Structure and assembly properties of the yeast prion Ure2p

The non-Mendelian phenotype [URE3] is due to a transmissible conformational change of the protein Ure2. The infectious protein form of Ure2p has lost its function and gained the capacity to transform the active form of the protein into an inactive form. The molecular basis of this conversion process is unknown. There are however indications that the conformational changes at the origin of the propagation of the inactive form of Ure2p in yeast cells are similar to those at the origin of the transition of PrPC into the scrapie-associated PrPSc form of the protein. To better understand the nature of the conformational changes at the origin of prion propagation, we have purified, characterized biochemically, examined the assembly properties and solved the crystal structure of Ure2p. Our data are presented below and a number of conclusions dealing with the molecular basis of the conversion of soluble Ure2p into its amyloid-forming state are derived.     

The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein

The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high beta-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, beta-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p.     

Small angle X-ray scattering study of the yeast prion Ure2p

The GdmCl-induced equilibrium unfolding and dissociation of the dimeric yeast prion protein Ure2, and its prion domain deletion mutants Delta 15-42Ure2 and 90Ure2, was studied by small angle X-ray scattering (SAXS) using synchrotron radiation and by chemical cross-linking with dithiobis(succinimidyl propionate) (DTSP). The native state is globular and predominantly dimeric prior to the onset of unfolding. R(g) values of 32 and 45A were obtained for the native and 5M GdmCl denatured states of Delta 15-42Ure2, respectively; the corresponding values for 90Ure2 were 2-3A lower. SAXS suggests residual structure in the 4M GdmCl denatured state and chemical cross-linking detects persistence of dimeric structure under these conditions. Hexamers consisting of globular subunits could be detected by SAXS at high protein concentration under partially denaturing conditions. The increased tendency of partially folded states to form small oligomers points to a mechanism for prion formation.     

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.     

Equilibrium folding properties of the yeast prion protein determinant Ure2.

The yeast non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The [URE3] phenotype is equivalent to loss of function of Ure2, a protein involved in regulation of nitrogen metabolism. The prion-like behaviour of Ure2 in vivo is dependent on the first 65 amino acid residues of its N-terminal region which contains a highly repetitive sequence rich in asparagine. This region has been termed the prion-determining domain (PrD). Removal of as little as residues 2-20 of the protein is sufficient to prevent occurrence of the [URE3] phenotype. Removal of the PrD does not affect the regulatory activity of Ure2. The C-terminal portion of the protein has homology to glutathione S -transferases, which are dimeric proteins. We have produced the Ure2 protein to high yield in Escherichia coli from a synthetic gene. The recombinant purified protein is shown to be a dimer. The stability, folding and oligomeric state of Ure2 and a series of N-terminally truncated or deleted variants were studied and compared. The stability of Ure2, DeltaGD-N, H2O, determined by chemical denaturation and monitored by fluorescence, is 12.1(+/-0.4) kcal mol-1at 25 degrees C and pH 8.4. A range of structural probes show a single, coincident unfolding transition, which is invariant over a 550-fold change in protein concentration. The stability is the same within error for Ure2 variants lacking all or part of the prion-determining domain. The data indicate that in the folded protein the PrD is in an unstructured conformation and does not form specific intra- or intermolecular interactions at micromolar protein concentrations. This suggests that the C-terminal domain may stabilise the PrD against prion formation by steric means, and implies that the PrD does not induce prion formation by altering the thermodynamic stability of the folded protein.     

Kinetic evidence of an on-pathway intermediate in the folding of lysozyme

By means of a kinetic test, it was demonstrated that one of the folding intermediates (Ialpha) of hen lysozyme with alpha-domain folded and beta-domain unfolded is on the folding pathway under the classical definition. Ialpha folds to the native (N) state directly (unfolded (U) <==> Ialpha <==> N) without having to unfold to U and then refold to N through alternative folding pathways as in Ialpha <==> U <==> N.     

The yeast prion protein Ure2: structure, function and folding

The Saccharomyces cerevisiae protein Ure2 functions as a regulator of nitrogen metabolism and as a glutathione-dependent peroxidase. Ure2 also has the characteristics of a prion, in that it can undergo a heritable conformational change to an aggregated state; the prion form of Ure2 loses the regulatory function, but the enzymatic function appears to be maintained. A number of factors are found to affect the prion properties of Ure2, including mutation and expression levels of molecular chaperones, and the effect of these factors on structure and stability are being investigated. The relationship between structure, function and folding for the yeast prion Ure2 are discussed.     

Protection of yeast lacking the Ure2 protein against the toxicity of heavy metals and hydroperoxides by antioxidants

The aim of this study was to examine the protection of the yeast lacking the “antioxidant-like” prion precursor protein (Ure2p), by antioxidants and to elucidate how modification of redox homeostasis affects toxicity of agents inducing oxidative stress in the Δure2 cells. We found a diverse ability of a range of antioxidants to ameliorate the hypersensitivity of the Δure2 disruptant to oxidants and heavy metal ions. Glutathione and then ascorbate were the most effective antioxidants; Tempol, Trolox and melatonin were much less effective or even hampered the growth of the Δure2 cells exposed to tested agents. The intracellular level of ROS was augmented in the Δure2 mutant under normal growth conditions (1.7-fold), and after treatment with H₂O₂ (2.3-fold) and Cd(II) (2.8-fold), with respect to its wild-type counterpart. Glutathione was unable to prevent the increase in ROS production caused by CdCl₂. The Δure2 disruptant was also hypersensitive to heat shock, like mutants lacking glutathione S-transferases.     

Packing of the prion Ure2p in protein fibrils probed by fluorescence X-ray near-edge structure spectroscopy at sulfur K-edge

The soluble protein Ure2p from the yeast Saccharomyces cerevisiae assembles in vitro into straight and insoluble protein fibrils, through subtle changes of conformation. Whereas the structure of soluble Ure2p has been revealed by X-ray crystallography, further characterization of the structure of insoluble Ure2p fibrils is needed. We performed X-ray absorption near-edge spectroscopy (XANES) at the sulfur K-edge to probe the state of Cys221 in the fibrillar form of Ure2pC221 and provide structural information on the structure of Ure2p within fibrils. Although the Ure2p dimer dissociation into its constituent monomers has proven to be a prerequisite for assembly into fibrils, we showed the ability of every Ure2pC221 monomer to establish disulfide bonds upon incubation of the fibrils under oxidizing conditions. Our result indicates either that the constituent unit of the fibrillar form of the protein is a dimeric Ure2p or that the fibrils are made of protofilaments assembled in such a way that the residue C221 from a Ure2p molecule in one protofilament is located in the vicinity of a C221 residue from another molecule belonging to a neighbor protofilament.     

Protection of yeast lacking the Ure2 protein against the toxicity of heavy metals and hydroperoxides by antioxidants

The aim of this study was to examine the protection of the yeast lacking the “antioxidant-like” prion precursor protein (Ure2p), by antioxidants and to elucidate how modification of redox homeostasis affects toxicity of agents inducing oxidative stress in the Δure2 cells. We found a diverse ability of a range of antioxidants to ameliorate the hypersensitivity of the Δure2 disruptant to oxidants and heavy metal ions. Glutathione and then ascorbate were the most effective antioxidants; Tempol, Trolox and melatonin were much less effective or even hampered the growth of the Δure2 cells exposed to tested agents. The intracellular level of ROS was augmented in the Δure2 mutant under normal growth conditions (1.7-fold), and after treatment with H₂O₂ (2.3-fold) and Cd(II) (2.8-fold), with respect to its wild-type counterpart. Glutathione was unable to prevent the increase in ROS production caused by CdCl₂. The Δure2 disruptant was also hypersensitive to heat shock, like mutants lacking glutathione S-transferases.     

Assembly of the yeast prion Ure2p into protein fibrils. Thermodynamic and kinetic characterization

The [URE3] phenotype in Saccharomyces cerevisiae propagates by a prion mechanism, involving the aggregation of the normally soluble and highly helical protein Ure2. Previous data have shown that the protein spontaneously forms in vitro long, straight, insoluble fibrils at neutral pH that are similar to amyloids in that they bind Congo red and show green-yellow birefringence and have an increased resistance to proteolysis. These fibrils are not amyloids as they are devoid of a cross-beta core. Here we further document the mechanism of assembly of Ure2p into fibrils. The critical concentration for Ure2p assembly is measured, and the minimal size of the nuclei that are the precursors of Ure2p fibrils is determined. Our data indicate that the assembly process is irreversible. As a consequence, the critical concentration is very low. By analyzing the elongation rates of preformed fibrils and combining the results with single-fiber imaging experiments of a variant Ure2p labeled by fluorescent dyes, we reveal the polarity of the fibrils and differences in the elongation rates at their ends. These results bring novel insight in the process of Ure2p assembly into fibrils and the mechanism of propagation of yeast prions.     

Folding barrier in horse cytochrome c: support for a classical folding pathway

Native-state structures and conformations of ferrocytochrome c, nitrosylcytochrome c, and carbonmonoxycytochrome c are very similar. They are, however, immensely different from each other in terms of thermodynamic stability. The dramatic destabilization of ferrocytochrome c to the extent of 12 kcal mol(-1) produces no effect on the folding rate, and this is so in spite of the fact that all three test-tube variants fold in an apparent two-state manner. For all three proteins the folding barrier is early in time, sizable in energy, and is of the same magnitude (approximately 6.5 kcal mol(-1)). These results raise some challenges to the "new view" of protein folding. An early transition state, the search for which consumes most of the observed folding time, is suggested.     

Probing the energy landscape of protein folding/unfolding transition states

Previous molecular dynamics (MD) simulations of the thermal denaturation of chymotrypsin inhibitor 2 (CI2) have provided atomic-resolution models of the transition state ensemble that is well supported by experimental studies. Here, we use simulations to further investigate the energy landscape around the transition state region. Nine structures within approximately 35 ps and 3 A C(alpha) RMSD of the transition state ensemble identified in a previous 498 K thermal denaturation simulation were quenched under the quasi-native conditions of 335 K and neutral pH. All of the structures underwent hydrophobically driven collapse in response to the drop in temperature. Structures less denatured than the transition state became structurally more native-like, while structures that were more denatured than the transition state tended to show additional loss of native structure. The structures in the immediate region of the transition state fluctuated between becoming more and less native-like. All of the starting structures had the same native-like topology and were quite similar (within 3.5 A C(alpha) RMSD). That the structures all shared native-like topology, yet diverged into either more or less native-like structures depending on which side of the transition state they occupied on the unfolding trajectory, indicates that topology alone does not dictate protein folding. Instead, our results suggest that a detailed interplay of packing interactions and interactions with water determine whether a partially denatured protein will become more native-like under refolding conditions.     

Equilibrium amide hydrogen exchange and protein folding kinetics

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (IUN). On the other hand, in an on-pathway three-state system (UIN), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.     

Extremely rapid folding of the C-terminal domain of the prion protein without kinetic intermediates.

The kinetics of folding of mPrP(121-231), the structured 111-residue domain of the murine cellular prion protein PrP(C), were investigated by stopped-flow fluorescence using the variant F175W, which has the same overall structure and stability as wild-type mPrP(121-231) but shows a strong fluorescence change upon unfolding. At 22 degrees C and pH 7.0, folding of mPrP(121-231)-F175W is too fast to be observable by stopped-flow techniques. Folding at 4 degrees C occurs with a deduced half-life of approximately 170 micros without detectable intermediates, possibly the fastest protein-folding reaction known so far. Thus, propagation of the abnormal, oligomeric prion protein PrP(Sc), which is supposed to be the causative agent of transmissible spongiform encephalopathies, is unlikely to follow a mechanism where kinetic folding intermediates of PrP(C) are a source of PrP(Sc) subunits.     

Thermal unfolding molecular dynamics simulation of Escherichia coli dihydrofolate reductase: thermal stability of protein domains and unfolding pathway

Temperature induced unfolding of Escherichia coli dihydrofolate reductase was carried out by using molecular dynamic simulations. The simulations show that the unfolding generally involves an initial end-to-end collapse of the adenine binding domain into partially extended loops, followed by a gradual breakdown of the remaining beta sheet core structure. The core, which consists of beta strands 5-7, was observed to be the most resistant to thermal unfolding. This region, which is made up of part of the N terminus domain and part of the large domain of the E. coli dihydrofolate reductase, may constitute the nucleation site for protein folding and may be important for the eventual formation of both domains. The unfolding of different domains at different stages of the unfolding process suggests that protein domains vary in stability and that the rate at which they unfold can affect the overall outcome of the unfolding pathway. This observation is compared with the recently proposed hierarchical folding model. Finally, the results of the simulation were found to be consistent with a previous experimental study (Frieden, Proc Natl Acad Sci USA 1990;87:4413-4416) which showed that the folding process of E. coli dihydrofolate reductase involves sequential formation of the substrate binding sites.     

Evidence for sequential barriers and obligatory intermediates in apparent two-state protein folding

Many small proteins fold fast and without detectable intermediates. This is frequently taken as evidence against the importance of partially folded states, which often transiently accumulate during folding of larger proteins. To get insight into the properties of free energy barriers in protein folding we analyzed experimental data from 23 proteins that were reported to show non-linear activation free-energy relationships. These non-linearities are generally interpreted in terms of broad transition barrier regions with a large number of energetically similar states. Our results argue against the presence of a single broad barrier region. They rather indicate that the non-linearities are caused by sequential folding pathways with consecutive distinct barriers and a few obligatory high-energy intermediates. In contrast to a broad barrier model the sequential model gives a consistent picture of the folding barriers for different variants of the same protein and when folding of a single protein is analyzed under different solvent conditions. The sequential model is also able to explain changes from linear to non-linear free energy relationships and from apparent two-state folding to folding through populated intermediates upon single point mutations or changes in the experimental conditions. These results suggest that the apparent discrepancy between two-state and multi-state folding originates in the relative stability of the intermediates, which argues for the importance of partially folded states in protein folding.     

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.     

Temperature and pressure denaturation of chignolin: Folding and unfolding simulation by multibaric-multithermal molecular dynamics method

A multibaric‐multithermal molecular dynamics (MD) simulation of a 10‐residue protein, chignolin, was performed. All‐atom model with the Amber parm99SB force field was used for the protein and the TIP3P model was used for the explicit water molecules. This MD simulation covered wide ranges of temperature between 260 and 560 K and pressure between 0.1 and 600 MPa and sampled many conformations without getting trapped in local‐minimum free‐energy states. Folding events to the native β‐hairpin structure occurred five times and unfolding events were observed four times. As the temperature and/or pressure increases, fraction of folded chignolin decreases. The partial molar enthalpy change ΔH and partial molar volume change ΔV of unfolding were calculated as ΔH = 24.1 ± 4.9 kJ/mol and ΔV = ?5.6 ± 1.5 cm³/mol, respectively. These values agree well with recent experimental results. Illustrating typical local‐minimum free‐energy conformations, folding and unfolding pathways were revealed. When chignolin unfolds from the β‐hairpin structure, only the C terminus or both C and N termini open first. It may undergo an α‐helix or 3₁₀‐helix structure and finally unfolds to the extended structure. Difference of the mechanism between temperature denaturation and pressure denaturation is also discussed. Temperature denaturation is caused by making the protein transferred to a higher entropy state and making it move around more with larger space. The reason for pressure denaturation is that water molecules approach the hydrophobic residues, which are not well hydrated at the folded state, and some hydrophobic contacts are broken. Proteins 2012;. © 2012 Wiley Periodicals, Inc.