fatal progressive neurodegenerative diseases including various prion and prion-like disorders are connected with the misfolding of specific proteins. and contingent inactivation.21 36 80 These color differences allow the straightforward quantitative assessment of the capacity of small molecules to convert [PSI+] cells into [psi?] cells in simple plating assays.60 61 Using a similar experimental approach we can also easily assess the capacity of small molecules to modulate different strains of [PSI+] in living cells.60 61 Importantly these distinct [PSI+] strains can be readily generated beginning with pure Sup35 in the test tube (Fig. 1A).30 33 36 61 77 Sup35 is a modular protein comprised of a C-terminal GTPase domain (C amino acids 254-685) which confers translation termination activity (Fig. 1A). Whether Sup35 adopts a prion or a non-prion state is determined by interplay between the middle domain (M amino acids 124-253) and the N-terminal domain (N amino acids 1-123). M is enriched in charged residues and confers solubility 81 whereas N is extremely amyloidogenic82 and of unusually low sequence complexity composed primarily of glutamine asparagine glycine and tyrosine. Together N and M (NM) confer all the properties needed to form a stable prion in yeast and are termed the prion domain.81 83 In isolation Sup35 NM and N can all spontaneously access infectious amyloid forms.30 33 36 61 77 84 That is if Sup35 NM or N are assembled into amyloid forms in vitro and then transformed into [psi?] cells then a large proportion of transformants (up to 80%) become [PSI+].30 33 36 61 77 84 This change in prion phenotype occurs because the introduced amyloid forms of Sup35 convert the endogenous Sup35 to the prion state which can then be propagated through successive generations.30 36 This transformation method of [PSI+] induction does not require cells to harbor another prion [PIN+] 30 36 61 77 84 which is usually comprised BINA of prion conformers of the protein Rnq1.85-88 By contrast [PSI+] can also be induced by overexpression of Sup35 or BINA NM but in this case [PSI+] induction depends on the presence of PIK3C2A [PIN+]86-88 because Rnq1 prions appear to provide a template for the initial formation of Sup35 prions.86 89 However once [PSI+] is established then [PIN+] is dispensable for [PSI+] propagation.86 87 Importantly using pure protein the precise strain ensemble that assembles can be controlled BINA by altering the assembly conditions. For example if pure NM is assembled into infectious BINA amyloid forms at 25°C termed NM25 and transformed into [psi?] [pin?] cells then the majority of transformants become weak [PSI+] (Fig. 1B).30 33 36 61 By contrast if pure NM is assembled into infectious amyloid forms at 4°C termed NM4 and transformed into [psi?] [pin?] cells then the majority of transformants become strong [PSI+]30 33 36 61 (Fig. 1B). Figure 1 Sup35 prion strains and small-molecule antagonists. (A) Sup35 is a modular protein comprised of a C-terminal GTPase domain (C amino acids 254-685 black) a highly charged middle domain (M amino acids 124-253 dark grey) and an N-terminal BINA … A collection of single cysteine NM mutants which behave like wild-type NM have allowed the strategic placement of fluorescent probes (e.g. pyrene or acrylodan) to provide position-specific information concerning the local environment of individual residues within NM in various conformational states.33 60 61 These tools have allowed definition of the intermolecular contacts that hold Sup35 prions together as well as the definition of..