proteins STRUCTURE O FUNCTION O BIOINFORMATICS Lipid membrane templates the ordering and induces the fibrillogenesis of Alzheimer’s disease amyloid-b peptide Eva Y. Chi, 1 Canay Ege, 1 Amy Winans, 1 Jaroslaw Majewski, 2 Guohui Wu, 1 Kristian Kjaer, 3,4 and Ka Yee C. Lee 1 * 1 Department of Chemistry, Institute for Biophysical Dynamics, and The James Franck Institute, The University of Chicago, Chicago, IL 60637 2 Manuel Lujan Jr. Neutron Scattering Center, Los Alamos Neutron Science Center, Los Alamos National Laboratory, Los Alamos, NM 87545 3 Max-Planck Institute of Colloids and Interfaces, Am Mu ¨hlenberg, Germany 4 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark Abbreviations: Ab, amyloid-b peptide; AD, Alzheimer’s disease; APP, amyloid precursor protein; CMC, critical micelle concentration; d62-DPPG, tail-deuterated DPPG; DLS, dynamic light scattering; DMSO, dimethyl sulfoxide; DMTAP, 1,2-dimyristoyl-3-trimethylammonium-propane; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocho- line; DPPG, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; DPTAP, 1,2-dipalmitoyl 3-trimethylammonium propane; FM, fluorescence microscopy; FWHM, full width at half maximum; GIXD, grazing-incident X-ray diffraction; MALDI, matrix assisted laser desorption/ionization mass spectrometry; NR, neutron reflectivity; PA, palmitic acid; PBS, phosphate buffered saline; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glyc- erol)]; RP-HPLC, reverse phase-HPLC; SE-HPLC, size-exclusion high performance liquid chromatography; SLD, scattering length density; TEM, transmission electron microscopy; ThT, Thioflavin-T; XR, X-ray specular reflectivity. Grant sponsor: Alzheimer’s Association; Grant number: IIRG-9901175; Grant sponsor: American Health Assistance Foundation; Grant number: A1999057; Grant spon- sor: US Department of Energy; Grant number: W-7405-ENG-36; Grant sponsor: National Institute of Health; Grant number: AG025649; Grant sponsor: National Science Foundation Materials Research and Engineering Centers Programs; Grant number: DMR-0213745; Grant sponsors: Packard Foundation, Beckman Scholars Program; GW acknowledges the support of Burroughs Wellcome Fund Interfaces No. 1001774. EYC and CE contributed equally to this work. *Correspondence to: Ka Yee C. Lee, Department of Chemistry, The University of Chicago, 929 E. 57th Street, Chicago, IL 60637. E-mail: kayeelee@uchicago.edu Received 27 July 2007; Revised 26 September 2007; Accepted 23 October 2007 Published online 10 January 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21887 ABSTRACT The lipid membrane has been shown to mediate the fibrillo- genesis and toxicity of Alzheimer’s disease (AD) amyloid-b (Ab) peptide. Electrostatic interactions between Ab40 and the phospholipid headgroup have been found to control the association and insertion of monomeric Ab into lipid mono- layers, where Ab exhibited enhanced interactions with charged lipids compared with zwitterionic lipids. To eluci- date the molecular-scale structural details of Ab-membrane association, we have used complementary X-ray and neutron scattering techniques (grazing-incidence X-ray diffraction, X- ray reflectivity, and neutron reflectivity) in this study to investigate in situ the association of Ab with lipid mono- layers composed of either the anionic lipid 1,2-dipalmitoyl- sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), the zwitter- ionic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or the cationic lipid 1,2-dipalmitoyl 3-trimethyl- ammonium propane (DPTAP) at the air-buffer interface. We found that the anionic lipid DPPG uniquely induced crystal- line ordering of Ab at the membrane surface that closely mimicked the b-sheet structure in fibrils, revealing an in- triguing templated ordering effect of DPPG on Ab. Further- more, incubating Ab with lipid vesicles containing the anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho- rac-(1-glycerol)] (POPG) induced the formation of amyloid fibrils, confirming that the templated ordering of Ab at the membrane surface seeded fibril formation. This study pro- vides a detailed molecular-scale characterization of the early structural fluctuation and assembly events that may trigger the misfolding and aggregation of Ab in vivo. Our results implicate that the adsorption of Ab to anionic lipids, which could become exposed to the outer membrane leaflet by cell injury, may serve as an in vivo mechanism of templated- aggregation and drive the pathogenesis of AD. Proteins 2008; 72:1–24. V V C 2008 Wiley-Liss, Inc. Key words: protein aggregation; fibril formation; protein– lipid interactions; protein conformation; lipid vesicles; lipid monolayer; Alzheimer’s disease; grazing-incidence X-ray diffraction; X-ray reflectivity. V V C 2008 WILEY-LISS, INC. PROTEINS 1