Single-molecule FRET monitors CLC transporter conformation and subunit independence

Abstract

CLC transporters catalyze the exchange of chloride ions for protons across cellular membranes. As secondary active transporters, CLCs must alternately allow ion permeation to the extracellular and intracellular sides of the membrane, adopting outward-facing and inward-facing conformational states. Here, we use single-molecule Forster resonance energy transfer (smFRET) to monitor the conformational state of CLC-ec1, an E. coli homolog for which high-resolution structures of occluded and outward-facing states are known. Since each subunit within the CLC homodimer contains its own transport pathways for chloride and protons, we developed a labeling strategy to follow conformational change within a subunit, without crosstalk from the second subunit of the dimer. Using this strategy, we evaluated smFRET efficiencies for labels positioned on the extracellular side of the protein, to monitor the status of the outer permeation pathway. When [H+] is increased to enrich the outward-facing state, the smFRET efficiencies for this pair decrease. In a triple-mutant CLC-ec1 that mimics the protonated state of the protein and is known to favor the outward-facing conformation, the lower smFRET efficiency is observed at both low and high [H+]. These results confirm that the smFRET assay is following the transition to the outward-facing state and demonstrate the feasibility of using smFRET to monitor the relatively small (~1 Angstrom) motions involved in CLC transporter conformational change. Using the smFRET assay, we show that the conformation of the partner subunit does not influence the conformation of the subunit being monitored by smFRET, thus providing evidence for the independence of the two subunits in the transport process.

Single‐Molecule FRET of Membrane Transport Proteins

“…[90][91][92][93][94][95][96] The impact of mutations on function can be monitored by ATPase assays [79,90,[97][98][99][100][101] or transport assays. [78,90,[105][106][107][108]93,94,96,97,100,[102][103][104] In transport assays, the protein is either overexpressed in a cell or incorporated into liposomes and the accumulation of fluorescent [78,97,105] or radiolabeled substrate [90,93,94,96,100,102,103,[106][107][108] in the compartment (cell or vesicle) is measured. Good examples for such careful controls are LmrP transporter variants whose activities were checked with the fluorescent ligand Hoechst, [105] or variants of the Glt Ph transporter in liposomes where the uptake of the radioactive substrate [ 3 H]-Asp was used to confirm activity.…”

“…[121] Thus numerous smFRET studies have been performed using detergents [121][122][123] and provided important insights. [78,79,108,[91][92][93][94]99,102,104,105] Yet, studies of transporters in membrane environments, which better mimic native conditions, showed that lipids play important roles in maintaining their structural and functional integrity. [120,[124][125][126][127][128] For certain transporters such as Glt Ph , dynamics in micelles do not differ significantly from those found in a lipid bilayer.…”

“…Representatives for each family and their respective substrates are illustrated in Figure 4. Some transporters such as LeuT [93][94][95][96] and Glt Ph [106][107][108]114] were subject of several studies while others such as AtSWEET13, [78] bcMAlT [102] and ClC-ec1 [104] were studied less frequently. In the following, we summarize the results obtained for the different transporter families and highlight individual examples.…”

“…SmFRET studies of a CLC transporter (CLC-ec1 from E. coli [191] ) became available just recently. [104] CLC transporters are secondary active anion/proton exchangers in the majority of intracellular membranes where they regulate ion concentrations. They are functional dimers where each unit takes up Cl À ions and simultaneously exports H + to the environment [192] in a stoichiometric 2 : 1 ratio.…”

“…Transporters with one inactive subunit and one FRET labeled subunit were immobilized while dimers with two FRET labeled subunits did not bind. [104] By using an inactive variant that mimicked the protonated monomer (QQQ-variant) known to adopt the outward-open state, [115] transitions from the outward-open to occluded state of a transporter unit could be observed. Remarkably, spatial changes associated with this transition were only in the range of 1-2.5 Å and were independent of whether the second monomer was active or inactive.…”