Lipid Membrane Interaction of Amyloidogenic Peptides

Abstract

Membrane interaction appears to be a key step in the toxic pathway of many amyloidogenic proteins, such as Amyloidβ (Aβ40/42, associated with Alzheimer's disease) and hIAPP (associated with Type II diabetes). While the oligomers are generally considered to be more toxic than either the monomers or the fibrils, the exact identity of the toxic oligomer has remained a matter of debate. How much the local structure and composition of the membrane influences the binding of the oligomers is another controversial issue.Here we use a combination of single‐molecule fluorescence techniques (e.g. single molecule photobleaching (smPB) and Fluorescence Correlation Spectroscopy (FCS)) and atomic force microscopy to quantitatively address these two issues. smPB measurements of freshly prepared hIAPP in physiological solution and on a supported lipid bilayer (composed of POPC: POPG: Cholesterol 1:1:1) allow us to measure the relative affinity of each oligomeric species. Our measurements show that both monomers and small oligomers (1–5 mers) have high affinity for the membrane, and dimers appear to have the highest affinity. However, FCS measurements show that the monomers have much lower affinity than the oligomers, Though FCS is unable to resolve the contribution of individual types of oligomers. It is likely that the smPB measurements are skewed by pre‐measurement photobleaching which is a known problem with this technique. We have now developed a method to measure pre‐bleaching, and are in the process of calibrating our results.We have also addressed the influence of the local membrane structure on binding of hIAPP oligomers. This aspect may throw light on the initiation of these diseases in the human body. We use a ternary neutral lipid mixture (DOPC: Sphingomyelin: Cholesterol in 2:2:1 mole ratio) to make stable phase separated bilayers. We use an AFM to map the bilayer surface topology, and a synchronized home‐built confocal microscope to simultaneously map the distribution of the fluorescently labeled peptides on this bilayer. We find that small oligomers of hIAPP bind preferentially to the disordered (Ld) phase, compared to the ordered phase Lo, with a binding ratio of Ld:Lo = 9:1. Our studies thus quantify the membrane interaction of small n‐mers of hIAPP as a function of n, and also show that local membrane organization can play a dominant role in determining membrane interactions.Support or Funding InformationTata Institute of Fundamental ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.