Measuring Reversible CLC-ec1 Dimerization In Membranes by Single Molecule Photobleaching

Abstract

ClC-ec1 is a bacterial Cl-/H+ antiporter that exists as a homodimer with each subunit containing a structurally independent pathway for ion transport. Mutations at the dimerization interface I201W/I422W (WW) destabilize the dimer in detergent while preserving function. In this study, we have applied single fluorescent molecule imaging and photobleaching to study the dimerization of an intermediately stable mutant; I422W (IW) in phospholipid membranes. WT CLC-ec1 and its mutants were purified from E. coli into detergent and labelled with Cy5-maleimide at a unique surface exposed cysteine residue. The dye/protein ratio for all samples were ∼0.66. Cy5 labelled proteins were reconstituted in Alexa Fluor 488 SDP-ester labelled 2:1 POPE/POPG liposomes at 0.03 % fluorophore/lipid. Dialyzed liposomes were then freeze-thawed 7 times to form large multilamellar membranes, and then incubated at 25°C to facilitate protein mixing. Prior to imaging on a multi-color TIR-FM these membranes were extruded through a 400 nm filter. Protein reconstitution in liposomes follows a Poisson distribution resulting in liposomes containing either 0, 1, 2 or more protein molecules. We counted the overall occupancy of liposomes using two-color colocalization and counting the number of labelled protein particles in each liposome using step-wise photobleaching of Cy5. We determined the photo-bleaching distribution for dimeric WT, monomeric WW and intermediately stable IW. Upon increasing protein/lipid mole fraction from 1.5 x 10-9 to 7.5 x 10-4, we find that IW evolves from a monomer to dimer distribution. By carrying out a least-squares fit to the monomer and expected dimer distribution, we calculate Fdimer, I422W(X) that shows a density dependent increase that fits to a dimerization isotherm with KD=2x10-6 protein/lipid. The dimers dissociate into monomers upon diluting proteoliposomes with empty membranes suggesting that we are observing the reversible dimerization reaction in membranes.