expression in e. coli , purification , and liposome reconstitution of clc - ec1 ( swiss - prot i d p37019 ) were performed as described14 , as were cl and h flux assays , except that we used lipid mixtures of egg phosphatidylcholine and 1-palmitoyl , 2-oleoyl phosphatidylyglycerol , 3/1 weight ratio .
ion flux rates in these lipids are 510-fold lower than observed in the e. coli phospholipids that we customarily use .
liposomes were formed at 20 mg / ml lipid , 1 g protein / mg lipid by dialysis or by centrifugation of 0.1 ml samples through 3-ml sephadex g-50 columns . clh exchange stoichiometry was determined as the ratio of initial transport rates16 , with cl efflux and h uptake recorded via cl and h electrodes using liposomes loaded with 300 mm kcl , 40 mm citrate - naoh ph 5.0 suspended in solutions of 1 mm kcl , 300 mm k - isethionate , 2 mm citrate - naoh ph 5.2 . for crystallography , clc protein ( 915 mg / ml ) in 100 mm nacl , ~40 mm dm,10 mm tris - hcl ph 7.5 was mixed with an equal volume of 100 mm lino3 , 4145% ( w / v ) peg400 , 100 mm glycine - naoh ph 9.5 , with ~10 mm 4-cyclohexyl-1-butyl--d - maltoside added to the 2 l drop .
crystals grown by vapor diffusion in sitting drop trays for 24 weeks at 20c were frozen in liquid nitrogen , and data were collected remotely at beamline 8.2.1 , advanced light source eastern annex , waltham , mass .
molecular replacement was done in phaser using residues 30450 of a single subunit of clc - ec1 as search model , and refinement was carried out in refmac5 .
 channels and transporters of the clc family bring about transmembrane movement of inorganic anions in service of a variety of biological tasks , from the arcane - generating the kilowatt pulses by which electric fish parboil their prey - to the quotidian - acidification of endosomes , vacuoles , and lysosomes1 . 
 the homodimeric architecture of clc proteins ( fig 1 ) , initially inferred from single - molecule studies of an elasmobranch cl channel2 and later confirmed by crystal structures of bacterial cl/h+ antiporters3,4 , appears to be universal . 
 moreover , the basic machinery enabling ion movement through these proteins - the aqueous pores for anion diffusion in the channels and the ion - coupling chambers that coordinate cl and h+ antiport in the transporters - are contained wholly within each subunit of the homodimer . the near - normal function of a bacterial clc transporter strait - jacketed by covalent crosslinks across the dimer interface and the behavior of a concatameric human homologue argue that the transport cycle resides within each subunit and does not require rigid - body rearrangements between subunits5,6 
 . however , this evidence is only inferential , and since examples are known in which quaternary rearrangements of extramembrane clc domains that contribute to dimerization modulate transport activity7 , we can not declare as definitive a  parallel pathways  picture in which the homodimer consists of two single - subunit transporters operating independently . 
 a strong prediction of such a view is that it should in principle be possible to obtain a monomeric clc . in this study , we exploit the known structure of a clc cl/h+ exchanger , clc - ec1 from e. coli , to design mutants that destabilize the dimer interface while preserving both structure and transport function of individual subunits . 
 the results demonstrate that the clc subunit alone is the basic functional unit for transport and that cross - subunit interaction is not required for cl/h+ exchange in clc transporters . 
Methods Summary
Supplementary Material