Membrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities,high-resolution structures are usually obtained in artificial detergents.T ou nderstand whether and how lipids guide membrane protein function, we use single-molecule FRET to probe the dynamics of DtpA, amember of the proton-coupled oligopeptide transporter (POT) family,i nv arious lipid environments.W es how that detergents trap DtpA in ad ynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity,a llowing an opening to the extracellular side and asampling of all relevant states.Bilayer compositions tune the abundance of these states. Anovel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups.Hence,chemical diversity of membranes translates into structural diversity,w ith the current POTs tructures only sampling ap ortion of the full structural space.

Uncovering the structure and function of biomolecules is a fundamental goal in structural biology. Membrane-embedded transport proteins are ubiquitous in all kingdoms of life. Despite structural flexibility, their mechanisms are typically studied by ensemble biochemical methods or by static high-resolution structures, which complicate a detailed understanding of their dynamics. Here, we review the recent progress of single molecule Förster Resonance Energy Transfer (smFRET) in determining mechanisms and timescales of substrate transport across membranes. These studies do not only demonstrate the versatility and suitability of state-of-the-art smFRET tools for studying membrane transport proteins but they also highlight the importance of membrane mimicking environments in preserving the function of these proteins. The current achievements advance our understanding of transport mechanisms and have the potential to facilitate future progress in drug design.

Membrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities,high-resolution structures are usually obtained in artificial detergents.T ou nderstand whether and how lipids guide membrane protein function, we use single-molecule FRET to probe the dynamics of DtpA, amember of the proton-coupled oligopeptide transporter (POT) family,i nv arious lipid environments.W es how that detergents trap DtpA in ad ynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity,a llowing an opening to the extracellular side and asampling of all relevant states.Bilayer compositions tune the abundance of these states. Anovel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups.Hence,chemical diversity of membranes translates into structural diversity,w ith the current POTs tructures only sampling ap ortion of the full structural space.

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… need to cycle through different conformations in order to perform function. Using state-of-the-art smFRET tools, H. Hofmann, C. Lçw, and co-workers reveal in their Research Article on page 19121 that the dynamics and conformational variety of a peptide transporter is sensitively regulated by the lipid environment. Analyses in different lipid compositions highlight the influence of the lipid head group while artificial detergents trap the transporter in a single conformation.