The acid-induced state of glucose oxidase exists as a compact folded intermediate

A systematic investigation of the acid-induced unfolding of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase) (GOD) from Aspergillus niger was made using steady-state tryptophan fluorescence, circular dichroism (CD), and ANS (1-anilino 8-naphthalene sulfonic acid) binding. Intrinsic tryptophan fluorescence studies showed a maximally unfolded state at pH 2.6 and the presence of a non-native intermediate in the vicinity of pH 1.4. Flavin adenine dinucleotide (FAD) fluorescence measurements indicate that the bound cofactors are released at low pH. In the pH range studied, near- and far-UV CD spectra show maximal loss of tertiary as well as secondary structure (40%) at pH 2.6 although glucose oxidase at this pH is relatively less denatured as compared to the conformation in 6M GdnHCl. Interestingly, in the vicinity of pH 1.4, glucose oxidase shows a refolded conformation (A-state) with approximately 90% of native secondary structure and native-like near-UV CD spectral features. ANS fluorescence studies, however, show maximal binding of the dye to the protein at pH 1.4, indicating a "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a more compact conformation at low pH. Thermal stability of this state was assessed by ellipticity changes at 222 nm relative to native protein. While native glucose oxidase showed a completely reversible thermal denaturation profile, the state at pH 1.4 showed approximately 50% structural loss and the denatured state appeared to be in a different conformation exhibiting prominent beta-sheet structure (around 85 degrees C) that was not reversible. To summarize; the A-state of GOD exists as a compact folded intermediate with "molten-globule"-like characteristics, viz., native-like secondary structure but with non-native cofactor environment, enhanced hydrophobic surface area and non-cooperative thermal unfolding. That the A-state also possesses significant tertiary structure is an interesting observation made in this study.     

Salt-induced folding of a rabbit muscle pyruvate kinase intermediate at alkaline pH

The effect of alkaline denaturation on the structural and functional characteristics of rabbit muscle pyruvate kinase (PK) was investigated using enzymatic activity measurements and a combination of optical methods such as circular dichroism, fluorescence, and ANS binding. At a critical pH, 10.5, PK exists in an intermediate state (alkaline unfolded state) with predominant secondary structure along with some of the tertiary interactions and a strong binding to the hydrophobic dye ANS. This intermediate retains the enzymatic activity and corresponds to a dimeric state of the molecule. Above pH 10.5, a sudden fall in the spectral properties and enzymatic activity occurs suggesting the dissociation of the molecule followed by unfolding at very high pH. Addition of salts such as NaCl, KCl, and Na₂SO₄ to the alkali-induced state induces both secondary and tertiary structure to a level equivalent to that of native tetramer (salt-induced state). Chemical- and temperature-induced unfolding of the alkali-induced state as well as the salt-induced refolded state of PK reveal the presence of intermediate conformations in the unfolding pathway. The unfolding transition curves are noncoinciding and noncooperative along with ANS binding at intermediate concentrations of denaturants during unfolding. The observations presented in this paper suggest that the native pyruvate kinase tetramer dissociates to an active dimer around pH 10.5 and further to inactive monomer before attaining a completely unfolded monomeric conformation.     

High-pressure SANS and fluorescence unfolding study of calmodulin

Apo-calmodulin, a small soluble mainly α protein, is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80 °C, whereas the calcium-saturated form is stable up to temperatures as high as 100 °C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al., BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000 bar (300 MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form have been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease have been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.     

Conformational States of Trifluoroacetic Acid–Treated Cytochrome c in the Presence of Salts and Alcohols

We have carried out a systematic investigation of salts- and alcohols-induced conformational alterations on the trifluoroacetic acid (TFA)-treated ferricytochrome c by soret absorption spectroscopy, far UV circular dichroism (CD), tryptophan fluorescence, and 1-anilino-8-naphthalene sulfonate (ANS) binding. TFA induces the unfolding of native cytochrome c obtained from horse heart leading to loss of secondary structure. The addition of increasing concentration of salts and alcohols leads to increase in MRE value at 222 and 208 nm indicating an increase in the alpha-helical content leading to formation of compact dimensional structure. Cytochrome c is a heme protein in which the resonance energy of tryptophan is transferred to heme resulting in quenched tryptophan fluorescence. Addition of alcohols leads to increase in tryptophan and ANS fluorescence. The tryptophan and ANS fluorescence in case of salts shows decreased fluorescence intensity. TFA-induced unfolded cytochrome c showed the soret absorption maximum at 394 nm. However, an intermediate state in presence of alcohols and salts showed the absorption maxima at 398 nm and 402 nm, respectively. Among all the salts and alcohols studied, K3Fe(CN)6 and butanol were found to be most effective as examined by the above-mentioned spectroscopic techniques. The order of effectiveness of alcohols was found to be butanol > propanol > ethanol > methanol. The following effective trend in the case of salts was obtained: K3Fe(CN)6 > K2SO4>KClO4 > KCl. These results suggest that alcohols induce an intermediate with molten globule-like conformation on the TFA unfolded state, whereas salts induce a refolded intermediate approaching native-like conformation.     

Two distinct intermediates of trigger factor are populated during guanidine denaturation

Trigger factor (TF) is an important catalyst of nascent peptide folding and possesses both peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities. TF has a modular structure, containing three domains with distinct structural and functional properties. The guanidine hydrochloride (GuHCl) induced unfolding of TF was investigated by monitoring Trp fluorescence, far-UV CD, second-derivative UV absorption, enzymatic and chaperone activities, chemical crosslinking and binding of the hydrophobic dye, 1-anilinonaphthalene-8-sulfonate (ANS); and was compared to the urea induced unfolding. The native state of TF was found to bind ANS in 1:1 stoichiometry with a K(d) of 84 microM. A native-like state, N', is stable around 0.5 M GuHCl, and shows increased ANS binding, while retaining PPIase activity and most secondary and tertiary structure, but loses chaperone and dimerization activities, consistent with slight conformational rearrangement. A compact denatured state, I, is populated around 1.0 M GuHCl, is inactive and does not show significant binding to ANS. The data suggest that TF unfolds in a stepwise manner, consistent with its modular structure. The ability of TF to undergo structural rearrangement to maintain enzymatic activity while reducing chaperone and dimerization abilities may be related to the physiological function of TF.     

Acid and chemical induced conformational changes of ervatamin B. Presence of partially structured multiple intermediates

The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the alpha + beta class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0- 2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly a beta-sheet conformation and shows a strong binding to 8-anilino-1- napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.     

The partially unfolded state of beta-momorcharin characterized with steady-state and time-resolved fluorescence studies

The specific conformation of partially unfolded state of beta-momorcharin was characterized through the steady-state and time-resolved fluorescence spectroscopic studies on a single Trp-190 which located adjacently to the active site. The content of secondary structure was retained, the binding of ANS was remarkably enhanced, and the correlation time of entire protein rotation was prolonged at the partially unfolded state formed by being equilibrated with the mild concentration of guanidine hydrochloride. The time-resolved fluorescence depolarization and excitation energy transfer analysis suggest that Trp-190 approached 2 A closer to Tyr-70 and was hidden from the exposure to the protein surface, while the rotational correlation time and freedom of its segmental motion were shortened and enhanced, respectively. These results suggest that the transient folding/unfolding intermediate state of beta-momorcharin adopt the specific conformation at the vicinity of the active site, although it exhibits very similar properties with those of the generally known molten-globule state.     

Acid-induced equilibrium folding intermediate of human platelet profilin

The acid-induced unfolding of human platelet profilin (HPP) can be minimally modeled as a three-state process. Equilibrium unfolding studies have been performed on human platelet profilin1 (HPP) and monitored by far-UV circular dichroism, tryptophan fluorescence, ANS binding, and NMR spectroscopy. Far-UV CD measurements obtained by acid titration demonstrate that HPP unfolds via a three-state mechanism (N --> I --> U), with a highly populated intermediate between pH 4 and 5. Approximately 80% of native helical secondary structural content remains at pH 4, as indicated by monitoring the CD signal at 222 nm. The stability (DeltaGH2O) of the native conformation at pH 7.0 (obtained by monitoring the change in tryptophan signal as a function of urea concentration) is 5.56 +/- 0.51 kcal mol-1; however, the DeltaGH2O for the intermediate species at pH 4 is 2.01 +/- 0.47 kcal mol-1. The calculated m-values for the pH 7.0 and pH 4.0 species were 1.64 +/- 0.15 and 1.34 +/- 0.17 kcal mol-1 M-1, respectively, which is an indication that the native and intermediate species are similarly compact. Additionally, translational diffusion measurements obtained by NMR spectroscopy and ANS binding studies are consistent with a globular and compact conformation at both pH 7.0 and 4.0. The pKa values for the two histidine (His) residues located on helix 4 of HPP were determined to be 5.6 and 5.7 pH units. These pKa values coincide with the midpoint of the far-UV CD acid titration curve and suggest that the protonation of one or both His residues may play a role in the formation of the unfolding intermediate. Stable intermediate species populate the 2D 1H-15N HSQC NMR spectra between pH 4 and 5. A number of backbone and side-chain resonances show significant perturbations relative to the native spectrum; however, considerable nativelike tertiary contacts remain. Interestingly, the residues on HPP that are significantly altered at low pH coincide with segments of the G-actin binding surface and poly-l-proline binding interface. The earlier reports that a decrease in pH below 6.0 induces structural alterations in profilin, favoring dissociation of the profilin-actin complex, corresponds with the structural alterations observed in the partially unfolded species. Our findings suggest that a novel mechanism for pH induced disruption of the profilin-G-actin complex involve a nativelike unfolding intermediate of profilin.     

Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi

In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T _m?~?69 °C at pH 7.0, monitored by change in MRE_{222 nm}). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C _m (~1 M), while urea is characterized by high C _m (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.     

Unfolding and refolding of Escherichia coli chaperonin GroES is expressed by a three-state model.

The guanidine-hydrochloride (Gdn-HCl) induced unfolding and refolding characteristics of the co-chaperonin GroES from Escherichia coli, a homoheptamer of subunit molecular mass 10,000 Da, were studied by using intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS) binding, and size-exclusion HPLC. When monitored by tyrosine fluorescence, the unfolding reaction of GroES consisted of a single transition, with a transition midpoint at around 1.0 M Gdn-HCl. Interestingly, however, ANS binding and size-exclusion HPLC experiments strongly suggested the existence of an intermediate state in the transition. In order to confirm the existence of an intermediate state between the native heptameric and unfolded monomeric states, a tryptophan residue was introduced into the interface of GroES subunits as a fluorescent probe. The unfolding reaction of GroES I48W as monitored by tryptophyl fluorescence showed a single transition curve with a transition midpoint at 0.5 M Gdn-HCl. This unfolding transition curve as well as the refolding kinetics were dependent on the concentration of GroES protein. CD spectrum and size-exclusion HPLC experiments demonstrated that the intermediates assumed a partially folded conformation at around 0.5 M Gdn-HCl. The refolding of GroES protein from 3 M Gdn-HCl was probed functionally by measuring the extent of inhibition of GroEL ATPase activity and the enhancement of lactate dehydrogenase refolding yields in the presence of GroEL and ADP. These results clearly demonstrated that the GroES heptamer first dissociated to monomers and then unfolded completely upon increasing the concentration of Gdn-HCl, and that both transitions were reversible. From the thermodynamic analysis of the dissociation reaction, it was found that the partially folded monomer was only marginally stable and that the stability of GroES protein is governed mostly by the association of the subunits.     

Evidence for the existence of an unfolding intermediate of thyroglobulin during denaturation by guanidine hydrochloride

The unfolding of bovine thyroglobulin (Tg) in guanidine hydrochloride (GuHCl) solution was studied by following the fluorescence and circular dichroism. With increasing GuHCl concentrations, the emission maximum of the intrinsic fluorescence clearly red-shifted in two stages. At concentrations of GuHCl less than 1.2 M or more than 1.6 M, the red shift showed a cooperative manner. At concentrations of GuHCl between 1.2 and 1.6 M, an unfolding intermediate was observed. It was further characterized by the increased binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid (ANS). No significant changes of the secondary structure were indicated by CD spectra at the concentrations of GuHCl between 1.2 and 1.6 M. The conformation of this state has properties similar to those of a molten globule state which may exist in the folding pathway of the protein. Further changes in fluorescence properties occurred at concentrations of denaturant higher than 1.6 M with a significant red shift of the emission maximum from 340 to 347 nm and a marked decrease in ANS binding. This in vitro study gave a clue to understand the biochemical mechanism for the occurrence of aggregation and molecular chaperone binding during Tg maturation in vivo.     

Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: spectroscopic description of the native, intermediate, and unfolded states

Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to alpha+beta class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in beta-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (I(A) state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in I(A) state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (I(UG) state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (I(A) state) identified at pH approximately 1.5 are different from those of the I(UG) state.     

Conformational plasticity of cryptolepain: accumulation of partially unfolded states in denaturants induced equilibrium unfolding

pH and chemical denaturant dependent conformational changes of a serine protease cryptolepain from Cryptolepis buchanani are presented in this paper. Activity measurements, near UV, far UV CD, fluorescence emission spectroscopy, and ANS binding studies have been carried out to understand the folding mechanism of the protein in the presence of denaturants. pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. The preliminary biophysical study on cryptolepain shows that major elements of secondary structure are beta-sheets. Under neutral conditions the enzyme was stable in urea while GuHCl-induced equilibrium unfolding was cooperative. Cryptolepain shows little ANS binding even under neutral conditions due to more hydrophobicity of beta-sheets. Multiple intermediates were populated during the pH-induced unfolding of cryptolepain. Temperature-induced denaturation of cryptolepain in the molten globule like state is non-cooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts, possibly domains, in the molecular structure of cryptolepain, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of A state (molten globule state) of cryptolepain is unique, as lower concentration of denaturant, not only induces structure but also facilitate transition from one molten globule like state (MG(1)) into another (MG(2)). The increase of pH drives the protein into alkaline denatured state characterized by the absence of any ANS binding. GuHCl- and urea-induced unfolding transition curves at pH 12.0 were non-coincidental indicating the presence of an intermediate in the unfolding pathway.     

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.     

Characterization of partially folded intermediates of papain in presence of cationic, anionic, and nonionic detergents at low pH

A systematic investigation of the effects of detergents [Sodium dodecyl sulphate (SDS), hexa decyltrimethyl ammonium bromide (CTAB) and Tween-20] on the structure of acid-unfolded papain (EC.3.4.22.2) was made using circular dichroism (CD), intrinsic tryptophan fluorescence, and 1-anilino 8-sulfonic acid (ANS) binding. At pH 2, papain exhibits a substantial amount of secondary structure and is relatively less denatured compared with 6 M GdnHCl (guanidine hydrochloride) but loses the persistent tertiary contacts of the native state. Addition of detergents caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity value at 208 and 222 nm. Near-UV CD spectra also showed the regain of native-like spectral features in the presence of 8 mM SDS and 3.5 mM CTAB. Induction of structure in acid-unfolded papain was greater in the presence SDS followed by CTAB and Tween-20. Intrinsic tryptophan fluorescence studies indicate the change in the environment of tryptophan residues upon addition of detergents to acid-unfolded papain. Addition of 8 mM SDS resulted in the loss of ANS binding sites exhibited by a decrease in ANS fluorescence intensity, suggesting the burial of hydrophobic patches. Maximum ANS binding was obtained in the presence of 0.1 mM Tween-20 followed by CTAB, indicating a compact "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acid-unfolded papain in the presence of detergents showed the partial recovery of enzymatic activity. These results suggest that papain at low pH and in the presence of SDS exists in a partially folded state characterized by native-like secondary structure and tertiary folds. While in the presence of Tween, acid-unfolded papain exists as a compact intermediate with molten-globule-like characteristics, viz. enhanced hydrophobic surface area and retention of secondary structure. While in the presence of CTAB it exists as a compact intermediate with regain of native-like secondary and partial tertiary structure as well as high ANS binding with the partially recovered enzymatic activity, i.e., a molten globule state with tertiary folds.     

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.     

Effects of aspartic acid and potassium chloride on arginine kinase from shrimp

The aspartic acid (Asp)-induced unfolding and the salt-induced folding of arginine kinase (AK) were studied in terms of enzyme activity, intrinsic fluorescence emission spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that Asp caused inactivation and unfolding of AK with no aggregation during AK denaturation. The unfolding of the whole molecule and the inactivation of AK in different Asp concentrations were compared. Much lower Asp concentration was required to induce inactivation than to produce significant conformational changes of the enzyme molecule. However, with further addition of Asp, the molar ellipticity at 222 and 208 nm, the wavelength shift and the emission intensity of ANS hardly changed. Asp denatured AK was reactivated by dilution. In addition, potassium chloride (KCl) induced the molten globule state with a compact structure after AK was denatured with 7.5 mM Asp. These results collectively elucidate the osmotic effect of Asp anions for the molten globule formed during unfolding process. They also suggest that the effect of Asp differed from that of other denaturants such as guanidine hydrochloride or urea during AK folding. The molten globule state indicates that intermediates exist during AK folding.     

More stable structure of wheat germ lipase at low pH than its native state

Wheat germ lipase is a cereal lipase which is a monomeric protein. In the present study we sought to structurally characterize this protein along with equilibrium unfolding in solution. Conformational changes occurring in the protein with varying pH, were monitored by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS) and dynamic light scattering (DLS). Our study showed that acid denaturation of lipase lead to characterization of multiple monomeric intermediates. Native protein at pH 7.0 showed far-UV spectrum indicating mixed structure with both alpha and beta-type of characteristics. Activity of lipase was found to fall on either sides of pH 7.0–8.0. Acid-unfolded state was characterized at pH 4.0 with residual secondary structure, disrupted tertiary spectrum and red-shifted fluorescence spectrum with decreased intensity. Further decrease in pH lead to formation of secondary structure and acid-induced molten globule state was found to be stabilized at pH 1.4, with exposed tryptophan residues and hydrophobic patches. Notably, interesting finding of this study was characterization of acid-induced state at pH 0.8 with higher secondary structure content than native lipase, regain in tertiary spectrum and induction of compact conformation. Although enzymatically inactive, acid-induced state at pH 0.8 was found to be structurally more stable than native lipase, as shown by chemical and thermal denaturation profiles.     

Protein folding intermediates of invasin protein IbeA from Escherichia coli

IbeA of Escherichia coli K1 was cloned, expressed and purified as a His(6)-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution. The far-UV CD spectrum of IbeA at pH 7.0 has a strong negative peak at 215 nm, indicating the existence of beta-sheet-like structure. The acidic unfolding curve of IbeA at pH 2.0 shows the existence of a partially unfolded molecule (molten globule-like structure) with beta-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid (ANS) binding. The pH dependent intrinsic fluorescence of IbeA was biphasic. At pH 2.0, IbeA exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is in extended beta-sheet conformation and exhibits strong ANS binding. Guanidine hydrochloride denaturation of IbeA in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two domains (possibly) in the molecular structure of IbeA, with differential unfolding stabilities. Furthermore, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. Acid denatured unfolding of IbeA monitored by far-UV CD is non-cooperative with two transitions at pH 3.0-1.5 and 1.5-0.5. At lower pH, IbeA unfolds to the acid-unfolded state, and a further decrease in pH to 2.0 drives the protein to the A state. The presence of 0.5 m KCl in the solvent composition directs the transition to the A state by bypassing the acid-unfolded state. Additional guanidine hydrochloride induced conformational changes in IbeA from the native to the A-state, as monitored by near- and far-UV CD and ANS-fluorescence.     

Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH.

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.