Figure 3.

Chromatographic elution profile of mixed lipid nanodiscs (E. coli total lipid extract + 20% cholesterol) obtained by HPLC-SEC in two separate experiments (absorbance was monitored at 280 nm). (A) Starting lipid: MSP1D1 is too small, resulting in formation of smaller, sub-optimal nanoparticles (at elution time ~26 to 28 min) together with nanodiscs (~24 min). As more lipid is added (with increasing lipid:MSP1D1 ratio), a more homogeneous, monodisperse peak for nanodiscs is observed at the appropriate elution time (shown in dotted line). (B) Reverse scenario when the starting lipid:MSP1D1 ratio is too large, resulting larger aggregates eluting at or close to the void volume. Note that different batches of reagents (lipid, MSP1D1, detergent) were used for the two reconstitution trials, which resulted in different optimized lipid to MSP1D1 ratios. Relatively non-Gaussian peak profile is observed, most likely because E. coli total lipid extract is a heterogeneous mixture of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CA) and other lipids.