Pressure denaturation of the yeast prion protein Ure2

Denaturation of the Saccharomyces cerevisiae prion protein Ure2 was investi gated using hydrostatic pressure. Pressures of up to 600 MPa caused only li mited perturbation of the structure of the 40-kDa dimeric protein. However, nondenaturing concentrations of GdmCl in combination with high pressure re sulted in complete unfolding of Ure2 as judged by intrinsic fluorescence. T he free energy of unfolding measured by pressure denaturation or by GdmCl d enaturation is the same, indicating that pressure does not induce dimer dis sociation or population of intermediates in 2 M GdmCl. Pressure-induced cha nges in 5 M GdmCl suggest residual structure in the denatured state. Cold d enaturation under pressure at 200 MPa showed at unfolding begins below -5 d egreesC and Ure2 is more susceptible to cold denaturation at low ionic stre ngth. Results obtained using two related protein constructs, which lack all or part of the N-terminal prion domain, were very similar. (C) 2001 Academ ic Press.