Ricardo Capone

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.

SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is essential to coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E into amphipol-class amphipathic polymer solutions. The physical properties of amphipols underpin their ability to solubilize and stabilize membrane proteins without disrupting membranes. Amphipol delivery of S2-E to pre-formed planar bilayers results in spontaneous membrane integration and formation of viroporin ion channels. Amphipol delivery of the S2-E protein to human cells results in membrane integration followed by retrograde trafficking to a location adjacent to the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC) and the Golgi, which are the sites of coronavirus replication. Delivery of S2-E to cells enables both chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and development of “Trojan Horse” anti-viral therapies. This work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

Alzheimer’s disease (AD) is the most common type of senile dementia in aging populations. Amyloid β (Aβ)-mediated dysregulation of ionic homeostasis is the prevailing underlying mechanism leading to synaptic degeneration and neuronal death. Aβ-dependent ionic dysregulation most likely occurs either directly via unregulated ionic transport through the membrane or indirectly via Aβ binding to cell membrane receptors and subsequent opening of existing ion channels or transporters. Receptor binding is expected to involve a high degree of stereospecificity. Here, we investigated whether an Aβ peptide enantiomer, whose entire sequence consists of d-amino acids, can form ion-conducting channels; these channels can directly mediate Aβ effects even in the absence of receptor–peptide interactions. Using complementary approaches of planar lipid bilayer (PLB) electrophysiological recordings and molecular dynamics (MD) simulations, we show that the d-Aβ isomer exhibits ion conductance behavior in the bilayer indistinguishable from that described earlier for the l-Aβ isomer. The d isomer forms channel-like pores with heterogeneous ionic conductance similar to the l-Aβ isomer channels, and the d-isomer channel conductance is blocked by Zn²⁺, a known blocker of l-Aβ isomer channels. MD simulations further verify formation of β-barrel-like Aβ channels with d- and l-isomers, illustrating that both d- and l-Aβ barrels can conduct cations. The calculated values of the single-channel conductance are approximately in the range of the experimental values. These findings are in agreement with amyloids forming Ca²⁺ leaking, unregulated channels in AD, and suggest that Aβ toxicity is mediated through a receptor-independent, nonstereoselective mechanism.

Alzheimer’s disease (AD) is a protein misfolding disease characterized by a buildup of β-amyloid (Aβ) peptide as senile plaques, uncontrolled neurodegeneration, and memory loss. AD pathology is linked to the destabilization of cellular ionic homeostasis and involves Aβ peptide–plasma membrane interactions. In principle, there are two possible ways through which disturbance of the ionic homeostasis can take place: directly, where the Aβ peptide either inserts into the membrane and creates ion-conductive pores or destabilizes the membrane organization, or, indirectly, where the Aβ peptide interacts with existing cell membrane receptors. To distinguish between these two possible types of Aβ-membrane interactions, we took advantage of the biochemical tenet that ligand–receptor interactions are stereospecific; l-amino acid peptides, but not their d-counterparts, bind to cell membrane receptors. However, with respect to the ion channel-mediated mechanism, like l-amino acids, d-amino acid peptides will also form ion channel-like structures. Using atomic force microscopy (AFM), we imaged the structures of both d- and l-enantiomers of the full length Aβ_1–42 when reconstituted in lipid bilayers. AFM imaging shows that both l- and d-Aβ isomers form similar channel-like structures. Molecular dynamics (MD) simulations support the AFM imaged 3D structures. Previously, we have shown that d-Aβ_1–42 channels conduct ions similarly to their l- counterparts. Taken together, our results support the direct mechanism of Aβ ion channel-mediated destabilization of ionic homeostasis rather than the indirect mechanism through Aβ interaction with membrane receptors.