Relationship between stability of folding intermediates and amyloid formation for the yeast prion Ure2p: a quantitative analysis of the effects of pH and buffer system.
The dimeric yeast protein Ure2 shows prion-like behaviour in vivo and forms amyloid fibrils in vitro. A dimeric intermediate is populated transiently during refolding and is apparently stabilized at lower pH, conditions suggested to favour Ure2 fibril formation. Here we present a quantitative analysis of the effect of pH on the thermodynamic stability of Ure2 in Tris and phosphate buffers over a 100-fold protein concentration range. We find that equilibrium denaturation is best described by a three-state model via a dimeric intermediate, even under conditions where the transition appears two-state by multiple structural probes. The free energy for complete unfolding and dissociation of Ure2 is up to 50 kcal mol(-1). Of this, at least 20 kcal mol(-1) is contributed by inter-subunit interactions. Hence the native dimer and dimeric intermediate are significantly more stable than either of their monomeric counterparts. The previously observed kinetic unfolding intermediate is suggested to represent the dissociated native-like monomer. The native state is stabilized with respect to the dimeric intermediate at higher pH and in Tris buffer, without significantly affecting the dissociation equilibrium. The effects of pH, buffer, protein concentration and temperature on the kinetics of amyloid formation were quantified by monitoring thioflavin T fluorescence. The lag time decreases with increasing protein concentration and fibril formation shows pseudo-first order kinetics, consistent with a nucleated assembly mechanism. In Tris buffer the lag time is increased, suggesting that stabilization of the native state disfavours amyloid nucleation.
Zhu
Li
L
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, People's Republic of China.
Zhang
Xu-Jia
XJ
Wang
Ling-Yun
LY
Zhou
Jun-Mei
JM
Perrett
Sarah
S
D000682:Amyloid / Q000737:chemistry*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002021:Buffers; D002942:Circular Dichroism; D019281:Dimerization; D005979:Glutathione Peroxidase; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008958:Models, Molecular; D010710:Phosphates / Q000737:chemistry; D011328:Prions / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014325:Tromethamine / Q000737:chemistry
D000682:Amyloid; D000817:Anilino Naphthalenesulfonates; D002021:Buffers; D010710:Phosphates; D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; D014325:Tromethamine; C027132:1-anilino-8-naphthalenesulfonate; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae