Water-Soluble Integral Membrane Protein Nanoparticles

Helical integral membrane proteins are assumed to require a lipid bilayer or model membrane (e.g. detergent micelles) to prevent aggregation and precipitation in aqueous solution. Here, we show that this is not always true and that single-span helical integral membrane proteins are capable of forming stable but reversible protein nanoparticle assemblies (detergent-limited particles — DLPs) when purified after removal of lipid and all or nearly all detergent. Residual detergent in DLPs is not exchangeable with the surrounding solution. This work focuses on DLPs formed by the transmembrane C-terminal domain of the human amyloid precursor protein (C99), although evidence of DLP formation by other membrane proteins is provided. Size exclusion chromatography, light scattering and electron microscopy showed that C99-DLPs are spherical with modest polydispersity (7-16 monomers) and are stable for months in solution. C99-DLPs revert to traditional protein-detergent complexes upon addition of detergent. Circular dichroism and 2D IR spectroscopies revealed that β-sheet content is significantly higher in C99-DLPs than that of C99 in detergent micelles. C99-DLPs were taken up by mammalian cells, trafficked to a perinuclear location and appeared to accumulate in or next to LC3B-positive punctae, which likely are stalled autophagosomes. C99-DLPs appeared to remain intact in cells as there was no evidence of protein degradation over a period of days. This work challenges the broad assumption that native single-span membrane proteins always require either a bilayer or membrane mimetic to prevent aggregation / precipitation from solutions. It also shows that protein nanoparticles are not always easily degraded by cellular proteases.