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.


Abstract

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. Study holds ProTherm entries: 15992, 15993, 15994, 15995, 15996, 15997, 15998, 15999, 16000, 16001, 16002, 16003, 16004, 16005, 16006, 16007, 16008, 16009, 16010, 16011, 16012, 16013, 16014, 16015, 16016, 16017, 16018, 16019, 16020, 16021, 16022, 16023, 16024, 16025, 16026, 16027, 16028, 16029, 16030, 16031, 16032, 16033, 16034, 16035, 16036, 16037, 16038, 16039, 16040, 16041, 16042, 16043, 16044, 16045, 16046, 16047, 16048, 16049, 16050, 16051, 16052, 16053, 16054, 16055, 16056, 16057, 16058, 16059, 16060, 16061, 16062, 16063, 16064, 16065, 16066, 16067, 16068, 16069, 16070, 16071, 16072, 16073, 16074, 16075, 16076, 16077, 16078, 16079, 16080, 16081, 16082, 16083, 16084, 16085 Extra Details: Transition 1; Residues 15-42 have been deleted. dissociation; first order kinetics; nucleation; protein folding; protein stability

Submission Details

ID: mQ6v7D944

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Zhu L;Zhang XJ;Wang LY;Zhou JM;Perrett S,J. Mol. Biol. (2003) 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. PMID:12684011
Additional Information

Number of data points 282
Proteins Transcriptional regulator URE2 ; Transcriptional regulator URE2
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Cm prot_conc:0.2 microM, ionic:NaCl: 0.2 M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: m ionic:NaCl: 0.2 M, prot_conc:0.2 microM, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O prot_conc:0.2 microM, ionic:NaCl: 0.2 M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm ionic:NaCl: 0.2 M, prot_conc:1 microM, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: m ionic:NaCl: 0.2 M, prot_conc:1 microM, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O ionic:NaCl: 0.2 M, prot_conc:1 microM, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm prot_conc:5 microM, ionic:NaCl: 0.2 M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: m prot_conc:5 microM, ionic:NaCl: 0.2 M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O prot_conc:5 microM, ionic:NaCl: 0.2 M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm prot_conc:20 microM, ionic:NaCl: 0.2 M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: m prot_conc:20 microM, ionic:NaCl: 0.2 M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O prot_conc:20 microM, ionic:NaCl: 0.2 M, pH:8.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm ionic:NaCl: 0.2 M, prot_conc:0.2 microM, pH:8.4, buffers:Tris-HCl: 50 mM ; Experimental Assay: m prot_conc:0.2 microM, pH:8.4, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O ionic:NaCl: 0.2 M, prot_conc:0.2 microM, pH:8.4, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm pH:8.4, prot_conc:1 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: m pH:8.4, prot_conc:1 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O pH:8.4, prot_conc:1 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm prot_conc:20 microM, pH:8.4, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: m prot_conc:20 microM, pH:8.4, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O prot_conc:20 microM, pH:8.4, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm prot_conc:0.2 microM, pH:7.0, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: m ionic:NaCl: 0.2 M, pH:7.0, prot_conc:0.2 microM, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O prot_conc:0.2 microM, pH:7.0, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm ionic:NaCl: 0.2 M, pH:7.0, prot_conc:1 microM, buffers:Tris-HCl: 50 mM ; Experimental Assay: m ionic:NaCl: 0.2 M, pH:7.0, prot_conc:1 microM, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O ionic:NaCl: 0.2 M, pH:7.0, prot_conc:1 microM, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm prot_conc:5 microM, ionic:NaCl: 0.2 M, pH:7.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: m prot_conc:5 microM, ionic:NaCl: 0.2 M, pH:7.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O prot_conc:5 microM, ionic:NaCl: 0.2 M, pH:7.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm prot_conc:20 microM, ionic:NaCl: 0.2 M, pH:7.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: m prot_conc:20 microM, ionic:NaCl: 0.2 M, pH:7.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O prot_conc:20 microM, ionic:NaCl: 0.2 M, pH:7.0, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm pH:7.5, prot_conc:0.2 microM, ionic:NaCl: 0.2 M, buffers:Tris-phosphate: 50 mM ; Experimental Assay: m pH:7.5, ionic:NaCl: 0.2 M, prot_conc:0.2 microM, buffers:Tris-phosphate: 50 mM ; Experimental Assay: dG_H2O pH:7.5, prot_conc:0.2 microM, ionic:NaCl: 0.2 M, buffers:Tris-phosphate: 50 mM ; Experimental Assay: Cm pH:7.5, ionic:NaCl: 0.2 M, prot_conc:1 microM, buffers:Tris-phosphate: 50 mM ; Experimental Assay: m pH:7.5, ionic:NaCl: 0.2 M, prot_conc:1 microM, buffers:Tris-phosphate: 50 mM ; Experimental Assay: dG_H2O pH:7.5, ionic:NaCl: 0.2 M, prot_conc:1 microM, buffers:Tris-phosphate: 50 mM ; Experimental Assay: Cm pH:7.5, prot_conc:5 microM, ionic:NaCl: 0.2 M, buffers:Tris-phosphate: 50 mM ; Experimental Assay: m pH:7.5, prot_conc:5 microM, ionic:NaCl: 0.2 M, buffers:Tris-phosphate: 50 mM ; Experimental Assay: dG_H2O pH:7.5, prot_conc:5 microM, ionic:NaCl: 0.2 M, buffers:Tris-phosphate: 50 mM ; Experimental Assay: Cm pH:7.5, prot_conc:20 microM, ionic:NaCl: 0.2 M, buffers:Tris-phosphate: 50 mM ; Experimental Assay: m pH:7.5, prot_conc:20 microM, ionic:NaCl: 0.2 M, buffers:Tris-phosphate: 50 mM ; Experimental Assay: dG_H2O pH:7.5, prot_conc:20 microM, ionic:NaCl: 0.2 M, buffers:Tris-phosphate: 50 mM ; Experimental Assay: Cm pH:7.5, ionic:NaCl: 0.2 M, prot_conc:1 microM, buffers:Tris-HCl: 50 mM ; Experimental Assay: m pH:7.5, ionic:NaCl: 0.2 M, prot_conc:1 microM, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O pH:7.5, ionic:NaCl: 0.2 M, prot_conc:1 microM, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm pH:7.5, prot_conc:3.5 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: m pH:7.5, prot_conc:3.5 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O pH:7.5, prot_conc:3.5 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm pH:7.5, prot_conc:5 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: m pH:7.5, prot_conc:5 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O pH:7.5, prot_conc:5 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm pH:7.5, prot_conc:20 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: m pH:7.5, prot_conc:20 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: dG_H2O pH:7.5, prot_conc:20 microM, ionic:NaCl: 0.2 M, buffers:Tris-HCl: 50 mM ; Experimental Assay: Cm prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: m prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: Cm ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, prot_conc:1 microM, pH:7.0 ; Experimental Assay: m ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, prot_conc:1 microM, pH:7.0 ; Experimental Assay: dG_H2O ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, prot_conc:1 microM, pH:7.0 ; Experimental Assay: Cm prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: m prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: Cm prot_conc:8 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: m prot_conc:8 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:8 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: Cm prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: m prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: dG_H2O prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:7.0 ; Experimental Assay: Cm pH:7.5, prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM ; Experimental Assay: m pH:7.5, prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM ; Experimental Assay: dG_H2O pH:7.5, prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM ; Experimental Assay: Cm pH:7.5, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, prot_conc:1 microM ; Experimental Assay: m pH:7.5, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, prot_conc:1 microM ; Experimental Assay: dG_H2O pH:7.5, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, prot_conc:1 microM ; Experimental Assay: Cm pH:7.5, prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM ; Experimental Assay: m pH:7.5, prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM ; Experimental Assay: dG_H2O pH:7.5, prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM ; Experimental Assay: Cm pH:7.5, prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM ; Experimental Assay: m pH:7.5, prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM ; Experimental Assay: dG_H2O pH:7.5, prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM ; Experimental Assay: Cm prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: m prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: dG_H2O prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: Cm ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, prot_conc:1 microM, pH:8.0 ; Experimental Assay: m ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, prot_conc:1 microM, pH:8.0 ; Experimental Assay: dG_H2O ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, prot_conc:1 microM, pH:8.0 ; Experimental Assay: Cm prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: m prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: dG_H2O prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: Cm prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: m prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: dG_H2O prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: Cm prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: m prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: dG_H2O prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.0 ; Experimental Assay: Cm prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5 ; Experimental Assay: m prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5 ; Experimental Assay: dG_H2O prot_conc:0.2 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5 ; Experimental Assay: Cm ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5, prot_conc:1 microM ; Experimental Assay: m ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5, prot_conc:1 microM ; Experimental Assay: dG_H2O ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5, prot_conc:1 microM ; Experimental Assay: Cm prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5 ; Experimental Assay: m prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5 ; Experimental Assay: dG_H2O prot_conc:5 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5 ; Experimental Assay: Cm prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5 ; Experimental Assay: m prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5 ; Experimental Assay: dG_H2O prot_conc:20 microM, ionic:NaCl: 0.15 M, buffers:Na2HPO4-KH2PO4: 50 mM, pH:8.5
Libraries Mutations for sequence MSHVEYSRITKFFQEQPLEGYTLFSHRSAPNGFKVAIVLSELGFHYNTIFLDFNLGEHRAPEFVSVNPNARVPALIDHGMDNLSIWESGAILLHLVNKYYKETGNPLLWSDDLADQSQINAWLFFQTSGHAPMIGQALHFRYFHSQKIASAVERYTDEVRRVYGVVEMALAERREALVMELDTENAAAYSAGTTPMSQSRFFDYPVWLVGDKLTIADLAFVPWNNVVDRIGINIKIEFPEVYKWTKHMMRRPAVIKALRGE
Sequence Assay Result Units