Progress in FY2007 has been in the following areas:? ? 1. STRUCTURAL MODEL FOR THREE-FOLD SYMMETRIC BETA-AMYLOID FIBRILS: In earlier work (Petkova et al., Science 2005), we discovered that the 40-residue beta-amyloid peptide can form more than one fibril structure, that the predominant fibril structure can be determined by subtle variations in growth conditions, that fibrils with distinct morphologies in electron microscope images have distinct underlying molecular structures, and that morphology and molecular structure are self-propagating when pre-existing fibril fragments are used as seeds for the growth of new fibrils. In earlier work (Petkova et al., Biochemistry 2006), we have also developed a full structural model for beta-amyloid fibrils that form under gentle agitation of a beta-amyloid solution and that have mass-per-length (MPL) values, determined by scanning transmission electron microscopy (collaboration with R.D. Leapman, DBEPS, NIBIB), corresponding to two molecular layers in a cross-beta structural motif. We found that fibrils grown under quiescent solution conditions have MPL values that correspond to THREE molecular layers, but our earlier solid state NMR data indicated that two or more quiescent fibril structures might coexist. Now, in FY2007, we have succeeded in preparing highly structurally homogeneous beta-amyloid fibrils with MPL values corresponding to three molecular layers, and we have succeeded in obtaining sufficient structural constraints from solid state NMR to develop a full molecular model. Whereas our previously published model for agitated fibrils had two-fold symmetry about the long fibril axis, the new model (which applies to a different fibril type) has three-fold symmetry. The beta-amyloid peptide conformations in two-fold and three-fold symmetric structures are similar, with essentially the same beta-strand segments and the same in-register parallel beta-sheet tertiary structure, but the conformations of non-beta-strand segments are different, and the contacts between beta-sheets are also different. A manuscript describing the new beta-amyloid fibril structure is currently in preparation. Additional measurements on three-fold symmetric fibrils are in progress, including cryo-EM studies in collaboration with U. Baxa and A.C. Steven of NIAMS.? ? 2. In collaboration with R.B. Wickner's group in NIDDK, we have carried out related studies of amyloid fibrils formed by the yeast prion proteins Ure2p and Sup35p. These are the first solid state NMR studies of full-length prion domains of yeast prions. In experiments on Sup35NM (the N-terminal and middle domains of Sup35p), we showed that Sup35NM fibrils are constructed from in-register parallel beta-sheets and do not have a beta-helical structure, which had been suggested by earlier x-ray fiber diffraction and fluorescence labeling experiments in other groups. Interestingly, our solid state NMR data indicate that the beta-sheets in Sup35NM are not localized exclusively in the N-terminal, Asn- and Gln-rich domain (which was expected to form the amyloid fibril core), but are also found in parts of the M domain. This work has been published in PNAS, vol. 103, p. 19754, year 2006. Beta-sheet formation in the M domain may be the distinguishing structural feature among PSI prion variants or strains, which are induced by intracellular Sup35p amyloid formation. This possibility is supported by recent H/D exchange data from the Weissman group at UCSF, which indicate different degrees of H/D exchange protection in the M domain for different Sup35NM amyloid variants. We have also carried out solid state NMR studies of the prion domain (residues 1-89) of Ure2p. Ure2p(1-89) fibrils also contain in-register parallel beta-sheets. This work is described in a manuscript that is currently under review for Biochemistry.
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