The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers

Abstract

Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states – monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.DOI: http://dx.doi.org/10.7554/eLife.17438.001

Occupancy distributions of membrane proteins in heterogeneous liposome populations

“…CLC-ec1 C85A/H234C including the hexahistidine tag has a molecular weight of 51,997 g/mole, MW POPE = 717.996 g/mole and MW POPG = 770.989 g/mole. Therefore, a proteoliposome sample reconstituted at 1 µg/mg in 2:1 POPE/POPG lipids corresponds to a mole fraction of: Assuming a 50% mole fraction recovery as was previously observed (14), then our observed mole fraction is χ observed = 0.7 x 10 −5 subunits/lipid.…”

“…MATLAB was used to simulate the random process of multi-species subunit capture into a heterogeneous liposome population, as described previously (14, 18). For each liposome sub-population with radius r , a matrix was created with size N liposomes (r) , and each N protein (r) sub-species was randomly assigned to the liposome population.…”

“…The methods of protein purification were carried out as described previously (14, 17). Purified C85A/H234C CLC-ec1 with a C-terminal hexahistidine tag in 150 mM NaCl, 20 mM 3-( N -morpholino)propanesulfonic acid (MOPS), pH 7.0 and 5 mM n-Decyl-β-D-Maltopyranoside, DM, (Anatrace, Maumee OH) was labeled with Cy5-malemide (Lumiprobe, Hunt Valley MD) for 10 minutes followed by quenching with 100 mM cysteine.…”

“…During this process, the large membrane is pushed through the pores, anywhere from 30 nm to 1 µm, forming a membrane bubble that breaks off into a vesicle that is approximate in diameter to the extrusion pore size (12). The probability of protein incorporation into that vesicle is a random process that depends on the density of protein in the overall membrane, and the number of vesicles in the resultant liposome population (8, 13, 14).…”

Cellular encoding of Cy dyes for single-molecule imaging

“…During incorporation, the Cy-ncAA may be exposed to reductive conditions in the cell that lead to formation of dark state adducts (Dempsey et al, 2009). Furthermore, imaging was performed in the absence of typical oxygen scavenger systems, as this has been shown to lead to improved photo-bleaching traces (Chadda et al, 2016) but could reduce the overall fluorescent yield. In this regard, future encoding experiments performed in the presence of oxygen quenchers or more stable fluorophores (Zheng et al, 2014) may be required for specific applications of the approach.…”