We suggest that antiparallel b-sheet structure might represent a distinctive sig- nature of amyloid oligomers (Cerf et al, Biochem J, 2009, 421:415-23) under- lying their common pathogenic action. 2236-Pos Board B6 Amyloid Beta Peptide: The Inﬂuence of Intrinsic Factors on Fibril Formation Risto Cukalevski 1 , Birgitta Frohm 1 , Barry Boland 2 , Sara Linse 1 . 1 Lund University, Lund, Sweden, 2 University College Dublin, Dublin, Ireland. Alzheimer’s disease (AD) is the most common form of dementia. Studying the amyloid b-peptide (Ab) involved in the pathogenesis and understanding the mechanism of the disease will hopefully lead to developing drug candidates that could cure or delay the onset of AD. One way of approaching this is to inhibit the ﬁbril formation. Mutating the pep- tide gives an insight on which amino acids are important in the aggregation mechanism. We have investigated the importance of the aromatic rings in the phenylalanine side chains in position 19 and 20 by substituting these with leucine. This way the hydrophobicity is preserved while aromatic stacking possibilities are removed. Single and double mutants were expressed in E. coli from synthetic genes. The kinetics of ﬁbril formation was followed using a highly reproducible thioﬂavin T assay and show that Ab(M1-42)F19L aggre- gates slower than the wild type, mainly observed as a longer lag time. Also the structural change, monitored by far-UV circular dichroism spectroscopy, dif- fers between wild type and all mutants. This indicates that the phenylalanine rings have a role in the amyloid formation. With cryo-TEM it was conﬁrmed that all peptides form ﬁbrils with similar morphology. The inﬂuence of the slower F19L mutant was further studied in co-aggregation experiments together with the wild type. Preliminary results show that the lag time of the mixture ends up in between those of the wild type and the F19L mu- tant, and that the lag time depends on the ratio of the two peptides as well as the total concentration. Further co-aggregation studies include a familial mutant peptide with faster aggregation kinetics than the wild type, as well as seeding experiments in which wild type is seeded with ﬁbrils from F19L or the familial mutant. 1(1) 2237-Pos Board B7 The Effect of a Membrane Mimicking Detergent on Alzheimer’s Amyloid Peptide Aggregation Studied by EPR Maryam Hashemi Shabestari 1 , Nico J. Meeuwenoord 2 , Dmitri V. Filippov 2 , Martina Huber 1 . 1 Department of Molecular Physics, Leiden University, Leiden, Netherlands, 2 Leiden Institute of Chemistry, Leiden University, Leiden, Netherlands. The aggregation of the b-Amyloid (Ab) peptide into ﬁbrils is the chief indicator of Alzheimer’s disease. However, it seems that small, oligomeric aggregates, rather than fully formed ﬁbrils could be the toxic species. Ab -membrane inter- action could also have an effect, so here effect of the membrane-mimicking de- tergent SDS is studied. We measure, by spin-label-mobility EPR, Ab aggregation at different SDS con- centrations. It monitors all peptide in the sample and is not limited to particular aggregate sizes. The high-SDS form found (C) is consistent with the one- peptide/micelle model [1-2]. In the absence of SDS (A) the high concentration of Ab (0.55 mM) most certainly enforces ﬁbrils. At intermediate SDS (B), EPR reveals an increase in particle size suggestive of oligomer formation, eventually involving detergents. Thus a ﬁrst glimpse into the behavior of SDS at sub- CMC (Critical Micelle Concentration) SDS con- centrations, where high res- olution techniques, such as NMR, fail because of parti- cle size limitation, [1-2] are accessible. [1] FEBS, Wahlstro ¨m et al. (2008) 275: 5117-5128. [2] J. Biomol NMR, Ju ¨ri et al. (2007) 39: 63-72. 2238-Pos Board B8 A Conventional and 2DCOS Infrared Study of Amyloid Fibril Formation Marcos Garcia Pacios 1,2 , Nagore Andraka 1,2 , Igor de la Arada 1,2 , Jose Luis R. Arrondo 1,2 . 1 University of Basque Country, Bilbao, Spain, 2 Unidad de Bioﬁsica, Bilbao, Spain. Amyloidosis is characterized by the abnormal self-assembly and deposition of proteinaceous material into insoluble ordered aggregates. There are several pathologies associated with this aggregates, known as ‘‘protein misfolding’’ disorders. In many proteins, when heated at high temperature and low pH, a se- ries of structural changes resulting in the formation of ﬁbrillar structures are produced. Insulin is a model of ﬁbril formation that has produced a wealth of biochemical and structural data. The time-course of ﬁbril formation can be fol- lowed by infrared spectroscopy looking at the appearance of a characteristic band in the amide I region. The kinetics is triggered by temperature at 70  C and pH at 2.3. The infrared spectrum shows, that after a lag time (concentra- tion-dependent), a-helix and b-turns bands decrease ﬁrst whereas the random coil component increases. Later, a band at 1626 cm-1, associated with extended chains, replaces the random coil component. Infrared 2D-COS has been applied to different stages of the process. Maps have been formed at different incuba- tion times: before random coil formation and at different stages in the random coil-ﬁbril exchange. The synchronous maps do not give in principle much in- formation because they are dominated by the appereance of the band due to the ﬁbril. In order to extract such information we have developed an approach in which we take a window and move it along the perturbation (2D-moving lapse). This, together with the asynchronous maps indicate a two-step process with a ﬁrst stage associated with an opening of the protein driven by destabili- zation of b-turns, located in the outer part of the protein, and a second part where the ﬁbril is formed. This work was supported in part by the Spanish Ministerio de Educacio ´n y Ciencia (grant No. BFU 2010-22103). 2239-Pos Board B9 Mechanism of Non-Speciﬁc Inhibitors of Amyloid Assembly: Interactions of Lacmoid with the Amyloid Beta Peptide Axel Abelein 1 , Benedetta Bolognesi 2 , Christopher M. Dobson 2 , Astrid Gra ¨slund 1 , Christofer Lendel 3 . 1 Stockholm University, Stockholm, Sweden, 2 University of Cambridge, Cambridge, United Kingdom, 3 Swedish University of Agricultural Sciences, Uppsala, Sweden. Increasing evidence shows a strong link between the self-assembly of the am- yloid beta peptide (Ab) and the pathogenesis of Alzheimer’s disease. Soluble oligomeric Ab assemblies are thought to be the toxic species causing synaptic and neuronal injury in the patient’s brain. Many inhibitors for the oligomeriza- tion and/or ﬁbrillation process of neurodegenerative diseases have been reported, yet only little is known about the mechanistic details of these com- pounds. The present studies concern the interaction of one such inhibitor, lacmoid. We investigated this interaction by a broad biophysical approach re- vealing similar binding characteristics to Ab as has been reported for deter- gents. Furthermore, we show that lacmoid has the ability to inhibit both oligomeric assembly and ﬁbrillation of Ab. Nuclear magnetic resonance exper- iments show an overall signal decrease upon addition of lacmoid while the chemical shifts only display small changes. High lacmoid concentration causes a loss of the major part of NMR signals including 1 H- 15 N-HSQC, 1 H- 15 N- TROSY and 1 H- 13 C-HSQC cross-peaks. Circular Dichroism spectroscopy was applied to monitor the kinetic aggregation process of Ab in presence of lacmoid. Low lacmoid concentrations slow down the conversion from a random coil-like towards a beta-sheet state while at high concentration lacmoid completely prohibits secondary structure changes. Taken together, these ﬁndings provide the basis for a simple model which could explain how non-speciﬁc interactions with small molecules interfere with amyloid formation. Understanding the mechanistic details is potentially helpful for future drug design of small molecule therapeutics targeting amyloid disor- ders, such as Alzheimer’s disease. 2240-Pos Board B10 Understanding Formation and Structure of Peptide Nanoﬁbers via Steered MD Simulations Ozge Engin, Beytullah Ozgur, Mehmet Sayar. Koc University, Istanbul, Turkey. Understanding the formation and structure of ﬁbers formed via peptide aggre- gation is a major challenge for both prevention of diseases and also for utilising such structures for nanostructure templating. Here, we propose that single mol- ecule pulling experiments can be used to characterise the mechanical and struc- tural properties of these ﬁbers. We demonstrate this idea via molecular dynamics simulations on ﬁbers formed by a library of triblock peptides. In these oligomers, hydrophobic residues in the central block are replaced to control the resulting ﬁbers. Our results suggest that, independent of the hydrophobic resi- due type, all these peptides yield an inter-dimer spacing of ~ 0.48 nm along the ﬁber axis. Hence, backbone hydrogen bonding alone dominates and the hydro- phobic residues simply squeeze into the hydrophobic core of these ﬁbers. We measure the relative strength of these ﬁbers via steered MD simulations. Our results suggest that hydrophobic residues such as tyrosine and tryptophan not Tuesday, February 28, 2012 441a