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

Quantification of protein dimerization energies in lipid bilayers is hard to achieve, largely due to methodological challenges. Chadda et al. present an expansion of the single-molecule subunit-capture approach that incorporates empirical benchmarks for monomers and dimers, simplifying the process.

Increasing antibiotic resistance, and a growing recognition of the importance of the human microbiome, demand that new therapeutic targets be identified. Characterization of metabolic pathways that are unique to enteric pathogens represents a promising approach. Iron is often the rate-limiting factor for growth, and Vibrio cholerae, the causative agent of cholera, has been shown to contain numerous genes that function in the acquisition of iron from the environment. Included in this arsenal of genes are operons dedicated to obtaining iron from heme and heme-containing proteins. Given the persistence of cholera, an important outstanding question is whether V. cholerae is capable of anaerobic heme degradation as was recently reported for enterohemorrhagic Escherichia coli O157:H7. In this work, we demonstrate that HutW from V. cholerae is a radical S-adenosylmethionine methyl transferase involved in the anaerobic opening of the porphyrin ring of heme. However, in contrast to the enzyme ChuW, found in enterohemorrhagic E. coli O157:H7, there are notable differences in the mechanism and products of the HutW reaction. Of particular interest are data that demonstrate HutW will catalyze ring opening as well as tetrapyrrole reduction and can utilize reduced nicotinamide adenine dinucleotide phosphate as an electron source. The biochemical and biophysical properties of HutW are presented, and the evolutionary implications are discussed.

9 10We previously reported the equilibrium dimerization reaction of the CLC-ec1 Cl -/H + transporter in 2:1 11POPE/POPG membranes (Chadda et al. 2016). This was determined by measuring the probability 21photobleaching data using this distribution shows that the protein is monomeric in the membrane and can 22serve as an experimental control. Dimer controls were constructed by glutaraldehyde cross-linking of 23C85A/H234C 'WT' or introducing R230C/L249C, which forms a spontaneous disulfide bond.

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