Formation of Toroidal Pores by Amyloid Proteins: Evidence of Lipid Transbilayer Exchange Induced by Islet Amyloid Polypeptide

Abstract

Recent computer simulations have indicated that amyloid peptides disrupt membranes by the formation of lipid-lined toroidal pores caused by excess membrane curvature, but experimental evidence for this mechanism has largely been lacking. A directly measurable consequence of this phenomenon is the significantly accelerated transbilayer exchange of lipids, which is a feature of the toroidal pore mechanism but not of other mechanisms of membrane disruption. Using vesicles asymmetrically labeled on the outer leaflet by pyrene-labeled lipids, we show that toxic versions of islet amyloid polypeptide, an amyloid peptide implicated in the pathogenesis of type II diabetes, induce rapid lipid flip-flop between bilayers. This manner is consistent with antimicrobial peptides known to disrupt membranes by the toroidal pore mechanism. We further demonstrate that a clear difference between toxic and non-toxic versions of IAPP can be observed in their binding to bicelles containing DMPC and the detergent DHPC, in which DHPC forms highly curved regions resembling toroidal pores. Using this model of a pre-constructed toroidal pore we show that toxic rat IAPP1-19 binds in the highly curved, pore-like DHPC enriched region while non-toxic rat IAPP1-37 binds to the flat lamellar DMPC enriched region away from the pore. Similarly, DSC indicates that toxic versions of the IAPP peptide strongly favor the formation of negative curvature in lipid bilayers, while the non-toxic rIAPP1-37 peptide does not. Further results on other amyloid peptides (including Aβ, calcitonin, and insulin) will also be presented as well as results from antimicrobial peptides.