Temperature Dependence of the CLC-ec1 Dimerization Kinetics in Lipid Bilayers

The mechanisms of membrane proteins assembly, folding, and operation in the oily solvent environment remain an important biological question. Our previous experiments show that the kinetics of the dissociation of membrane protein, CLC-ec1, were extremely slow at room temperature, as the samples took weeks to converge with the equilibrium end-points. This slow dissociation is surprising because it is slower than most biological reactions. We study the questions of why membrane proteins exhibit much slower dissociation kinetics even when the complexes are overall less stable than other soluble proteins and the majority of the binding interface is hydrophobic and the oily environment of the membrane is expected to facilitate dissociation. To understand this further, I am measuring CLC-ec1 subunit-exchange kinetics by examining CLC-ec1 FRET pairs in fused CLC-ec1-Cy3 and CLC-ec1-Cy5 proteo-liposomes. Monitoring the kinetics of FRET signals arising from the formation of new Cy3/Cy5 homodimers under equilibrium conditions allows us to determine the dissociation rate constant of the reaction. With this approach, I will examine how the temperature affects the dissociation kinetics of CLC-ec1 and estimate the activation energy for dissociation by carrying out an Arrhenius analysis of the kinetic data to understand how the activation energy relates to protein assembly reactions and examine which parameters of the systems affect the dissociation kinetics.