Single-Molecule AFM Force Spectroscopy Reveals the Difference in the Folding Patterns between Amyloid β 40 and 42 Within Dimers

OPEN SUBJECT AREAS: SINGLE-MOLECULE BIOPHYSICS ALZHEIMER’S DISEASE Received 26 July 2013 Accepted 20 September 2013 Published 7 October 2013 Correspondence and requests for materials should be addressed to Y.L.L. (ylyubchenko@ unmc.edu) Mechanism of amyloid b2protein dimerization determined using single2molecule AFM force spectroscopy Zhengjian Lv 1 , Robin Roychaudhuri 2 , Margaret M. Condron 2 , David B. Teplow 2,3 & Yuri L. Lyubchenko 1 Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198, United States, 2 Department of Neurology, 3 Brain Research Institute and Molecular Biology Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, United States. Ab42 and Ab40 are the two primary alloforms of human amyloid b2protein (Ab). The two additional C2terminal residues of Ab42 result in elevated neurotoxicity compared with Ab40, but the molecular mechanism underlying this effect remains unclear. Here, we used single2molecule force microscopy to characterize interpeptide interactions for Ab42 and Ab40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Ab42 and Ab40 monomers within dimers. Although the sequence difference between the two peptides is at the C2termini, the N2terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N2terminal was considered as disordered segment with no effect on the Ab peptide aggregation. These novel properties of Ab proteins suggests that the stabilization of N2terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments. A berrant folding (misfolding) and aggregation of amyloid proteins are pathological hallmarks of a large class of neurodegenerative diseases, including Alzheimer’s disease (AD) (reviewed in 1,2 ). In AD, amyloid precursor proteins are sequentially cleaved by b2secretase and c2secretase, producing the amyloid b2protein (Ab) 3 . Ab peptides contain several alloforms with varying sequences of amino acids, dependent on the cleavage sites of c2secretase. While Ab40 is the most abundant species among these alloforms, Ab42 is the most amyloidogenic species, and also appears to be the most toxic 4–6 . It was shown that the two extra C2terminal residues of Ab42 dramatically change the peptide’s oligomerization properties compared to Ab40 7,8 . Ab42 prefers to form a pentamer/hexamer structure (paranucleus) that can nucleate fibril formation 7 . By contrast, Ab40 reaches rapid equilibrium with coexisting monomers, dimers, trimers, and tetramers 7 . According to computer simulations, Ab42 has a turn2like fold at the C2terminus that involves Met352Gly38 (referred to as a C2turn), suggesting that the more structured C2terminus of Ab42 generates greater intramolecular contacts than Ab40 9 . The hypothesis that the C2terminal residues play an important role in the Ab aggregation process was recently tested by 10 , in which a set of Ab peptides with defined alterations in the C2terminus were studied. Specifically, it was shown that VPV substitutions (Gly33Val2Val36Pro2Gly38Val) stabilized the b2hairpin structure by reducing the flexibility of the peptide backbone and by strengthening hydrophobic interactions between the putative b2strands. A similar VPV motif in an Ab40 mutant changed the oligomerization pattern to one similar to Ab42, resulting in increased toxicity of the oligomers compared with the toxicity of wild type (WT) Ab40 10 . pP substitutions (Val36/D2Pro)2(Gly37/L2Pro) destabilized the putative C2turn structure and thereby made [pP]Ab42 ‘‘Ab402like’’ 10 . We have developed a single2molecule atomic force microscopy (SMFS) approach enabling us to probe and characterize the interactions of the peptides and proteins in transient misfolded conformations 11–15 . In this approach, the protein is end2immobilized on the AFM tip and substrate, allowing us to measure the interprotein interactions at the single2molecule level. The dynamics force spectroscopy (DFS) approach has been quite informative with respect to the lifetimes of transiently formed misfolded dimers (reviewed in 16 ). These studies, applied to Ab40 12 , Ab42 14 , a2synuclein 13 and Sup35 15 , revealed that misfolded dimers have an unexpectedly high stability. Their lifetimes exceeded the characteristic lifetimes of monomers by orders of magnitudes, suggesting SCIENTIFIC REPORTS | 3 : 2880 | DOI: 10.1038/srep02880