Equilibrium unfolding of <Emphasis Type="Italic">A. niger</Emphasis> RNase: pH dependence of chemical and thermal denaturation

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A_MG1) into another (I_MG2).     

Different molten globule-like folding intermediates of hen egg white lysozyme induced by high pH and tertiary butanol

We have provided evidence that hen egg white lysozyme (HEWL) existed in alpha helical and beta structure dominated molten globule (MG) states at high pH and in the presence of tertiary butanol, respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide-induced fluorescence quenching techniques have been used to investigate alkali-induced unfolding of HEWL and the effect of tertiary butanol on the alkaline-induced state. At pH 12.75, HEWL existed as molten globule like intermediate. The observed MG-like intermediate was characterized by (i) retention of 77% of the native secondary structure, (ii) enhanced binding of ANS (approximately 5 times) compared to native and completely unfolded state, (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near-UV, CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover, structural properties of the protein at isoelectric point (pI) and denatured states have also been described. We have also shown that in the presence of 45% tertiary butanol (t-butanol), HEWL at pH 7.0 and 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another MG state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly beta sheets. On further increasing the t-butanol concentration alpha helix was found to reform. We have proposed that formation of both alpha helical and beta sheet dominated intermediate may be possible in the folding pathway of alpha + beta protein.     

2,2,2-Trifluoroethanol-Induced structural change of peanut agglutinin at different pH: A comparative account

Peanut Agglutinin (PNA) is a legume lectin with a unique open quarternary structure. It is a homotetrameric protein, the monomeric subunit of which is made up of 3 beta sheets. The structural change in this protein has been induced by 2,2,2-trifluoroethanol (TFE) at two different pH. At neutral pH, PNA exists as a homotetramer, while at pH 2.5, it is known to dissociate to a dimer. The effect of TFE has been studied at both the pH by intrinsic tryptophan fluorescence, far and near UV Circular Dichroism, ANS binding and dynamic light scattering. At low pH, 15% TFE is found to induce a molten globule like state that shows maximum ANS binding. Increasing concentration of TFE increases alpha helical content and the compactness of the protein. The compact PNA at higher concentration of TFE is structurally different from the native structure. The effect of TFE at neutral pH on PNA is somewhat different from that observed at low pH. TFE does not induce molten globule like state at this pH. The detailed study of the structural change of PNA by TFE has been presented.     

Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large deltaCp's.

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274-329 K. At 298 K, values of deltaGdegrees , deltaCp, and Cm were 3.1+/-0.2 kcal mol(-1), 5.9+/-0.8 kcal mol(-1) K(-1) (15.9 cal (mol-residue)(-1) K(-1)), and 0.8 M, respectively, at pH 3.0 and 14.5+/-0.4 kcal mol(-1), 8.3+/-0.7 kcal mol(-1) K(-1) (22.4 kcal (mol-residue)(-1) K(-1)), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of deltaGdegrees and deltaCp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of deltaCp per mol-residue for the molten globule is comparable to corresponding values of deltaCp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of deltaCp per mol-residue for the unfolding of the native state is among the highest currently known for any protein.     

Characterization of acid-induced unfolding intermediates of glucose/xylose isomerase.

Acid-induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1-anilino-8-naphthalene-sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule-like characteristics, such as the presence of native-like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N-->PFI-->MG-->U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 M GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric alpha/beta barrel protein.     

A pH-dependent conformational change in EspA, a component of the Escherichia coli O157:H7 type III secretion system

pH-Dependent structural changes for Escherichia coli O157:H7 EspA were characterized by CD, 8-anilino-2-naphthyl sulfonic acid (ANS) fluorescence, and sedimentation equilibrium ultracentrifugation. Far- and near-UV CD spectra, recorded between pH 2.0 and 7.0, indicate that the protein has significant amounts of secondary and tertiary structures. An increase in ANS fluorescence intensity (in the presence of EspA) was observed at acidic pH; whereas, no increased ANS fluorescence was observed at pH 7.0. These results suggest the presence of a partially unfolded state. Interestingly, urea-induced unfolding transitions, monitored by far-UV CD spectroscopy, showed that the protein is destabilized at pH 2.0 as compared with EspA at neutral pH. Although increased ANS fluorescence was observed at pH 3.0, the urea-induced unfolding curve is similar to that found at pH 7.0. This result suggests the presence, at pH 3.0, of an ordered, but partially unfolded state, which differs from typical molten globule. The results of analytical ultracentrifugation and infrared spectroscopy indicate that EspA molecules associate at pH 7.0, suggesting the formation of short filamentous oligomers containing alpha-helical structures, whereas the protein tend to form nonspecific aggregates containing intermolecular beta-sheets at pH 2.0. Our experiments indicate that EspA has the potential to spontaneously form filamentous oligomers at neutral pH; whereas the protein is partially unfolded, assuming different conformations, at acidic pH.     

Differences in the unfolding of procerain induced by pH, guanidine hydrochloride, urea, and temperature

The structural and functional aspects along with equilibrium unfolding of procerain, a cysteine protease from Calotropis procera, were studied in solution. The energetic parameters and conformational stability of procerain in different states were also estimated and interpreted. Procerain belongs to the alpha + beta class of proteins. At pH 2.0, procerain exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is predominantly a beta-sheet conformation and exhibits strong ANS binding. GuHCl and temperature denaturation of procerain in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts in the molecular structure of procerain, possibly domains, with different stability that unfolds in steps. Moreover, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. At lower pH, procerain unfolds to the acid-unfolded state, and a further decrease in the pH drives the protein to the A state. The presence of 0.5 M salt in the solvent composition directs the transition to the A state while bypassing the acid-unfolded state. GuHCl-induced unfolding of procerain at pH 3.0 seen by various methods is cooperative, but the transitions are noncoincidental. Besides, a strong ANS binding to the protein is observed at low concentrations of GuHCl, indicating the presence of an intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8 M), procerain retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to unfolding by urea at lower pH, and the transitions are cooperative and coincidental. Further, the properties of the molten globule-like state and the intermediate state are different, but both states have the same conformational stability. This indicates that these intermediates may be located on parallel folding routes of procerain.     

Stabilization of molten globule state of papain by urea

Papain exists in molten globule (MG) state at pH 2.0 and in this state protein tends to aggregate in the presence of lower concentrations of guanidine hydrochloride (GuHC1). Such aggregation is prevented if a low concentration of urea is also present in the buffer; in addition, stabilization of the protein is also induced. Intrinsic fluorescence properties of papain as well as ANS binding suggest significant changes in the structure of papain, in the presence of urea with the absence of major changes in the secondary structure of the protein. The GuHCl- and temperature-induced unfolding of papain, in the presence of urea, indicates stabilization of the protein as seen from the higher transition midpoints, when monitored by fluorescence and circular dichroism (CD). However, a similar phenomenon is not seen under neutral conditions in the presence of urea either at low or high concentrations. The utility of prevention of aggregation by urea is also discussed.     

Deciphering the molecular structure of cryptolepain in organic solvents

Solvent composition plays a major role in stabilizing/destabilizing the forces that are responsible for the native structure of a protein. Often, the solvent composition drives the protein into non-native conformations. Elucidation of such non-native structures provides valuable information about the molecular structure of the protein, which is unavailable otherwise. Inclusion of methanol (non-fluorinated alcohol) or TFE (fluorinated alcohol) in the solvent composition drove cryptolepain, a serine protease and an all-β-protein, into a non-native structure with an enhanced β-sheet or induction of α-helix. These solvents did not much affect cryptolepain under neutral conditions, even at higher concentrations, but the effects were predominant at lower pH, when the protein molecule is under stress. The organic solvent-induced state is partially unfolded with similar characteristics to the molten globule state seen with protein under a variety of conditions. Chemical- or temperature-induced unfolding of cryptolepain in the presence of organic solvent is distinctly different from that in the absence of organic solvent. Such different unfolding provided evidence of two structural variants in the molecular structure of the protein as well as the differential stabilization/destabilization of such structural variants and their sequential unfolding.     

1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain

The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0–7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.     

Structural characterization of a highly stable cysteine protease ervatamin C.

Ervatamin C, a novel cysteine protease, belongs to alpha + beta class of proteins, probably with two domains, and retains both secondary and tertiary structures along with biological activity over a wide range of pH (2-12). Under neutral conditions, GuHCl and temperature-induced unfolding was cooperative with high transition midpoints and shows no structural changes in the presence of urea reflecting a remarkable stability. The fluorescence emission maximum at 350 nm suffers a blue shift of 4-5 nm upon lowering the pH and a red shift of 5 nm under denatured conditions. Unfolding transition curves at pH 2.0 are non-coincidental indicating the presence of intermediates in the unfolding pathway. At extremely low pH, the enzyme loses all the tertiary structure and proteolytic activity but retains a predominant secondary structure and a strong binding to ANS. GuHCl-induced unfolding of the enzyme in this intermediate state is noncooperative and indicates sequential unfolding of the domains.     

Equimolar Mixture of 2,2,2-Trifluoroethanol and 4-Chloro-1-butanol is a Stronger Inducer of Molten Globule State: Isothermal Titration Calorimetric and Spectroscopic Studies

A mixture of 4-chloro-1-butanol and 2,2,2-Trifluoroethanol (TFE) has been used to generate Molten globule (MG) state of structurally homologous but functionally different proteins bovine α-lactalbumin and hen egg-white lysozyme. The thermal denaturation was done using UV–Visible spectroscopy. From UV–Visible profile, thermal transition was not observed beyond a particular concentration. There was an indication of molten globule state in case of α-lactalbumin from circular dichroism experiments. By intrinsic tryptophan fluorescence, acrylamide and potassium iodide quenching, 8-anilino-naphthalene sulfonic acid (ANS) binding and energy transfer studies the presence of molten globule state was confirmed. Quantitative characterization of MG state and determining the binding thermodynamics of ANS to the MG state was done using Isothermal Titration Calorimetry (ITC). Results show that α-lactalbumin exists in MG state at a particular concentration but lysozyme does not show features of MG state.     

Role of electrostatic repulsion in the acidic molten globule of cytochrome c.

The molten globule has been assumed to be a major intermediate state of protein folding. To extend our understanding of protein folding it is important to elucidate the thermodynamic mechanism of conformational stability of the molten globule. To clarify the role of electrostatic charge repulsion in the stability of the acidic molten globule state, we prepared a series of acetylated horse ferricytochrome c species with various degrees of charge repulsion. On the basis of circular dichroism measurement, we show that the stability of the acidic molten globule is determined by a balance of electrostatic repulsions between positive residues, which favor the extended conformation, and the opposing forces, which stabilize the molten globule. These results provide a clear example of charge repulsions producing unfolding of the compact protein structure, and suggest that the reversibly denatured conformation of ferricytochrome c under physiological conditions (i.e. neutral pH, ambient temperature and no denaturant) is the molten globule.     

Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: a comparison with catalytically inactive form

We report the accumulation of an acid unfolded (UA) state and a molten globule (MG) state in the acid induced unfolding pathway of unmodified preparation of stem bromelain (SB) [EC 3.4.22.32], a cystein protease from Ananas cosmosus. The conformation of SB was examined over the pH 0.8-3 regions by circular dichroism, tryptophanyl fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, and tryptophanyl fluorescence quenching study. The pH 0.8-3.0 regions were selected to study the acid induced unfolding of SB because no autolysis of the enzyme was observed in these pH regions. The results show that SB at pH 2.0 is maximally unfolded and characterizes by significant loss of secondary structure ( approximately 80%) and almost complete loss of tertiary contacts. However, on further decreasing the pH to 0.8 a MG state was observed, with secondary structure content similar to that of native protein but no tertiary structure. We also made a comparative study of these acid induced states of SB with acid induced states of modified stem bromelain (mSB), reported by our group earlier [Eur. J. Biochem. (2002) 269, 47-52]. We have shown that modification of SB for inactivation significantly affects the N-UA transition but neither affects the UA-MG transition nor the stability of the MG state.     

Dissociation and unfolding of bovine odorant binding protein at acidic pH

Dissociation of bovine odorant binding protein (bOBP) dimers to monomers at pH 2.5 has been confirmed through size exclusion chromatography experiments. Moreover, structural and binding properties of the acidic monomer and neutral dimer have been compared using a combination of experimental (circular dichroism and fluorescence) and computational (molecular dynamics) techniques. The secondary and tertiary structures of bOBP are largely maintained at acidic pH, but molecular dynamics simulations suggest the loop regions (N-terminal residues, Omega-loop and C-terminal segments) are more relaxed and Phe36 and Tyr83 residues are involved in the regulation of the binding cavity entrance. The formation of a molten globule state at acidic pH, suggested by the strong enhancement of fluorescence of 8-anilino-1-naphtalenesulphonic acid (ANS), is not confirmed by any significant change in the near UV circular dichroism spectrum. Functionality measurements, deduced from the interaction of bOBP with 1-amino-anthracene (AMA), show that the binding capacity of the protein at acidic pH is preserved, though slightly looser than at neutral pH. Unfolding of acidic bOBP, induced by guanidinium chloride (GdnHCl), was investigated by means of CD spectroscopy, steady state fluorescence, fluorescence anisotropy and light scattering. The stability of the acidic monomer is lower than that of the neutral dimer, owing to the loss of the swapping interactions, but renaturation is completely reversible. Finally, in contrast with the neutral dimer, at low denaturant concentration some aggregation of the acidic monomer, which vanishes before the unfolding transition, has been observed.     

Effect of hydrostatic pressure on unfolding of alpha-lactalbumin: volumetric equivalence of the molten globule and unfolded state

The effect of pressure on the unfolding of bovine alpha-lactalbumin was investigated by ultraviolet absorption methods. The change of molar volume associated with unfolding, deltaV, was measured in the presence or absence of guanidine hydrochloride at pH 7. The deltaV was estimated to be -63 cm3/mol in the absence of a chemical denaturant. While in the presence of guanidine hydrochloride (GuHCl), it was found that deltaV was -66 cm3/mol at 25 degrees C and was independent of the concentration of GuHCl, despite the fact that the molten globule fraction in the total unfolding product decreased with the increase of GuHCl concentration. The results indicate that the volume of alpha-lactalbumin only changes at the transition from a native to a molten globule state, and almost no volume change has been found during the transition from a molten globule to the unfolded state.     

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme.

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.     

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.     

pH-Dependent Conformational Transitions in Conalbumin (Ovotransferrin), a Metalloproteinase from Hen Egg White

Acid unfolding pathway of conalbumin (CA), a monomeric glycoprotein from hen egg white, has been investigated using far- and near-UV CD spectroscopy, intrinsic fluorescence emission, extrinsic fluorescence probe 1-anilino-8-napthalene sulfonate (ANS) and dynamic light scattering (DLS). We observe pH-dependent changes in secondary and tertiary structure of CA. It has native-like α-helical secondary structure at pH 4.0 but loss structure at pH 3.0. The CA existed exclusively as a pre-molten globule state and molten globule state in solution at pH 4.0 and pH 3.0, respectively. The effect of pH on the conformation and thermostability of CA points toward its heat resistance at neutral pH. DLS results show that MG state existed as compact form in aqueous solutions with hydrodynamic radii of 4.7 nm. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states.     

Trichloroacetic acid-induced molten globule state of aminoacylase from pig kidney

The trichloroacetic acid (TCA)-induced unfolding of aminoacylase was investigated by measurement of aggregation, enzyme activity, intrinsic fluorescence, 8-anilino-1-naphthalene sulfonate (ANS) binding, circular dichroism, and native polyacrylamide gel electrophoresis. The results showed that TCA caused inactivation and unfolding of aminoacylase. Intrinsic fluorescence results demonstrated that the TCA-induced transition of aminoacylase was characterized by two distinct stages during which the fluorescence emission maxima first redshifted to 338 nm and then blueshifted to 332 nm, close to that of native protein. ANS binding measurements revealed that TCA-denatured aminoacylase had a large hydrophobic area for TCA concentration near 2 mM. Comparison of the relative changes in wavelength shift and in the ANS intensity suggested the formation of a stable molten globule state of aminoacylase with a slightly disrupted tertiary structure and more hydrophobic surface than the native protein. Far-UV circular dichroism results provided further support that TCA induced the formation of two partially folded intermediates each with an enhanced native-like secondary structure. The results collectively suggest that a TCA-induced molten globule state is formed and stabilized during unfolding of aminoacylase and that association of the molten globule state may account for precipitation of the protein when denatured by TCA.