a previously proposed hypothesis that Met-35/Gly-33 can be oxidized [4] by Cu 2þ to produce ROS by SSNMR. 2367-Pos Detecting the Aggregation of Amyloid Peptides by Spin Label EPR Maryam Hashemi Shabestari 1 , Irina Sepkhanova 1 , Malte Drescher 1 , Nico J. Meeuwenoord 2 , Dimitri V. Filippov 2 , Ronald Limpens 3 , Roman I. Koning 3 , Martina Huber 1 . 1 Leiden University, Leiden, Netherlands, 2 Department of Chemistry, Leiden University, Leiden, Netherlands, 3 Leiden University Medical Center, Leiden, Netherlands. Plaques containing aggregated b-Amyloid (Ab) peptide in the brain are the main indication of Alzheimers disease. These plaques consist of Ab fibrils. Oligomers of Ab have been implicated as infective agents in the disease and may also be intermediates of fibril formation. Therefore, methods to study olig- omers on the timescale of aggregation are sought. We show that by EPR the dynamics of spin-labeled Ab in solutions in which fibrils are formed can be de- termined. The EPR experiments were performed on solutions of the Ab peptide with 42 residues (1-42 Ab) containing an N-terminal cysteine, which was spin labeled with the MTSL spin label (1-oxyl-2,2,5,5-tetramethyl-D-pyrroline-3- methyl]methanethiosulfonate) (SL- Ab). For diamagnetic dilution, SL- Ab was mixed with unlabeled Ab. Fibril-formation in these solutions is shown by Congo-red binding and electron microscopy. Continuous wave, 9 GHz EPR reveals three fractions of different spin-label mobility, a fast one attributed to monomeric Ab, one with a mobility that corresponds to a multimer of eight to 15 monomers, and a slow one due to larger aggregates or fibrils. The approach, in principle, allows detection of oligomers on the timescale of aggregation. 2368-Pos Structural Characterization of Amyloids Comprised of Anchorless Prion Proteins Jan Stoehr, David Colby, Kurt Giles, Stanley B. Prusiner, Holger Wille. UCSF, San Francisco, CA, USA. Prion diseases are fatal, neurodegenerative diseases that afflict sheep, cows, and humans. The key event in prion diseases is the conversion of the a-helical, cel- lular isoform of the prion protein (PrP C ) to an insoluble, b-sheet-rich, infectious isoform (PrP Sc ). Host-encoded PrP C is anchored to the cell membrane by a gly- cosylphosphatidyl inositol (GPI) anchor and converts to GPI-anchored PrP Sc during prion infection. Here we describe prion infection in transgenic mice (Tg8015) that lack the signal sequence for attachment of the GPI anchor and therefore express non-anchored, free-floating, PrP C (DGPI-PrP). The lack of the GPI anchor leads to incomplete modification of PrP, resulting in an ungly- cosylated molecule. Therefore, Tg8015 mice produce unglycosylated DGPI- PrP Sc upon infection with the Rocky Mountain Laboratory (RML) prion strain. These mice did not show clinical signs of disease until >300 days post-infec- tion (dpi), but their brains harbored DGPI-PrP Sc and macroscopic amyloid de- posits. In contrast, RML infection of wild-type (wt) mice resulted in clinical signs of disease within 120 dpi but no macroscopic amyloid formation. DGPI-PrP Sc and wt PrP Sc in the brains of Tg8015 and wt mice, respectively, were purified by two different protocols, both involving N-terminal truncation by proteinase K. The resulting, highly concentrated DGPI-PrP 27-30 and wt PrP 27-30 preparations were analyzed and compared by negative-stain electron microscopy to characterize possible differences in fibril morphology. Addi- tional analyses were performed using Fourier transform infrared spectroscopy and X-ray fiber diffraction to compare the structures of DGPI-PrP 27-30 amy- loid and wt PrP 27-30 amyloid preparations. Additionally, we tested whether DGPI-PrP Sc could self-propagate in vitro by evaluating its seeding capacity in a prion-specific amyloid seeding assay and examined its biological activity in vivo using mouse-based bioassays. 2369-Pos Oligomerization of Amphipathic Peptides in a Membrane Studied by Coarse-Grained Molecular Dynamics Simulations Myunggi Yi 1,2 , Huan-Xiang Zhou 1 . 1 Florida State University, Tallahassee, FL, USA, 2 National High Magnetic Field Laboratory, Tallahassee, FL, USA. To gain insight into the aggregation and oligomerization of antimicrobial or amyloidogenic peptides, we carried out molecular dynamics simulations of a 26-residue amphipathic peptide at different concentrations (8, 16, and 32 cop- ies) in a fully hydrated bilayer composed of 1600 POPC lipid molecules. With a coarse-grained representation of the molecules, >28 microseconds of simu- lations were accumulated for each system. Oligomers of various orders were observed to form. The system with 32 copies of the peptide was finally com- prised of a 4-, 7-, 8-, and 13-mer. The final compositions of the systems with 16 and 8 copies of the peptide were 3-, 4-, and 9-mer for one and 3- and 5-mer for the other. Higher oligomers were formed by addition of mono- mers and by association of pre- formed lower oligomers (see Figure). Dissociation was not observed. In the lower oligomers (up to 4-mer) only the hydrophilic side of each copy was buried, but in the higer oligomers the hydrophobic sides of some copies were also buried. These simulations provide molecular insight into oligo- merization of peptides inside membranes. 2370-Pos Clues for the Mechanism of SEVI HIV Enhancement from Structural Studies of the SEVI Precursor Peptide PAP 248-286 in a Membrane Environ- ment by NMR Ravi Prakash Reddy Nanga, Jeffrey R. Brender, Vivekanandan Subramanian, Nataliya Popovych, Ramamoorthy Ayyalusamy. University of Michigan, Ann Arbor, MI, USA. Despite the rapid progress of the AIDS pandemic, the HIV virus is a surprisingly weak pathogen in vitro. The large difference between in vitro and in vivo infec- tion rates suggests that cofactors absent in vitro but essential for the natural transmission of the virus may be responsible for this discrepancy. A recently identified peptide in human semen, PAP 248-286 , has emerged as a clear candi- date for the missing cofactor as it dramatically enhances the infectivity of HIV by up to five orders of magnitude. PAP 248-286 appears to enhance HIV in- fection by forming amyloid fibers known as SEVI, which are believed to en- hance the attachment of the virus by bridging interactions between virion and host-cell membranes. To understand the unique ability of SEVI to enhance HIV infection, we have solved the atomic-level resolution structure of the SEVI precursor PAP 248-286 using NMR spectroscopy in SDS micelles. In contrast to other toxic amyloid peptides that generally penetrate into the core of the mem- brane, non-toxic PAP 248-286 binds superficially to the surface of the micelle. Unlike most amyloid peptides that bind to the membrane in an a-helical state, PAP 248-286 is mostly disordered when bound to the surface of the micelle. The highly disordered nature of the SEVI peptide may explain the high ability of SEVI to enhance HIV infection, as partially disordered amyloid fibers will have a greater capture radius for the virus than more compact amyloid fibers. Two regions of nascent structure match the prediction of highly amyloidogenic sequences and may serve as nuclei for aggregation and amyloid fibril forma- tion. NMR studies of the binding of PAP 248-286 to the anti-amyloid agent ECGC will also be presented. 2371-Pos Formation of Toroidal Pores by Amyloid Proteins: Evidence of Lipid Transbilayer Exchange Induced by Islet Amyloid Polypeptide Daniel W. Youngstrom, Jeffrey R. Brender, Pieter E.S. Smith, Kevin Hartman, Ayyalusamy Ramamoorthy. University of Michigan, Ann Arbor, MI, USA. 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 fea- ture of the toroidal pore mechanism but not of other mechanisms of mem- brane disruption. Using vesicles asymmetrically labeled on the outer leaflet by pyrene-labeled lipids, we show that toxic versions of islet amyloid poly- peptide, 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-con- structed 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 for- mation of negative curvature in lipid bilayers, while the non-toxic rIAPP1-37 peptide does not. Further results on other amyloid peptides (including Ab, cal- citonin, and insulin) will also be presented as well as results from antimicro- bial peptides. 458a Tuesday, February 23, 2010