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short β strands. In contrast, numerous analogs of KKLTVS-IXGK-KITVSA and otherhairpin scaffolds with 5-7 residue long β strands, led to accelerated production of insolubleaggregates. This was always accompanied by a decrease in the ThT fluorescence signal andintensity of the β CD signature (versus the uninhibited control) at the 16 h point in theassay. Peptides WW2 and YY2 were the most effective “inhibitors”. We attributed theseobservations to the formation of insoluble non-amyloid species. To verify this, we relied onTEM imaging and Congo red (CR) staining of the final assay mixtures with completedispersion of the precipitates so that all species present would be observed in the images.The right panel of Figure 1 shows the amyloid fibrils observed in an uninhibited control. Insuch controls (and in the presence of μPro1 and other peptides that did not reduce the ThTfluorescence response), CR staining is observed with the bright green birefringence that isdiagnostic of amyloid species. Aggregation experiments with WW2 and YY2 inihibitionfailed to display fibrils of normal morphology by TEM and displayed little CR staining andno green birefringence. TEM images appear below (Figure 2 - compare with Figure 1).Fig. 2. TEM image for: WW2 inhibition (left); YY2 inhibition (middle); CD spectra withKKLTVWI (right).The limited formation of short thick fibrils was observed for most hairpins that decreasedthe time to cloudiness in the assay: illustrated for peptide WW2 which produced immediatecloudiness in assays initiation by the addition of HFIP (to a 1.5% final concentration).Peptide YY2, in contrast, afforded circular (spherical?) structures in the TEM images.Of interest, KKLTVWI, the single strand control for hairpin WW2, which increasesthe lag time to fibril formation but eventually yields fibrils of normal morphology and CRstaining characteristics, displayed a different time course of CD spectral changes(Figure 2). At intermediate times in the assay, the CD spectrum indicates the formation of ahighly helical state. We anticipate that both solution-state NMR and 2D IR [3] studies ofamyloidogenesis in the presence of hairpins and other peptides will provide detailsregarding the initial binding sites for the inhibitors and the alterations in theamyloidogenesis pathway that result.ConclusionsFor the amyloidogenesis inhibition with either hAM or α-syn, hairpins with strand lengthsof a least 5 residues are required. In the case of hAM, the presence of cross-strand W/W (orless effectively Y/Y) pairs at non-H-bonded sites is required for inhibition as detected byeither lag time prolongation or reduced fibril yield (TEM and ThT assays). In the case ofα-syn, long hairpins result in the formation of non-amyloid aggregates. This diversion ofthe preamyloid state is enhanced by Trp and Tyr residues in the hairpin strands, but canalso be observed with hairpins lacking the aromatic sidechains.AcknowledgmentsWork at U.W. was supported by grant 3R01GM59658-8S1 (NIH); some of the hairpins were designedin and were available from prior NSF supported (CHE-0650318) studies.References1. Huggins, K.N.L., Andersen, N.H. In Lankinen H., (Ed.) Peptides 2008 (Proceedings of the 30thEuropean Peptide Symposium) 2010, p. 590.2. Cao, P., Meng, F., Abedini, A., Raleigh, D.P. Biochemistry 49, 872-881 (2009).3. Shim, S.-H., Gupta, R., Ling, Y.L., Strasfeld, D.B., Raleigh, D.P., Zanni, M.T. PNAS U.S.A. 106,6614-6619 (2009).23