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

doi:10.1016/S0022-2836(03)00249-3

Journal of Molecular Biology

Volume 328, Issue 1, 18 April 2003, Pages 235-254

https://doi.org/10.1016/S0022-2836(03)00249-3 Get rights and content

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.

Introduction

Ure2p is termed a yeast prion¹ by analogy to the mammalian prion protein,² due to its ability to convey a heritable phenotype change by undergoing a structural change at the protein level to an aggregated form.³ The prion function of Ure2 is determined by the prion domain (PrD), which consists of the N-terminal approximately 80 amino acid residues and contains a high proportion of asparagine and glutamine.3., 4. The PrD also conveys the ability to form amyloid in vitro.⁵ The “normal” function of Ure2 in vivo is regulation of nitrogen metabolism⁶ and this activity is carried out in the C-terminal region of the protein.³ The C-terminal region has been crystallised7., 8. confirming the homology between Ure2 and the glutathione S-transferase (GST) family of enzymes.⁹ Ure2 binds glutathione (GSH),¹⁰ but does not show classic GST activity.⁹ Like all known GSTs, Ure2 is a dimer in solution.5., 11. Removal of all or parts of the PrD has no detectable effect on the oligomeric state, thermodynamic stability, kinetics of folding or folding pathway of Ure2 over a wide range of conditions.11., 12., 13., 14.

Models for the role of intermediates in Ure2 amyloid formation have recently been proposed,14., 15. but the molecular mechanism of assembly is not yet understood. Further characterisation of the kinetics and thermodynamics of Ure2 folding and fibril formation is required. Ure2 folds via a dimeric intermediate and there is evidence for kinetic partitioning between correct folding (via the dimeric intermediate) and aggregation (via a monomeric intermediate).11., 14. To date, quantitative thermodynamic or kinetic data have only been measured in Tris buffer at pH 8.4.11., 12., 14. Lowering the pH reduces the solubility of Ure2¹¹ and increases amorphous aggregation.¹³ However, lowered pH has also been suggested to favour in vitro fibril formation and to induce a change in the denaturation profile from two-state to three-state due to population of an intermediate.¹³ Therefore analysis of the effect of pH on the folding behaviour of Ure2 is an important step towards understanding the mechanism of prion and amyloid formation.

The purposes of this study are threefold: (1) to present a quantitative analysis of the effect of pH on stability and folding of Ure2, so that the role of intermediates in amyloid formation can be assessed. (2) To investigate the switch between two-state and three-state unfolding and to determine the basis for this change in mechanism. (3) To investigate the mechanism of amyloid formation by relating the above findings to the effects of pH, buffer, protein concentration and temperature on the rate and quality of amyloid formation.

Section snippets

Results

The equilibrium unfolding of Ure2 was investigated by following the changes in intrinsic tryptophan fluorescence, far-UV CD at 222 nm and 1-anilino-naphthalene-8-sulphonate (ANS) binding fluorescence. The conditions sampled here are pH 7.0–8.5, a 100-fold range of protein concentrations (0.2–20 μM of monomer), at a constant temperature of 25 °C. Two different buffer systems were examined: 50 mM Na₂HPO₄–KH₂PO₄ containing 0.15 M NaCl (referred to as phosphate) and 50 mM Tris–HCl containing 0.2 M NaCl

Ure2 stability and folding

Understanding the stability and folding behaviour of the prion proteins is a prerequisite to elucidating the molecular mechanism of prion formation. Identification and characterisation of their folding intermediates is a crucial step in evaluating plausible mechanisms for prion conversion and propagation. However, the prion proteins, by definition, have a tendency to misfold and aggregate, making them challenging candidates for rigorous protein folding studies. Thermodynamic parameters measured

Materials

Ultrapure GdmCl was obtained from ICN Biochemicals. ANS, Tris and thioflavin T were from Sigma. All other reagents were local products of analytical grade. Twice-deionised water was used throughout. Solutions were made volumetrically.

Protein production and purification

Ure2 and N-terminal variants, 90Ure2 and Δ15–42Ure2, were produced in E. coli and purified as described:¹¹ 90Ure2 lacks the glutamine/asparagine-rich region from residues 1–89 and therefore lacks the entire PrD as defined either by biological activity⁴¹ or

Acknowledgements

We thank Dr L.S. Itzhaki and Professor X.M. Pan for helpful discussions. This work was supported by the National Natural Science Foundation of China (30070163) and the 973 project of the Chinese Ministry of Science and Technology (G1999075608). S.P acknowledges support from the Chinese Academy of Sciences, the Royal Commission for the Exhibition of 1851 and the Royal Society.

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Journal of Molecular Biology

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

Introduction

Section snippets

Results

Ure2 stability and folding

Materials

Protein production and purification

Acknowledgements

Structure

J. Mol. Biol.

Biochem. Biophys. Res. Commun.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Curr. Opin. Struct. Biol.

J. Biol. Chem.

Cell

Methods Enzymol.

J. Biol. Chem.

Curr. Opin. Struct. Biol.

J. Biol. Chem.

Advan. Protein Chem.

URE3. as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae

Science

Prions

Proc. Natl Acad. Sci. USA

Prion-inducing domain of yeast Ure2p and protease resistance of Ure2p in prion-containing cells

Science

The prion model for URE3. of yeast: spontaneous generation and requirements for propagation

Proc. Natl Acad. Sci. USA

Prion domain initiation of amyloid formation in vitro from native Ure2p

Science

Non-mendelian mutation allowing ureidosuccinic acid uptake in yeast

J. Bacteriol.

The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

Proc. Natl Acad. Sci. USA

The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to the nitrogen source and has homology to glutathione S-transferases

Mol. Cell. Biol.

Crystal structures of the yeast prion Ure2p functional region in complex with glutathione and related compounds

Biochemistry