Aggregation of the Acylphosphatase from Sulfolobus solfataricus: the folded and partially unfolded states can both be precursors for amyloid formation

Protein aggregation is associated with a number of human pathologies including Alzheimer's and Creutzfeldt-Jakob diseases and the systemic amyloidoses. In this study, we used the acylphosphatase from the hyperthermophilic Archaea Sulfolobus solfataricus (Sso AcP) to investigate the mechanism of aggregation under conditions in which the protein maintains a folded structure. In the presence of 15-25% (v/v) trifluoroethanol, Sso AcP was found to form aggregates able to bind specific dyes such as thioflavine T, Congo red, and 1-anilino-8-naphthalenesulfonic acid. The presence of aggregates was confirmed by circular dichroism and dynamic light scattering. Electron microscopy revealed the presence of small aggregates generally referred to as amyloid protofibrils. The monomeric form adopted by Sso AcP prior to aggregation under these conditions retained enzymatic activity; in addition, folding was remarkably faster than unfolding. These observations indicate that Sso AcP adopts a folded, although possibly distorted, conformation prior to aggregation. Most important, aggregation appeared to be 100-fold faster than unfolding under these conditions. Although aggregation of Sso AcP was faster at higher trifluoroethanol concentrations, in which the protein adopted a partially unfolded conformation, these findings suggest that the early events of amyloid fibril formation may involve an aggregation process consisting of the assembly of protein molecules in their folded state. This conclusion has a biological relevance as globular proteins normally spend most of their lifetime in folded structures.     

Mutational analysis of acylphosphatase suggests the importance of topology and contact order in protein folding.

Muscle acylphosphatase (AcP) is a small protein that folds very slowly with two-state behavior. The conformational stability and the rates of folding and unfolding have been determined for a number of mutants of AcP in order to characterize the structure of the folding transition state. The results show that the transition state is an expanded version of the native protein, where most of the native interactions are partially established. The transition state of AcP turns out to be remarkably similar in structure to that of the activation domain of procarboxypeptidase A2 (ADA2h), a protein having the same overall topology but sharing only 13% sequence identity with AcP. This suggests that transition states are conserved between proteins with the same native fold. Comparison of the rates of folding of AcP and four other proteins with the same topology, including ADA2h, supports the concept that the average distance in sequence between interacting residues (that is, the contact order) is an important determinant of the rate of protein folding.     

A model for the aggregation of the acylphosphatase from Sulfolobus solfataricus in its native-like state

Evidence is accumulating that normally folded proteins retain a significant tendency to form amyloid fibrils through a direct assembly of monomers in their native-like conformation. However, the factors promoting such processes are not yet well understood. The acylphosphatase from Sulfolobus solfataricus (Sso AcP) aggregates under conditions in which a native-like state is initially populated and forms, as a first step, aggregates in which the monomers maintain their native-like topology. An unstructured N-terminal segment and an edge beta-strand were previously shown to play a major role in the process. Using kinetic experiments on a set of Sso AcP variants we shall show that the major event of the first step is the establishment of an inter-molecular interaction between the unstructured segment of one Sso AcP molecule and the globular unit of another molecule. This interaction is determined by the primary sequence of the unstructured segment and not by its physico-chemical properties. Moreover, we shall show that the conversion of these initial aggregates into amyloid-like protofibrils is an intra-molecular process in which the Sso AcP molecules undergo conformational modifications. The obtained results allow the formulation of a model for the assembly of Sso AcP into amyloid-like aggregates at a molecular level.     

The folding process of acylphosphatase from Escherichia coli is remarkably accelerated by the presence of a disulfide bond

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (k_F^H2O = 72,600 s^− 1), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms.     

Characterizing intermolecular interactions that initiate native-like protein aggregation

Folded proteins can access aggregation-prone states without the need for transitions that cross the energy barriers for unfolding. In this study we characterized the initial steps of aggregation from a native-like state of the acylphosphatase from Sulfolobus solfataricus (Sso AcP). Using computer simulations restrained by experimental hydrogen/deuterium (H/D) exchange data, we provide direct evidence that under aggregation-promoting conditions Sso AcP populates a conformational ensemble in which native-like structure is retained throughout the sequence in the absence of local unfolding (N¹), although the protein exhibits an increase in hydrodynamic radius and dynamics. This transition leads an edge strand to experience an increased affinity for a specific unfolded segment of the protein. Direct measurements by means of H/D exchange rates, isothermal titration calorimetry, and intermolecular relaxation enhancements show that after formation of N¹, an intermolecular interaction with an antiparallel arrangement is established between the edge strand and the unfolded segment of the protein. However, under conditions that favor the fully native state of Sso AcP, such an interaction is not established. Thus, these results reveal a novel (to our knowledge) self-assembly mechanism for a folded protein that is based on the increased flexibility of highly aggregation-prone segments in the absence of local unfolding.     

Comparison of the folding processes of distantly related proteins. Importance of hydrophobic content in folding

The N-terminal domain of HypF from Escherichia coli (HypF-N) is a 91 residue protein module sharing the same folding topology and a significant sequence identity with two extensively studied human proteins, muscle and common-type acylphosphatases (mAcP and ctAcP). With the aim of learning fundamental aspects of protein folding from the close comparison of so similar proteins, the folding process of HypF-N has been studied using stopped-flow fluorescence. While mAcP and ctAcP fold in a two-state fashion, HypF-N was found to collapse into a partially folded intermediate before reaching the fully folded conformation. Formation of a burst-phase intermediate is indicated by the roll over in the Chevron plot at low urea concentrations and by the large jump of intrinsic and 8-anilino-1-naphtalenesulphonic acid-derived fluorescence immediately after removal of denaturant. Furthermore, HypF-N was found to fold rapidly with a rate constant that is approximately two and three orders of magnitudes faster than ctAcP and mAcP, respectively. Differences between the bacterial protein and the two human counterparts were also found as to the involvement of proline isomerism in their respective folding processes. The results clearly indicate that features that are often thought to be relevant in protein folding are not highly conserved in the evolution of the acylphosphatase superfamily. The large difference in folding rate between mAcP and HypF-N cannot be entirely accounted for by the difference in relative contact order or related topological metrics. The analysis shows that the higher folding rate of HypF-N is in part due to the relatively high hydrophobic content of this protein. This conclusion, which is also supported by the highly significant correlation found between folding rate and hydrophobic content within a group of proteins displaying the topology of HypF-N and AcPs, suggests that the average hydrophobicity of a protein sequence is an important determinant of its folding rate.     

Thermodynamics and kinetics of folding of common-type acylphosphatase: comparison to the highly homologous muscle isoenzyme

The thermodynamics and kinetics of folding of common-type acylphosphatase have been studied under a variety of experimental conditions and compared with those of the homologous muscle acylphosphatase. Intrinsic fluorescence and circular dichroism have been used as spectroscopic probes to follow the folding and unfolding reactions. Both proteins appear to fold via a two-state mechanism. Under all the conditions studied, common-type acylphosphatase possesses a lower conformational stability than the muscle form. Nevertheless, common-type acylphosphatase folds more rapidly, suggesting that the conformational stability and the folding rate are not correlated in contrast to recent observations for a number of other proteins. The unfolding rate of common-type acylphosphatase is much higher than that of the muscle enzyme, indicating that the differences in conformational stability between the two proteins are primarily determined by differences in the rate of unfolding. The equilibrium m value is markedly different for the two proteins in the pH range of maximum conformational stability (5. 0-7.5); above pH 8.0, the m value for common-type acylphosphatase decreases abruptly and becomes similar to that of the muscle enzyme. Moreover, at pH 9.2, the dependencies of the folding and unfolding rate constants of common-type acylphosphatase on denaturant concentration (mf and mu values, respectively) are notably reduced with respect to pH 5.5. The pH-induced decrease of the m value can be attributed to the deprotonation of three histidine residues that are present only in the common-type isoenzyme. This would decrease the positive net charge of the protein, leading to a greater compactness of the denatured state. The folding and unfolding rates of common-type acylphosphatase are not, however, significantly different at pH 5.5 and 9.2, indicating that this change in compactness of the denatured and transition states does not have a notable influence on the rate of protein folding.     

Kinetic partitioning of protein folding and aggregation.

We have systematically studied the effects of 40 single point mutations on the conversion of the denatured form of the alpha/beta protein acylphosphatase (AcP) into insoluble aggregates. All the mutations that significantly perturb the rate of aggregation are located in two regions of the protein sequence, residues 16-31 and 87-98, each of which has a relatively high hydrophobicity and propensity to form beta-sheet structure. The measured changes in aggregation rate upon mutation correlate with changes in the hydrophobicity and beta-sheet propensity of the regions of the protein in which the mutations are located. The two regions of the protein sequence that determine the aggregation rate are distinct from those parts of the sequence that determine the rate of protein folding. Dissection of the protein into six peptides corresponding to different regions of the sequence indicates that the kinetic partitioning between aggregation and folding can be attributed to the intrinsic conformational preferences of the denatured polypeptide chain.     

Relative influence of hydrophobicity and net charge in the aggregation of two homologous proteins

A potentially amyloidogenic protein has to be at least partially unfolded to form amyloid aggregates. However, aggregation of the partially or totally unfolded state of a protein is modulated by at least three other factors: hydrophobicity, propensity to form secondary structure, and net charge of the polypeptide chain. We propose to evaluate the relative importance of net charge, as opposed to the other factors, on protein aggregation and amyloidogenicity. For this aim, we have used two homologous proteins that were previously shown to be able to form amyloid fibrils in vitro, the N-terminal domain of HypF from Escherichia coli (HypF-N) and human muscle acylphosphatase (AcP). The aggregation process from an ensemble of partially unfolded conformations is ca. 1000-fold faster for HypF-N than for AcP. This difference can mainly be attributed to a higher hydrophobicity and a lower net charge for HypF-N than for AcP. By using protein engineering methods, we have decreased the net charge of AcP to a value identical to that of wild-type HypF-N and increased the net charge of HypF-N to a value identical to that of wild-type AcP. Amino acid substitutions were selected to minimize changes in hydrophobicity and secondary structure propensities. We were able to estimate that the difference in net charge between the two wild-type proteins contributes to 20-25% of the difference in their aggregation rates. An understanding of the relative influences of these forces in protein aggregation has implications for elucidating the complexity of the aggregation process, for predicting the effect of natural mutations, and for accurate protein design.     

Amyloid fibril formation can proceed from different conformations of a partially unfolded protein

Protein misfolding and aggregation are interconnected processes involved in a wide variety of nonneuropathic, systemic, and neurodegenerative diseases. More generally, if mutations in sequence or changes in environmental conditions lead to partial unfolding of the native state of a protein, it will often aggregate, sometimes into well-defined fibrillar structures. A great deal of interest has been directed at discovering the characteristic features of metastable partially unfolded states that precede the aggregated states of proteins. In this work, human muscle acylphosphatase (AcP) has been first destabilized, by addition of urea or by means of elevated temperatures, and then incubated in the presence of different concentrations of 2,2,2, trifluoroethanol ranging from 5% to 25% (v/v). The results show that AcP is able to form both fibrillar and nonfibrillar aggregates with a high beta-sheet content from partially unfolded states with very different structural features. Moreover, the presence of alpha-helical structure in such a state does not appear to be a fundamental determinant of the ability to aggregate. The lack of ready aggregation under some of the conditions examined here is attributable primarily to the intrinsic properties of the solutions rather than to specific structural features of the partially unfolded states that precede aggregation. Aggregation appears to be favored when the solution conditions promote stable intermolecular interactions, particularly hydrogen bonds. In addition, the structures of the resulting aggregates are largely independent of the conformational properties of their soluble precursors.     

Acid-Induced Formation of Molten Globule States in the Wild Type <Emphasis Type="Italic">Escherichia coli</Emphasis> 5-Enolpyruvylshikimate 3-Phosphate Synthase and its Three Mutated Forms: G96A,A183T and G96A/A183T

Recent advances in protein chemistry have led to progress in the understanding of protein folding and properties of possible intermediates during the folding of proteins. The molten globule (MG) state, a major intermediate of protein folding, has a denatured state with native-like secondary structure. In the present work, the acid-induced unfolding of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and its three different variants (G96A, A183T and G96A/A183T) were studied by far- and near-UV circular dichroism (CD), intrinsic fluorescent emission spectroscopy and 1-anilino naphthalene-8-sulfonate (ANS) binding. At pH < 3.0, these EPSPS variants acquire partially folded state, which show the characteristics of the MG state, e.g., a drastic reduction of defined tertiary structure and almost no change in the secondary structure. ANS binding experiments show that hydrophobic surface of these variants is exposed to a greater extent in comparison to the native form, at acidic pH. Wild type, G96A, A183T and G96A/A183T acquire MG states at pH 2.0, 1.5, 3.0 and 3.0, respectively, which show that pH stability of MG state of G96A has increased in comparison to wild type; and pH stability of MG states of two other mutants is lower than that of the wild type. The results suggest that there is a direct relationship between stability of protein and pH stability of its folding intermediates.     

Stabilisation of alpha-helices by site-directed mutagenesis reveals the importance of secondary structure in the transition state for acylphosphatase folding

The effects of stabilising mutations on the folding process of common-type acylphosphatase have been investigated. The mutations were designed to increase the helical propensity of the regions of the polypeptide chain corresponding to the two alpha-helices of the native protein. Various synthetic peptides incorporating the designed mutations were produced and their helical content estimated by circular dichroism. The most substantial increase in helical content is found for the peptide carrying five mutations in the second alpha-helix. Acylphosphatase variants containing the corresponding mutations display, to different extents, enhanced conformational stabilities as indicated by equilibrium urea denaturation experiments monitored by changes of intrinsic fluorescence. All the protein variants studied here refold with apparent two-state kinetics. Mutations in the first alpha-helix are responsible for a small increase in the refolding rate, accompanied by a marked decrease in the unfolding rate. On the other hand, multiple mutations in the second helix result in a considerable increase in the refolding rate without any significant effect on the unfolding rate. Addition of trifluoroethanol was found to accelerate the folding of the acylphosphatase variants, the extent of the acceleration being inversely proportional to the intrinsic rate of folding of the corresponding mutant. The trifluoroethanol-induced acceleration is far less marked for those variants whose alpha-helical structure is efficiently stabilised by amino acid replacements. This observation suggests that trifluoroethanol acts in a similar manner to the stabilising mutations in promoting native-like secondary structure. Analysis of the kinetic data indicates that the second helix is fully consolidated in the transition state for folding of acylphosphatase, whereas the first helix is only partially formed. These data suggest that the second helix is an important element in the folding process of the protein.     

Acid- and pressure-induced (un)folding of yeast glutathione reductase: competition between protein oligomerization and aggregation

Glutathione reductase (GR) is a homodimeric flavoenzyme involved in cellular defense against oxidative stress. In the present study, we have used a combination of acidic pH and hydrostatic pressure to investigate the (un)folding transition of yeast GR. Our results indicate that at pH 2 a distinct partially folded state is stabilized, as judged by intrinsic fluorescence, bis ANS binding and circular dichroism (CD) analysis. Further characterization of this partially folded state by size exclusion chromatography revealed that it corresponds to expanded GR monomers. CD analysis at pH 2 showed a significant loss of secondary structure. The partially folded GR monomers stabilized at pH 2 were fully and reversibly unfolded using hydrostatic pressure (up to 3.5kbar) as a thermodynamic perturbant. By contrast, return to physiological pH after exposure to acidic pH led to a competing reaction between refolding dimerization and aggregation of GR. These results support the notion that a partially folded intermediate state is not only critical for folding of GR but also appears to be a seed for protein aggregation.     

Thermodynamics of core hydrophobicity and packing in the hyperthermophile proteins Sac7d and Sso7d

The influence of core hydrophobicity and packing on the structure and stability of the hyperthermophile proteins Sac7d and Sso7d have been studied by calorimetry, circular dichroism, and NMR. Valine 30 is positioned in Sac7d to allow a cavity-filling Val --> Ile substitution which occurs naturally in the homologous more thermostable Sso7d. The cavity-filling mutation in Sac7d has been characterized and compared to the reciprocal Ile --> Val mutation in Sso7d. A detailed analysis of the stability of the proteins was obtained by globally fitting the variation of DSC parameters and circular dichroism intensities as a function of temperature (0-100 degrees C), salt (0-0.3 M), and pH (0-8). A global analysis over such a range of conditions permitted an unusually precise measure of the thermodynamic parameters, as well as the separation of the thermodynamics of the intrinsic unfolding reaction from the linked effects of protonation and chloride binding associated with acid-induced folding. The results indicate differences in the energetics of unfolding Sac7d and Sso7d that would not be apparent from an analysis of DSC data alone using conventional methods. The sign and magnitude of the changes in DeltaG, DeltaH, TDeltaS, and DeltaC(P) of unfolding resulting from core Ile/Val substitutions in the two proteins were consistent with differences in hydrophobicity of Val and Ile and negligible changes in packing (van der Waals) interactions. The benefit of increased hydrophobicity of the core increased with temperature, with maximal effect around 116 degrees C. Increased hydrophobicity of the core achieved not only an increase in the free energy of unfolding, but also a lateral shift of the temperature of maximal stability to higher temperature.     

Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by (1)H-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 A resolution, R-factor 13.7%) were obtained on the full-length protein and on an N-truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same betaalphabetabetaalphabeta topology previously described for other members of the AcP family from mesophilic sources. The unstructured N-terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25-37 degrees C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C-terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, Tm, of 100.8 degrees C and an unfolding free energy, DeltaG(U-F)H2O, at 28 degrees C and 81 degrees C of 48.7 and 20.6 kJ mol(-1), respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcP's display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcP's revealed the importance of a ion pair network stabilizing particularly the beta-sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher alpha-helical propensity.     

Characterization of the non-native trifluoroethanol-induced intermediate conformational state of the Shiga toxin B-subunit

The effect of increasing concentrations of 2,2,2-trifluoroethanol (TFE) on the conformational stability of the Shiga toxin B-subunit (STxB), a bacterial homopentameric protein involved in cell-surface binding and intracellular transport, has been studied by far-, near-UV circular dichroism (CD), intrinsic fluorescence, analytical ultracentrifugation, and differential scanning calorimetry (DSC) under equilibrium conditions. Our data show that the native structure of STxB is highly perturbed by the presence of TFE. In fact, at concentrations of TFE above 20% (v/v), the native pentameric conformation of the protein is cooperatively transformed into a helix-rich monomeric and partially folded conformational state with no significant tertiary structure. Additionally, no cooperative transition was detected upon a further increase in the TFE concentration (above 40% (v/v)). The thermal stability of STxB was investigated at several different TFE concentrations using DSC and CD spectroscopy. Thermal transitions at TFE concentrations of up to 20% (v/v) were successfully fitted to the two-state folding/unfolding coupled to oligomerization model consistent with the transition between a pentameric folded conformation to a monomeric state of the protein, which the presence of TFE stabilizes as a partially folded conformation.     

Shortening a loop can increase protein native state entropy

Protein loops are essential structural elements that influence not only function but also protein stability and folding rates. It was recently reported that shortening a loop in the AcP protein may increase its native state conformational entropy. This effect on the entropy of the folded state can be much larger than the lower entropic penalty of ordering a shorter loop upon folding, and can therefore result in a more pronounced stabilization than predicted by polymer model for loop closure entropy. In this study, which aims at generalizing the effect of loop length shortening on native state dynamics, we use all‐atom molecular dynamics simulations to study how gradual shortening a very long or solvent‐exposed loop region in four different proteins can affect their stability. For two proteins, AcP and Ubc7, we show an increase in native state entropy in addition to the known effect of the loop length on the unfolded state entropy. However, for two permutants of SH3 domain, shortening a loop results only with the expected change in the entropy of the unfolded state, which nicely reproduces the observed experimental stabilization. Here, we show that an increase in the native state entropy following loop shortening is not unique to the AcP protein, yet nor is it a general rule that applies to all proteins following the truncation of any loop. This modification of the loop length on the folded state and on the unfolded state may result with a greater effect on protein stability. Proteins 2015; 83:2137–2146. © 2015 Wiley Periodicals, Inc.     

Alcohol-induced versus anion-induced states of alpha-chymotrypsinogen A at low pH

Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various alcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) and salts (K(3)FeCN(6), Na(2)SO(4), KClO(4) and KCl) on the acid-induced state of alpha-chymotrypsinogen A, a predominantly beta-sheet protein, at pH 2.0 by near-UV circular dichroism (CD), far-UV CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements. Addition of alcohols led to an increase in ellipticity value at 222 nm indicating the formation of alpha-helical structure. The order of effectiveness of alcohols was shown to be TFE>butanol>propanol>ethanol>methanol. ANS fluorescence data showed a decrease in fluorescence intensity on alcohol addition, suggesting burial of hydrophobic patches. The near-UV CD spectra showed disruption of tertiary structure on alcohol addition. No change in ellipticity was observed on addition of salts at pH 2.0, whereas in the presence of 2 M urea, salts were found to induce a molten globule-like state as evident from the increases in ellipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-like state, i.e. both increase in ellipticity at 222 nm and increase in ANS fluorescence, followed the order K(3)FeCN(6)>Na(2)SO(4)>KClO(4)>KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disordering of tertiary structure results suggested that the decrease in ANS fluorescence intensity may be attributed to the unfolding of the ANS binding sites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfolded state. Characterization of such partially folded states has important implications for protein folding.     

8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions.

Hydrophobic interaction of 8-anilino-1-naphthalene sulfonic acid (ANS) with proteins is one of the widely used methods for characterizing/detecting partially folded states of proteins. We have carried out a systematic investigation on the effect of ANS, a charged hydrophobic fluorescent dye, on structural properties of acid-unfolded horse heart cytochrome c at pH 2.0 by a combination of optical methods and electrospray ionization mass spectroscopy (ESI MS). ANS was found to induce, a secondary structure similar to native protein and quenching of fluorescence of tryptophan residue, in the acid-unfolded protein. However, the tertiary structure was found to be disrupted thus indicating that ANS stabilizes a molten globule state in acid-unfolded protein. To understand the mechanism of ANS-induced folding of acid-unfolded cytochrome c, comparative ESI MS, soret absorption, and tryptophan fluorescence studies using nile red, a neutral hydrophobic dye, and ANS were carried out. These studies suggested that, at low pH, electrostatic interactions between negatively charged ANS molecules and positively charged amino acid residues present in acid-unfolded cytochrome c are probably responsible for ANS-induced folding of acid-unfolded protein to partially folded compact state or molten globule state. This is the first experimental demonstration of ANS induced folding of unfolded protein and puts to question the usefulness of ANS for characterization/determination of partially folded intermediates of proteins observed under low pH conditions.     

Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.