Molecular orientation determination in Nanodiscs at the single-molecule level

The function of membrane-bound proteins often depends on their interactions with the lipid bilayer. Bulk absorption-based linear dichroism has been historically used to investigate molecular orientations in the phospholipid bilayer but cannot resolve the actual distribution of molecules embedded in the membrane and is often limited by a poor signal-to-noise ratio. Here, we present Single-molecule Orientation determination by fluorescence Linear dichroism Visualization In Nanodisc Grids or SOLVING, to determine the molecular orientation of molecules assembled into nanoscale lipid bi-layers. We provide a proof-of-concept by using SOLVING to quantitate the orientation distribution of two commonly used fluorescent dyes, DiO and BODIPY, in 10-nm Nanodiscs. Besides confirming the mean orientation determined by bulk absorption measurement, SOLVING provides the actual distribution of orientations and promises to provide key molecu-lar insights into the topology and interactions of multi-protein complexes such as ...