The dimerization equilibrium of a ClC Cl(-)/H(+) antiporter in lipid bilayers.

Rahul Chadda,Kacey Mersch,Janice L Robertson,Ankita Chadda,Jeff Gelles,Marley Brimberry,Larry J Friedman,Venkatramanan Krishnamani,Jason Wong,Ludmila Kolmakova-Partensky

doi:10.7554/elife.17438

Rahul Chadda, Kacey Mersch + Show 8 more

Open Access

https://doi.org/10.7554/elife.17438

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Journal: eLife	Publication Date: Aug 3, 2016
Citations: 53	License type: CC BY 4.0

Affiliation: University of Iowa, Brandeis University, University of Bath

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.

Highlights

Membrane protein folding involves the favorable association of non-polar protein interfaces amidst an excess of non-polar lipid solvent (Popot and Engelman, 1990)
The membranes are fractionated by extrusion (Figure 1C), forming small liposomes that are imaged on a total internal reflection fluorescence (TIRF) microscope (Figure 1D) for single-molecule photobleaching analysis (Figure 1—figure supplement 3D)
The liposome extrusion step captures the monomerdimer equilibrium in the prior multilamellar vesicles (MLVs) membrane state and ignores any changes in protein density or lipid composition that might arise during the extrusion process

Summary

Introduction

Membrane protein folding involves the favorable association of non-polar protein interfaces amidst an excess of non-polar lipid solvent (Popot and Engelman, 1990). The relatively small change in solvent accessible surface area upon dimerization (MacKenzie et al, 1997) limits their potential to study protein-specific van der Waals interactions and lipid-solvent-dependent effects, the two driving forces hypothesized to be major players within the membrane environment (Popot and Engelman, 1990; White and Wimley, 1999; Bowie, 2005). One example that appears well suited is the homodimeric ClC-ec Cl-/H+ antiporter native to Escherichia coli (Maduke et al, 1999; Dutzler et al, 2002) This is a 50-kDa membrane protein that dimerizes via a membrane embedded, non-polar interface lined mainly by isoleucines and leucines (Figure 1—figure supplement 1A).

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