Presence Of GdnHCl Research Articles

Biochemical Characterization and Structural Modeling of Fused Glucose-6-Phosphate Dehydrogenase-Phosphogluconolactonase from Giardia lamblia.

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway and is highly relevant in the metabolism of Giardia lamblia. Previous reports suggested that the G6PD gene is fused with the 6-phosphogluconolactonase (6PGL) gene (6pgl). Therefore, in this work, we decided to characterize the fused G6PD-6PGL protein in Giardia lamblia. First, the gene of g6pd fused with the 6pgl gene (6gpd::6pgl) was isolated from trophozoites of Giardia lamblia and the corresponding G6PD::6PGL protein was overexpressed and purified in Escherichia coli. Then, we characterized the native oligomeric state of the G6PD::6PGL protein in solution and we found a catalytic dimer with an optimum pH of 8.75. Furthermore, we determined the steady-state kinetic parameters for the G6PD domain and measured the thermal stability of the protein in both the presence and absence of guanidine hydrochloride (Gdn-HCl) and observed that the G6PD::6PGL protein showed alterations in the stability, secondary structure, and tertiary structure in the presence of Gdn-HCl. Finally, computer modeling studies revealed unique structural and functional features, which clearly established the differences between G6PD::6PGL protein from G. lamblia and the human G6PD enzyme, proving that the model can be used for the design of new drugs with antigiardiasic activity. These results broaden the perspective for future studies of the function of the protein and its effect on the metabolism of this parasite as a potential pharmacological target.

Exploring the role of L209 residue in the active site of NDM-1 a metallo-β-lactamase.

BackgroundNew Delhi Metallo-β-Lactamase (NDM-1) is one of the most recent additions to the β-lactamases family. Since its discovery in 2009, NDM-1 producing Enterobacteriaceae have disseminated globally. With few effective antibiotics against NDM-1 producers, there is an urgent need to design new drug inhibitors through the help of structural and mechanistic information available from mutagenic studies.Results/ConclusionsIn our study we focus the attention on the non-catalytic residue Leucine 209 by changing it into a Phenylalanine. The L209F laboratory variant of NDM-1 displays a drastic reduction of catalytic efficiency (due to low kcat values) towards penicillins, cephalosporins and carbapenems. Thermofluor-based assay demonstrated that NDM-1 and L209F are stable to the temperature and the zinc content is the same in both enzymes as demonstrated by experiments with PAR in the presence of GdnHCL. Molecular Dynamics (MDs) simulations, carried out on NDM-1 and L209F both complexed and uncomplexed with Benzylpenicillin indicate that the point mutation produces a significant mechanical destabilization of the enzyme and also an increase of water content. These observations clearly show that the single mutation induces drastic changes in the enzyme properties which can be related to the observed different catalytic behavior.

Denaturation induced aggregation in α-crystallin: differential action of chaotropes.

α-Crystallin is a member of small heat shock proteins and is believed to play an exceptional role in the stability of eye lens proteins. The disruption or denaturation of the protein arrangement or solubility of the crystallin proteins can lead to vision problems including cataract. In the present study, we have examined the effect of chemical denaturants urea and guanidine hydrochloride (GdnHCl) on α-crystallin aggregation, with special emphasis on protein conformational changes, unfolding, and amyloid fibril formation. GdnHCl (4 M) induced a 16 nm red shift in the intrinsic fluorescence of α-crystallin, compared with 4 nm shift by 8 M urea suggesting a major change in α-crystallin structure. Circular dichroism analysis showed marked increase in the ellipticity of α-crystallin at 216 nm, suggesting gain in β-sheet structure in the presence of GdnHCl (0.5-1 M) followed by unfolding at higher concentration (2-6 M). However, only minor changes in the secondary structure of α-crystallin were observed in the presence of urea. Moreover, 8-anilinonaphthalene-1-sulfonic acid fluorescence measurement in the presence of GdnHCl and urea showed changes in the hydrophobicity of α-crystallin. Amyloid studies using thioflavin T fluorescence and congo red absorbance showed that GdnHCl induced amyloid formation in α-crystallin, whereas urea induced aggregation in this protein. Electron microscopy studies further confirmed amyloid formation of α-crystallin in the presence of GdnHCl, whereas only aggregate-like structures were observed in α-crystallin treated with urea. Our results suggest that α-crystallin is susceptible to unfolding in the presence of chaotropic agents like urea and GdnHCl. The destabilized protein has increased likelihood to fibrillate. Copyright © 2016 John Wiley & Sons, Ltd.

Subtilase from Beauveria sp.: conformational and functional investigation of unusual stability

Retention of total activity of the subtilisin-like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50% methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the protein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chromatography in the presence of GdnHCl (1.0-3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentration of denaturant (1.0-4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k unf of native protein to be 5.407 × 10(-5) s(-1) and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resistant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interesting structural transitions.

Polyproline fold—In imparting kinetic stability to an alkaline serine endopeptidase

Polyproline II (PPII) fold, an unusual structural element was detected in the serine protease from Nocardiopsis sp. NCIM 5124 (NprotI) based on far UV circular dichroism spectrum, structural transitions of the enzyme in presence of GdnHCl and a distinct isodichroic point in chemical and thermal denaturation. The functional activity and conformational transitions of the enzyme were studied under various denaturing conditions. Enzymatic activity of NprotI was stable in the vicinity of GdnHCl upto 6.0M concentration, organic solvents viz. methanol, ethanol, propanol (all 90% v/v), acetonitrile (75% v/v) and proteases such as trypsin, chymotrypsin and proteinase K (NprotI:protease 10:1). NprotI seems to be a kinetically stable protease with a high energy barrier between folded and unfolded states. Also, an enhancement in the activity of the enzyme was observed in 1M GdnHCl upto 8h, in organic solvents (75% v/v) for 72h and in presence of proteolytic enzymes. The polyproline fold remained unaltered or became more prominent under the above mentioned conditions. However, it diminished gradually during thermal denaturation above 60°C. Thermal transition studies by differential scanning calorimetry (DSC) showed scan rate dependence as well as irreversibility of denaturation, the properties characteristic of kinetically stable proteins. This is the first report of PPII helix being the global conformation of a non structural protein, an alkaline serine protease, from a microbial source, imparting kinetic stability to the protein.

Protein-Ligand Interactions of the D-Galactose/D-Glucose-Binding Protein as a Potential Sensing Probe of Glucose Biosensors

In this paper we have studied peculiarities of protein-ligand interaction under different conditions. We have shown that guanidine hydrochloride (GdnHCI) unfolding-refolding of GGBP in the presence of glucose (Glc) is reversible, but the equilibrium curves of complex refolding-unfolding have been attained only after 10-day incubation of GGBP/Glc in the presence of GdnHCl. This effect has not been revealed at heat-induced GGBP/Glc denaturation. Slow equilibration between the native protein in GGBP/Glc complex and the unfolded state of protein in the GdnHCl presence is connected with increased viscosity of solution at moderate and high GdnHCl concentrations which interferes with diffusion of glucose molecules. Thus, the limiting step of the unfolding-refolding process of the complex GGBP/Glc is the disruption/tuning of the configuration fit between the protein in the native state and the ligand.

The effect of zinc oxide nanoparticles on the structure of the periplasmic domain of the Vibrio cholerae ToxR protein

Proteins adsorbed on nanoparticles (NPs) are being used as biosensors and in drug delivery. However, our understanding of the effect of NPs on the structure of proteins is still in a nascent state. In this work we report the unfolding behavior of the periplasmic domain of the ToxR protein (ToxRp) of Vibrio cholerae on zinc oxide (ZnO) nanoparticles with a diameter of 2.5 nm. This protein plays a crucial role in regulating the expression of several virulence factors in the pathogenesis of cholera. Thermodynamic analysis of the equilibrium of unfolding, induced both by urea and by guanidine hydrochloride (GdnHCl), and measured by fluorescence spectroscopy, revealed a two-state process. NPs increased the susceptibility of the protein to denaturation. The midpoints of transitions for the free and the NP-bound ToxRp in the presence of GdnHCl were 1.5 and 0.5 m respectively, whereas for urea denaturation, the values were 3.3 and 2.4 m, respectively. Far-UV CD spectra showed a significant change in the protein conformation upon binding to ZnO NPs, which was characterized by a substantial decrease in the α-helical content of the free protein. Isothermal titration calorimetry, used to quantify the thermodynamics of binding of ToxRp with ZnO NPs, showed an exothermic binding isotherm (ΔH = -9.8 kcal·mol(-1) and ΔS = -5.17 cal·mol(-1)·K(-1)).

Conformational plasticity of DM43, a metalloproteinase inhibitor from Didelphis marsupialis: Chemical and pressure-induced equilibrium (un)folding studies

We have investigated the folding of DM43, a homodimeric metalloproteinase inhibitor isolated from the serum of the South American opossum Didelphis marsupialis. Denaturation of the protein induced by GdnHCl (guanidine hydrochloride) was monitored by extrinsic and intrinsic fluorescence spectroscopy. While the equilibrium (un)folding of DM43 followed by tryptophan fluorescence was well described by a cooperative two-state transition, bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid) fluorescence measurements revealed an intensity maximum at the midpoint of the unfolding transition (2 M GdnHCl), indicating a partially folded intermediate state. We further investigated the DM43 intermediate stabilized at 2 M GdnHCl using size exclusion chromatography. This analysis revealed that the folding intermediate can be best described as partially folded DM43 monomers. Thermodynamic analysis of the GdnHCl-induced denaturation of DM43 revealed Gibbs free-energy changes of 13.57 kcal/mol for dimer dissociation and 1.86 kcal/mol for monomer unfolding, pointing to a critical role of dimerization as a determinant of the structure and stability of this protein. In addition, by using hydrostatic pressure (up to 3.5 kbar) we were able to stabilize partially folded states different from those stabilized in the presence of GdnHCl. Taken together, these results indicate that the conformational plasticity of DM43 could provide this protein with the ability to adapt its conformation to a variety of different environments and biological partners during its biological lifetime.

Mechanism of Unfolding of Goat Lung Cystatin During Urea and Guanidine Hydrochloride Induced Denaturation

Cystatins essentially regulate lysosomal cysteine protease besides affecting several physiological processes. In the present study, denaturation of a high molecular weight cystatin (Mr 66.4 kDa) purified from goat lung (GLC-I) has been studied by monitoring its inhibitory activity, intrinsic fluorescence, circular dichroism (CD), and binding of ANS. It was found that increasing concentration of GdnHCl significantly enhances the inactivation and unfolding of the purified inhibitor (GLC-I) with complete loss of inhibitory activity at 4 M GdnHCl. Denaturation of GLC-I in the presence of GdnHCl is accompanied by red shift (15 nm) of the emission maximum as shown by intrinsic fluorescence. The inhibitory activity of GLC-I was increased by 1.5 fold at 2 M urea; however, it decreased with further increased of the urea concentration. Intrinsic fluorescence studies of GLC-I in the presence of 0–3 M urea shows blue shift of 5 nm, suggesting stabilization of the inhibitor followed by 5 nm red shift at higher concentration. ANS binding studies in the presence of urea indicate significant changes in the tertiary structure of the inhibitor. Thus, our result shows denaturation profile of GLC-I following simple two state transitions in the presence of GdnHCl while it proceeds through an intermediate state in the presence of urea.

Catalysis by Triton X-100 micelles at neutral pH

Hydrolysis of nitrophenyl esters in micelles has served as an excellent model for studies on micellar catalysis. Non-ionic micelles do not support ester hydrolysis due to the lack of charged species in the micelles. p-Nitrophenyloleate dissolved in Triton X-100 micelles does not undergo hydrolysis at pH 7.2; however, the hydrolysis increases with increasing concentration of guanidinium chloride (GdnHCl) up to 6M. Such hydrolysis was not observed in the presence of urea (8M). The chaotropes, GdnHCl and urea, destabilize the micelles by increasing the CMC. The decrease in micelle radius, measured by dynamic light scatter, and increase in polarity inside the palisade layer, measured by emission of 2-anthroyloxy fatty acids, was significant in GdnHCl but not in urea. Since hydrolysis was observed exclusively in the presence of GdnHCl, we have investigated the interaction of guanidinium ion with Triton X-100 micelle by using the chemical shift analysis of protons in ethylene oxide (PEO) using 1H NMR. A significant shift in the resonance frequency of the protons of the polyethylene oxide of Triton X-100 in GdnHCl, and not in urea, shows a hydrogen bonding between guanidinium ion and the PEO chains. The altered partitioning of the counter ion, hydroxyl, inside the micelles in the presence of GdnHCl was suggested to be responsible for enhanced hydrolysis of PNPO in Triton X-100. Such behavior of a neutral micelle, Triton X-100, as functional micelles was not demonstrated at neutral pHs earlier and this phenomenon is discussed with respect to the binding ability of PEO chains with cations.

Hydrophobic interactions and ionic networks play an important role in thermal stability and denaturation mechanism of the porcine odorant‐binding protein

Despite the fact that the porcine odorant-binding protein (pOBP) possesses a single tryptophan residue (Trp 16) that is characterized by a high density microenvironment (80 atoms in a sphere with radius 7 A) with only one polar group (Lys 120) and three bound water molecules, pOBP displayed a red shifted fluorescence emission spectrum (lambda(max) = 340 nm). The protein unfolding in 5M GdnHCl was accompanied by the red shift of the fluorescence emission spectrum (lambda(max) = 353 nm), by the increase of fluorescence quantum yield, and by the decrease of lifetime of the excited state (from 4.25 ns in native state to 3.15 ns in the presence of 5M GdnHCl). Taken together these data indicate the existence of an exciplex complex (Trp 16 with Lys 120 and/or with bound molecules of water) in the protein native state. Heat-induced denaturation of pOBP resulted in significant red shifts of the fluorescence emission spectra: the value of the ratio (I(320)/I(365)) upon excitation at lambda(ex) = 297 nm (parameter A) decreases from 1.07 to 0.64 passing from 60 to 85 degrees C, and the calculated midpoint of transition was centered at 70 degrees C. Interestingly, even at higher temperature, the values of the parameter A both in the absence and in the presence of GdnHCl did not coincide. This suggests that a portion of the protein structure is still preserved upon the temperature-induced denaturation of the protein in the absence of GdnHCl. CD experiments performed on pOBP in the absence and in the presence of GdnHCl and at different temperatures were in agreement with the fluorescence results. In addition, the obtained experimental data were corroborated by the analysis of the 3D structure of pOBP which revealed the amino acid residues that contribute to the protein dynamics and stability. Finally, molecular dynamics simulation experiments pointed out the important role of ion pair interactions as well as the molecular motifs that are responsible for the high thermal stability of pOBP, and elucidated the reasons of the protein aggregation that occurred at high temperature.

Pressure and Denaturants in the Unfolding of Triosephosphate Isomerase: The Monomeric Intermediates of the Enzymes from Saccharomyces cerevisiae and Entamoeba histolytica

Triosephosphate isomerase (TIM) is a dimeric enzyme formed by two identical (beta/alpha)8 barrels. In this work, we compare the unfolding and refolding of the TIMs from Entamoeba histolytica (EhTIM) and baker's yeast (yTIM). A monomeric intermediate was detected in the GdnHCl-induced unfolding of EhTIM. The thermodynamic, spectroscopic, catalytic, and hydrodynamic properties of this intermediate were found to be very similar to those previously described for a monomeric intermediate of yTIM observed in GdnHCl. Monomer unfolding was reversible for both TIMs; however, the dissociation step was reversible in yTIM and irreversible in EhTIM. Monomer unfolding induced by high pressure in the presence of GdnHCl was a reversible process. DeltaGUnf, DeltaVUnf, and P1/2 were obtained for the 0.7-1.2 M GdnHCl range. The linear extrapolation of these thermodynamic parameters to the absence of denaturant showed the same values for both intermediates. The DeltaVUnfH2O values calculated for EhTIM and yTIM monomeric intermediates are the same within experimental error (-57 +/- 10 and -76 +/- 14 mL/mol, respectively). These DeltaVUnf H2O values are smaller than those reported for the unfolding of monomeric proteins of similar size, suggesting that TIM intermediates are only partially hydrated. |DeltaVUnf| increased with denaturant concentration; this behavior is probably related to structural changes in the unfolded state induced by GdnHCl and pressure. From the thermodynamic parameters that were obtained, it is predicted that in the absence of denaturants, pressure would induce monomer unfolding (P1/2 approximately 140 MPa) prior to dimer dissociation (P1/2 approximately 580 MPa). Therefore, dimerization prevents the pressure unfolding of the monomer.

Annealing Prion Protein Amyloid Fibrils at High Temperature Results in Extension of a Proteinase K-resistant Core

Amyloids are highly ordered, rigid beta-sheet-rich structures that appear to have minimal dynamic flexibility in individual polypeptide chains. Here, we demonstrate that substantial conformational rearrangements occur within mature amyloid fibrils produced from full-length mammalian prion protein. The rearrangement results in a substantial extension of a proteinase K-resistant core and is accompanied by an increase in the beta-sheet-rich conformation. The conformational rearrangement was induced in the presence of low concentrations of Triton X-100 either by brief exposure to 80 degrees C or, with less efficacy, by prolonged incubation at 37 degrees C at pH 7.5 and is referred to here as "annealing." Upon annealing, amyloid fibrils acquired a proteinase K-resistant core identical to that found in bovine spongiform encephalopathy-specific scrapie-associated prion protein. Annealing was also observed when amyloid fibrils were exposed to high temperatures in the absence of detergent but in the presence of brain homogenate. These findings suggest that the amyloid fibrils exist in two conformationally distinct states that are separated by a high energy barrier and that yet unknown cellular cofactors may facilitate transition of the fibrils into thermodynamically more stable state. Our studies provide new insight into the complex behavior of prion polymerization and highlight the annealing process, a previously unknown step in the evolution of amyloid structures.

Δ98Δ, a Functional All-β-Sheet Abridged Form of Intestinal Fatty Acid Binding Protein

Intestinal fatty acid binding protein (IFABP) is a 15 kDa intracellular lipid-binding protein exhibiting a beta-barrel fold that resembles a clamshell. The beta-barrel, which encloses the ligand binding cavity, consists of two perpendicular five-stranded beta-sheets with an intervening helix-turn-helix motif between strands A and B. Delta98delta (fragment 29-126 of IFABP) was obtained either in its recombinant form or by limited proteolysis with clostripain. Despite lacking extensive stretches involved in the closure of the beta-barrel, delta98delta remains soluble and stable in solution. Spectroscopic analyses by circular dichroism, ultraviolet absorption, and intrinsic fluorescence indicate that the fragment retains substantial beta-sheet content and tertiary interactions. In particular, the environment around W82 is identical in both delta98delta and IFABP, a fact consistent with the conservation in the former of all the critical amino acid residues belonging to the hydrophobic core. In addition, the Stokes radius of delta98delta is similar to that of IFABP and 16% larger than that calculated from its molecular weight (11 kDa). The monomeric status of delta98delta was further confirmed by chemical cross-linking experiments. Although lacking 25% of the amino acids of the parent protein, in the presence of GdnHCl, delta98delta unfolds through a cooperative transition showing a midpoint at 0.90 M. Remarkably, it also preserves binding activity for fatty acids (Kd = 5.1 microM for oleic acid and Kd = 0.72 microM for trans-parinaric acid), a fact that exerts a stabilizing effect on its structure. These cumulative evidences show that delta98delta adopts a monomeric state with a compact core and a loose periphery, being so far the smallest structure of its kind preserving binding function.

The Native Energy Landscape for Interleukin-1β. Modulation of the Population Ensemble through Native-state Topology

A minimalist Go-model, with no energetic frustration in the native conformation, has been shown to describe accurately the folding pathway of the beta-trefoil protein, interleukin-1beta (IL-1beta). While it appears that these models successfully model transition states and intermediates between the unfolded and native ensembles, it is unclear how accurately they capture smaller, yet biologically relevant, structural changes within the native ensemble after energetic perturbation. Here, we address the following questions. Can a simple Go-model of interleukin-1beta, based on native topology, describe changes in structural properties of the native ensemble as the protein stability is changed? Or is it necessary to include a more explicit representation of atoms, electrostatic, hydrogen bonding, and van der Waals forces to describe these changes? The native ensemble of IL-1beta was characterized using a variety of experimental probes under native (0 M NaCl, guanidine hydrochloride (Gdn-HCl)), moderately destabilized (0 M NaCl, 0.8 M Gdn-HCl), and in moderate salt concentration (0.8 M NaCl, 0 M Gdn-HCl). Heteronuclear (1)H-(15)N nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) NMR spectra confirmed that the beta-trefoil global fold was largely intact under these three conditions. However, 25 of the 153 residues throughout the chain did demonstrate (13)C and (1)H-(15)N chemical shifts when perturbed with 0.8 M NaCl or Gdn-HCl. Despite large differences in protection factors from solvent hydrogen-deuterium exchange for all residues between stable (0 M Gdn-HCl) and destabilized (0.8 M Gdn-HCl) IL-1beta, no difference in steady-state (15)N-(1)H NOE enhancements were measured. Thus, the chemical shifts correlate with a global but limited increase in residue flexibility in the presence of Gdn-HCl. Minimalist simulations highlight the regions of greatest position shift between native and 0.8 M Gdn-HCl, which were determined experimentally. This correlation demonstrates that structural changes within the native ensemble of IL-1beta are, at least partially, governed by the principle of minimal energetic frustration.

Effect of the polypeptide binding on the thermodynamic stability of the substrate binding domain of the DnaK chaperone

The effect of polypeptide binding on the stability of the substrate binding domain of the molecular chaperone DnaK has been studied by thermodynamic analysis. The calorimetric scan of the fragment of the substrate binding domain DnaK384–638, consisting of a β-domain and an α-helical lid, showed two transitions centered at 56.2 and 76.0 °C. On the other hand, the thermal unfolding of the shorter fragment DnaK386–561, which lacks half of the α-helical lid, exhibited a single transition at 57.0 °C. Therefore, the transition of DnaK384–638 at 56.2 °C is mainly attributed to the unfolding of the β-domain. The calorimetric scan of DnaK384–638D526N showed that the unfolding of the β-domain was composed of two transitions. The polypeptide bound DnaK384–638 exhibited a symmetrical DSC peak at 58.6 °C, indicating that the substrate binding shifts the β-domain toward a single cooperative unit. A low concentration of GdnHCl (<1.0 M) induced a conformational change in the β-domain of DnaK384–638 without changes in the secondary structure. While the thermal unfolding of the β-domain of DnaK384–638 was composed of two transitions in the presence of GdnHCl, the β-domain of the substrate bound DnaK384–638 exhibited a single symmetrical DSC peak in the same condition. All together, our results indicate that complex between DnaK384–638 and substrate forms a rigid conformation in the β-domain.

Predissociated Dimers and Molten Globule Monomers in the Equilibrium Unfolding of Yeast Glutathione Reductase

The equilibrium unfolding of dimeric yeast glutathione reductase (GR) by guanidine hydrochloride (GdnHCl) was investigated. Unfolding was monitored by a variety of techniques, including intrinsic fluorescence emission, anisotropy and iodide quenching measurements, far-ultraviolet circular dichroism and thiol reactivity measurements. At 1 M GdnHCl, one thiol group of GR became accessible to modification with 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB), whereas no changes could be detected in the spectroscopic properties (fluorescence, circular dichroism) of the protein. Between 2 and 3 M GdnHCl, two partially folded intermediate states possessing flexible tertiary structures (revealed by fluorescence data) but compact secondary structures (as indicated by circular dichroism measurements) were identified. The quaternary structure of GR in the presence of GdnHCl was also investigated by size-exclusion liquid chromatography. These results indicated the presence of an expanded predissociated dimer at 2.5 M GdnHCl and partially folded monomers at 3 M GdnHCl. Taken together, these results suggest the existence of two molten-globule-like intermediate species (one dimeric and one monomeric) in the unfolding of GR. The results are discussed in terms of the mechanism of GR folding and dimerization.

Guanidine hydrochloride inhibits the generation of prion "seeds" but not prion protein aggregation in yeast.

[PSI(+)] strains of the yeast Saccharomyces cerevisiae replicate and transmit the prion form of the Sup35p protein but can be permanently cured of this property when grown in millimolar concentrations of guanidine hydrochloride (GdnHCl). GdnHCl treatment leads to the inhibition of the replication of the [PSI(+)] seeds necessary for continued [PSI(+)] propagation. Here we demonstrate that the rate of incorporation of newly synthesized Sup35p into the high-molecular-weight aggregates, diagnostic of [PSI(+)] strains, is proportional to the number of seeds in the cell, with seed number declining (and the levels of soluble Sup35p increasing) in the presence of GdnHCl. GdnHCl does not cause breakdown of preexisting Sup35p aggregates in [PSI(+)] cells. Transfer of GdnHCl-treated cells to GdnHCl-free medium reverses GdnHCl inhibition of [PSI(+)] seed replication and allows new prion seeds to be generated exponentially in the absence of ongoing protein synthesis. Following such release the [PSI(+)] seed numbers double every 20 to 22 min. Recent evidence (P. C. Ferreira, F. Ness, S. R. Edwards, B. S. Cox, and M. F. Tuite, Mol. Microbiol. 40:1357-1369, 2001; G. Jung and D. C. Masison, Curr. Microbiol. 43:7-10, 2001), together with data presented here, suggests that curing yeast prions by GdnHCl is a consequence of GdnHCl inhibition of the activity of molecular chaperone Hsp104, which in turn is essential for [PSI(+)] propagation. The kinetics of elimination of [PSI(+)] by coexpression of a dominant, ATPase-negative allele of HSP104 were similar to those observed for GdnHCl-induced elimination. Based on these and other data, we propose a two-cycle model for "prionization" of Sup35p in [PSI(+)] cells: cycle A is the GdnHCl-sensitive (Hsp104-dependent) replication of the prion seeds, while cycle B is a GdnHCl-insensitive (Hsp104-independent) process that converts these seeds to pelletable aggregates.

The elimination of the yeast [PSI+] prion by guanidine hydrochloride is the result of Hsp104 inactivation.

In the yeast Saccharomyces cerevisiae, Sup35p (eRF3), a subunit of the translation termination complex, can take up a prion-like, self-propagating conformation giving rise to the non-Mendelian [PSI+] determinant. The replication of [PSI+] prion seeds can be readily blocked by growth in the presence of low concentrations of guanidine hydrochloride (GdnHCl), leading to the generation of prion-free [psi-] cells. Here, we provide evidence that GdnHCl blocks seed replication in vivo by inactivation of the molecular chaperone Hsp104. Although growth in the presence of GdnHCl causes a modest increase in HSP104 expression (20-90%), this is not sufficient to explain prion curing. Rather, we show that GdnHCl inhibits two different Hsp104-dependent cellular processes, namely the acquisition of thermotolerance and the refolding of thermally denatured luciferase. The inhibitory effects of GdnHCl protein refolding are partially suppressed by elevating the endogenous cellular levels of Hsp104 using a constitutive promoter. The kinetics of GdnHCl-induced [PSI+] curing could be mimicked by co-expression of an ATPase-negative dominant HSP104 mutant in an otherwise wild-type [PSI+] strain. We suggest that GdnHCl inactivates the ATPase activity of Hsp104, leading to a block in the replication of [PSI+] seeds.

Guanidine hydrochloride inhibits Hsp104 activity in vivo: a possible explanation for its effect in curing yeast prions.

The presence of millimolar concentrations of guanidine hydrochloride (Gdn-HCl) in growth media causes efficient loss of the normally stable [PSI+] element from yeast cells. Although it has become common practice to include 5 mm Gdn-HCl in growth media to cure [PSI+] and other prions of yeast, the biochemical mechanism by which it cures is unknown. We find that 5 mm Gdn-HCl significantly reduces Hsp104-mediated basal and acquired thermotolerance. Gdn-HCl also reduced the ability of Hsp104 to restore activity of thermally denatured luciferase in vivo. The abundance of Hsp104 was not reduced in cells grown in the presence of Gdn-HCl, ruling out negative effects on expression or stability of Hsp104. We therefore conclude that Gdn-HCl inhibits Hsp104 activity in vivo. Since replication of yeast prions is dependent on Hsp104, our results suggest that Gdn-HCl cures prions by inhibiting Hsp104 activity.