Abstract
Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed 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 one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, 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 structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.
Highlights
Keen interest has been shown during the past several years to a phenomenon of protein misfolding and aggregation, due to studies of various amyloidosis and prion diseases [1,2,3,4,5,6]
Since yeast expression systems are well known not to be as efficient as that of E. coli, we developed an approach to overexpress the yeast prion-like protein Ure2p in E. coli
Structure of Ure2p—Soluble Ure2p was found to emerge from a sizing column with an apparent molecular mass of 130,000 incompatible with that expected for a monomeric Ure2p molecule that would have a globular shape
Summary
Keen interest has been shown during the past several years to a phenomenon of protein misfolding and aggregation (inside the cells), due to studies of various amyloidosis and prion diseases [1,2,3,4,5,6]. There are some significant differences between prion diseases and amyloid diseases, such as transmissibility of prion diseases, but it is clear that conversion of a soluble form of a protein into insoluble aggregate is a key mechanism involved in all the cases [1,2,3,4,5,6] Such aggregates reveal high resistance to proteases, escaping different common degradation pathways, e.g. proteosomal complexes [7, 8]. It is widely accepted that formation of insoluble aggregates is a result of a shift in equilibrium between native soluble conformer of a prion protein and aggregation-competent molecules [6] Reasons for such a shift are rather obscure, the basis for partitioning between different conformers seems to be provided (at least in case of mammalian prion proteins) by their specific structural properties [4, 14, 15]. These changes affect essentially the COOH-terminal part of the molecule
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