Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates 1 1Edited by J. Karn

The yeast prion protein Ure2: Structure, function and folding

“…The conditions found to be 'ideal' for folding experiments (Tris-HCl buffer pH 8.4, 0.2 M NaCl, 25°C and 1 μM protein concentration) unsurprisingly resemble the conditions adopted for Ure2 purification in a variety of labs [26,27,32,109], and coincide with maximal stabilization of the native state of Ure2 with respect to partially-folded intermediates [36]. Nevertheless, under these conditions, the folding kinetics of Ure2 is extremely complex and a variety of folding intermediates are transiently populated during unfolding or refolding [27,35,36].…”

“…Although the N-terminal domain does not contribute to the stability of the Ure2 protein in vitro [27], or affect the pathway of folding [35], the presence of the prion domain has a marked effect on the solubility of the protein [27]. Interestingly, conditions where Ure2 is relatively destabilized, such as at lower pH, or in phosphate rather than Tris buffer, also coincide with conditions where the protein more readily forms fibrils, implying that the mechanism of fibril formation may involve partial unfolding [33,36].…”

“…The lower propensity of mutants such as Δ15-42Ure2 (see Fig. 2) to undergo non-specific aggregation and to form amyloid makes them particularly useful for use in rigorous folding studies [27,35,36,105,112].…”

“…Subsequent biophysical analysis of the purified Ure2 protein has shown that the structural properties of the N-terminal and C-terminal regions are also distinct. The N-terminal domain is extremely sensitive to protease digestion [26][27][28]104] and progressive deletion of the N-terminal domain has no effect on the dimerisation, stability or folding kinetics of the protein in vitro [27,33,35,36,105] (see Fig. 6).…”

“…The N-terminal region of Ure2 is required for its prion properties in vivo [31] and to form amyloid-like filaments in vitro [32][33][34]. However, deletion of the N-terminal region has no detectable effect on the stability or folding of the protein in vitro [27,33,35,36]. The N-terminal approximately 90 residues of Ure2 are rich in asparagine and glutamine (see Fig.…”