Structural characterization of Saccharomyces cerevisiae prion-like proteinUre2

Sacchromyces cerevisiae prion-like protein Urea was expressed in Escherichi a coli and was purified to homogeneity, We show here that Ure2p is a solubl e protein that can assemble into fibers that are similar to the fibers obse rved in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where on e can distinguish a lag phase followed by an elongation phase preceding a p lateau. A combination of size exclusion chromatography, sedimentation veloc ity, and electron microscopy demonstrates that the soluble form of Ure2p co nsists at least of three forms of the protein as follows: a monomeric, dime ric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two s tructural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be com pactly folded. Finally, we show that only slight conformational changes acc ompany Ure2p assembly into insoluble high molecular weight oligomers, These changes essentially affect the COOH-terminal part of the molecule. The pro perties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.