Progress in FY2009 was in the following areas: 1. DETERMINATION OF BETA-SHEET STRUCTURES IN YEAST PRIONS. We showed that three yeast prion proteins adopt in-register parallel beta-sheet structures in their prion-active, amyloid fibril states. These include the Ure2p protein that produces the URE3 phenotype, the Sup35 protein that produces the PSI+ phenotype, and the Rnq1 protein that produces the PIN+ phenotype. We showed that the in-register parallel beta-sheet structure is maintained in mutants of Ure2p and Sup35 that have scrambled amino acid sequences in their prion domains, and that this structure is not dependent on hydration. These results should quell suggestions in the literature that yeast prions have beta-helix-like structures, rather than in-register parallel beta-sheet structures. 2. INITIAL INVESTIGATIONS OF FUNCTIONAL AMYLOIDS. We carried out initial studies of two proteins that are believed to have biological functions in their amyloid state, namely CsgA (which forms so-called curli fibrils on the surface of bacteria, which have an adhesive function) and Pmel (which catalyzes melanin production in mammalian melanocytes). Neither CsgA nor Pmel fibrils appear to have the in-register parallel beta-sheet structures found in yeast prion fibrils and in beta-amyloid fibrils. Instead, they may have beta-helical structures. Pmel fibrils give very high-quality solid state NMR spectra, indicating that full molecular structure determination for Pmel fibrils may be possible in the next fiscal year. 3. METHOD FOR MASS-PER-LENGTH DETERMINATION IN AMYLOID AND PRION FIBRILS. We demonstrated that the mass-per-length (kDa/nm) of a fibril can be determined quantitatively from dark field electron microscope images obtained with the tilted-beam mode of a conventional transmission electron microscope. This makes mass-per-length determination (an essential component of molecular structure determination) possible with readily-available instrumentation. We are now applying the new method in studies of functional amyloids, prion fibrils, and related systems.
Showing the most recent 10 out of 18 publications
