Single‐molecule studies of conformational states and dynamics in the ABC importer OpuA

Abstract

The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule F€ orster resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the osmoregulatory type I ABC importer OpuA from Lactococcus lactis. We present data probing both intradomain distances that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC, and interdomain distances between SBDs or transmembrane domains. Using this methodology, we studied ligand-binding mechanisms, as well as ATP and glycine betaine dependences of conformational changes. Our work expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

“…Both crystallography and single-molecule Förster resonance energy transfer (smFRET) experiments on the single SBD1 and SBD2 showed that substrate binding is linked to a conformational change of the corresponding SBD from an open apo conformation to a closed liganded conformation [18][19][20][21]. The results also implied an induced-fit-type ligandbinding mechanism, where conformational dynamics are induced by ligand-SBD interactions similar to later demonstrated for other SBPs [22][23][24][25][26]. Additionally, it was shown that the opening of the SBDs and ligand release can be one rate-limiting step in the transport cycle and that the closed conformation triggers ATP-hydrolysis and transport [18].…”

Single‐Molecule FRET of Membrane Transport Proteins

“…These assays include antibodies that recognize structural epitopes, [89] thermal unfolding assays of the protein in the presence and absence of ligands, [89] binding assays via bilayer interferometry [89] or Surface Plasmon Resonance [90] or other biophysical and biochemical techniques. [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.…”

“…[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.…”

“…[142] SmFRET studies were carried out on prokaryotic (MsbA, McjD, BtuCD from E. coli and OpuA from Lactococcus lactis) and eukaryotic (PgP from Mus musculus and MRP1 from Bos Taurus) ABC transporters. [90,[97][98][99][100][101] Out of these, OpuA and BtuCD are characterized as importers whereas all others are exporters.…”

“…[90] Finally, we would like to note that homology modeling had been successfully used to design labeling positions as shown for OpuA. [100] In general, the strength of smFRET in elucidating dynamic processes benefits strongly from structure-determination methods such as x-ray crystallography or cryo-electron microscopy.…”