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Phospholipid bilayers host and support the function of membrane proteins and may be stabilized in disk-like nano structures allowing for unprecedented solution studies of assembly, structure and function of membrane proteins. Based on small-angle neutron scattering in combination with variable temperature studies of synchrotron small-angle x-ray scattering on nanodiscs1 in solution we show that the fundamental nanodisc unit, consisting of a lipid bilayer surrounded by amphiphilic scaffold proteins, posses intrinsically an elliptical shape. The temperature dependence of the curvature of the nanodiscs prepared with two different phospholipid types (DLPC and POPC) shows that it is the scaffold protein that determines the overall elliptical shape and that the nanodiscs become more circular with increasing temperature. Our data also show that the hydrophobic bilayer thickness is to a large extent dictated by the scaffolding protein and adjusted to minimize the hydrophobic mismatch between protein and phospholipid. Our conclusions result from a new comprehensive and molecular based model of the nanodisc structure and the use of this to analyze the experimental scattering profile from nanodiscs. The model paves the way for future detailed structural studies of functional membrane proteins encapsulated in nanodiscs.

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Post-staining of extracellular vesicles (EVs) with lipid-anchored fluorophores (LAFs) such as PKH67 is a widely used strategy for studying EVs but it is associated with several pitfalls. The pitfalls discussed in this commentary are related to LAF labelling of non-EV species due to (1) lipoprotein contamination in EV samples, (2) desorption of the LAF reporters from vesicles into proteins and lipoproteins in blood and serum, and (3) the capability of the amphiphilic LAF compounds to form EV-like particles. Awareness of these challenges and developing solutions to overcome these are important to ensure that we make relevant interpretations when using LAFs to track EVs.