixed lipid bilayers that include smaller lipoidal molecules will lead to a reduced cavity volume along the reaction pathway, decreasing the transition state energy barrier, which will be observed by speeding up of the reaction kinetics. To test this, CLC-ec1-Cy3 and CLC-ec1-Cy5 subunit exchange equilibration rates are measured by bulk Förster Resonance Energy Transfer at 37 ºC in 2:1 POPE/POPG while titrating benzene, n-Octyl-β-D-Glucoside, and 2:1 PE/PG lysolipids. In parallel, coarse-grained molecular dynamics simulations are carried out on the same mixed lipid systems to calculate the solvent-inaccessible cavity volumes between subunits as they are separated from 8 to 24 Å. The results from this investigation will provide a quantitative analysis offering insight into why membrane protein complexes are often kinetically trapped in lipid bilayers.