Induction of 'molten globule' like state in acid-denatured state of unmodified preparation of stem bromelain: implications of disulfides in protein folding

A denatured state of unmodified preparation of stem bromelain representing a structureless form has been characterized at pH 2.0 and the effect of increasing concentration of TFE on the acid-denatured state has been investigated by circular dichroism (CD), fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show considerable accumulation of secondary structure when the acid-denatured bromelain is subjected to 70% (v/v) TFE and exhibited close resemblance to spectral features of those of pH 7.0 preparation. Interestingly, the acid-denatured state also regained some tertiary structure/interactions, with increasing concentration of TFE and at 60% (v/v) TFE, these approached almost those of the native like state. However, further increase to 70% (v/v) TFE resulted in complete loss of tertiary structure/interactions. Tryptophan fluorescence emission studies also suggested the induction of significant compact structure at 60% (v/v) concentration of TFE. In addition the acid-denatured state showed enhanced binding of ANS in presence of 60% (v/v) TFE. Taken together these observations suggest the existence of a molten globule state in acid-denatured bromelain between 60 and 70% (v/v) TFE. A similar molten globule state under identical conditions has been identified in reduced and carboxymethylated preparation of stem bromelain as reported in our earlier communication [Arch. Biochem. Biophys. 413 (2003) 199]. Comparison suggests unfolding/folding behavior of the bromelain to be independent of the intactness of the disulfide bonds.     

Formation of a molten globule like state in bovine serum albumin at alkaline pH

Little work has been done to understand the folding profiles of multi-domain proteins at alkaline conditions. We have found the formation of a molten globule-like state in bovine serum albumin at pH 11.2 with the help of spectroscopic techniques; like far and near ultra-violet circular dichroism, intrinsic and extrinsic fluorescence spectroscopy. Interestingly, this state has features similar to the acid-denatured state of human serum albumin at pH 2.0 reported by Muzammil et al. (Eur J Biochem 266:26–32, 1999). This state has also shown significant increase in 8-anilino-1-naphthalene-sulfonate (ANS) binding in compare to the native state. At pH 13.0, the protein seems to acquire a state very close to 6 M guanidinium hydrochloride (GuHCl) denatured one. But, reversibility study shows it can regain nearly 40% of its native secondary structure. On the contrary, tertiary contacts have disrupted irreversibly. It seems, withdrawal of electrostatic repulsion leave room for local interactions, but disrupted tertiary contacts fail to regain their original states.     

Pressure-induced molten globule state of cholinesterase

The denaturing effect of pressure on the structure of human butyrylcholinesterase was examined by gel electrophoresis under pressure and by 8-anilino-1-naphthalene sulfonate (ANS) binding. It was found that the fluorescence intensity of bound ANS is increased by pressure between 0.5 and 1.5 kbar and that the hydrodynamic volume of the enzyme swells when pressures around 1.5 kbar are applied. These findings indicate that pressure denaturation of butyrylcholinesterase is a multi-step process and that the observed transient pressure-denatured states have characteristics of molten globules.     

Fluoroalcohols induced unfolding of Succinylated Con A: native like beta-structure in partially folded intermediate and alpha-helix in molten globule like state

Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2-40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0-90% v/v TFE and HFIP) and resulted in stable alpha-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both beta-sheet and alpha-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from beta-sheet to alpha-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.     

Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point

Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T_ms), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.     

Low versus high molecular weight poly(ethylene glycol)-induced states of stem bromelain at low pH: stabilization of molten globule and unfolded states

The effect of low, medium, and high molecular weight poly(ethylene glycol) (e.g., PEG-400, -6000, and -20,000) on the structure of the acid unfolded state of unmodified stem bromelain (SB) obtained at pH 2.0 has been studied by various spectroscopic methods. The conformation of stem bromelain at pH 2.0 exhibits substantial loss of secondary structure and almost complete loss of native tertiary contacts and has been termed the acid unfolded state (A(U)). Addition of PEG-400 to A(U) led to an increase in the mean residue ellipticity (MRE) value at 222 nm, indicating formation of alpha-helical structure. On the other hand, PEG-6000 and 20,000 led to a decrease in the MRE value at 222 nm, indicating unfolding of the A(U) state. Interestingly, at 70% (w/v) PEG-400 and 40% (w/v) PEG-20,000, MRE values at 222 nm almost approach the native state at pH 7.0 and the unfolded state (6 M GnHCl) of stem bromelain, respectively. The probes for tertiary structure showed formation of nonnative tertiary contacts in the presence of 70% (w/v) PEG-400, while 40% (w/v) PEG-6000 and 20,000 were found to stabilize the unfolded state of SB. An increase in binding of 1-anilino 8-naphthalene sulfonic acid and a decrease in fractional accessibility of tryptophan residues (f(a)) compared to A(U) in the presence of 70% PEG-400 indicate that the PEG-400-induced state has a significant amount of exposed hydrophobic patches and is more compact than A(U). The results imply that the PEG-400-induced state has characteristics of molten globule, and higher molecular weight PEGs led to the unfolding of the A(U) state.     

Characterization of acid-induced molten globule like state of ficin

Effect of pH on the conformational behaviour of ficin (EC 3.4.22.3), a cysteine protease from the latex of Ficus carica was monitored by circular dichroism, fluorescence spectroscopy, ANS binding and hydrodynamic studies. The results obtained from near- and far-UV CD, intrinsic fluorescence and ANS binding studies demonstrate that ficin exhibits the characteristic properties of molten globule at acidic conditions between pH 1.4 and 2.0. Ficin at pH 1.4 retained about 74% secondary structure with a substantial loss of tertiary structure. The acid-induced state was found to have a compact shape as measured by Stokes radius on size exclusion chromatography.     

Effect of pH,temperature and alcohols on the stability of glycosylated and deglycosylated stem bromelain

The biological significance of the carbohydrate moiety of a glycoprotein has been a matter of much speculation. In the present work, we have chosen stem bromelain fromAnanas comosus as a model to investigate the role of glycosylation of proteins. Stem bromelain is a thiol protease which contains a single hetero-oligosaccharide unit per molecule. Here, the deglycosylated form of the enzyme was obtained by periodate oxidation. The differences in the glycosylated and deglycosylated forms of the glycoprotein have been studied at various temperatures and pH values, using probes such as loss of enzyme activity and by the changes in fluorescence and circular dichroism spectra. Deglycosylated bromelain showed decreased enzyme activity and perturbed fluorescence and circular dichroism spectra. In addition to this, a comparative study of their activities in different organic solvents showed a marked decrease in case of deglycosylated form of the enzyme. It is thus concluded that glycosylation contributes towards the functional stability of glycoenzymes.     

Partially folded intermediate state of concanavalin A retains its carbohydrate specificity

A systematic investigation of the effect of polyethylene glycol (PEG) 200 and 400 on the solution conformation of concanavalin A (con A) was made using circular dichroism (CD), tryptophan fluorescence, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, and size-exclusion chromatography. Far-UV CD spectra of con A at 30%(v/v) PEGs show the retention of ordered secondary structure as compared to 70%(v/v) PEGs. Near-UV CD spectra showed the retention of native-like spectral features in the presence of 30%(v/v) PEGs. Intrinsic tryptophan fluorescence studies indicate a change in the environment of tryptophan residues on the addition of PEG. ANS binding was maximum at 30%(v/v) PEGs suggesting the compact "molten-globule"-like state with enhanced exposure of hydrophobic surface area. Size-exclusion chromatography indicates an intermediate hydrodynamic size at 30%(v/v) PEGs. GdnHCl denaturation of these states was a single-step, two-state transition. To study the possible minimum structural requirement in the specific binding, the effect of PEGs on the interaction of con A with ligand was investigated by turbidity measurements. The C50 value was less in PEG 400 suggesting the more inhibitory ability of PEG 400. The C50 value of PEGs was highest for dextran followed by glycogen, ovalbumin, and ovomucoid. From percentage inhibition of con A-ligands at 30%(v/v) PEG, maximum inhibition was in ovalbumin followed by ovomucoid, glycogen, and dextran. To summarize: con A at 30%(v/v) PEGs exists as compact intermediate with molten-globule-like characteristics, viz., enhanced hydrophobic surface area, retention of compact secondary as well as tertiary structure, and a considerable degree of carbohydrate binding specificity and activity. This result has significant implications on the molten globule state during the folding pathway(s) of proteins in general and quaternary association in the legume lectin in particular, where precise topology is required for their biological activities.     

Structural energetics of the molten globule state

Certain partly ordered protein conformations, commonly called “moltenglobule states,” are widely believed to represent protein folding intermediates. Recentstructural studies of molten globule states ofdifferent proteins have revealed features whichappear to be general in scope. The emergingconsensus is that these partly ordered forms exhibit a high content of secondary structure, considerable compactness, nonspecific tertiary structure, and significant structural flexibility. These characteristics may be used to define ageneral state of protein folding called “the molten globule state,” which is structurally andthermodynamically distinct from both the native state and the denatured state. Despite exaatensive knowledge of structural features of afew molten globule states, a cogent thermodynamic argument for their stability has not yetbeen advanced. The prevailing opinion of thelast decade was that there is little or no enthalpy difference or heat capacity differencebetween the molten globule state and the unfolded state. This view, however, appears to beat variance with the existing database of protein structural energetics and with recent estimates of the energetics of denaturation of α-lactalbumin, cytochrome c, apomyoglobin, and T4 lysozyme. We discuss these four proteins at length. The results of structural studies, together with the existing thermodynamic values for fundamental interactions in proteins, provide the foundation for a structural thermodynamic framework which can account for the observed behavior of molten globule states. Within this framework, we analyze the physical basis for both the high stability of several molten globule states and the low probability of other protential folding intermediates. Additionally, we consider, in terms of reduced enthalpy changes and disrupted cooperative interactions, the thermodynamic basis for the apparent absence of a thermally induced, cooperative unfolding transition for some molten globule states. © 1993 Wiley-Liss, Inc.     

Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c.

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric alpha-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion- and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric alpha-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)-1 K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)-1 K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.     

Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule state

The conformational transitions starting with the native protein, passing the molten globule state and finally approaching the unfolded state of proteins was investigated for bovine carbonic anhydrase B (BCAB) and human -lactalbumin (-HLA) by means of fluorescence decay time measurements of the dye 8-anilinonaphthalene-1-sulphonic acid (8-ANS). Stepwise denaturation was realized by using the denaturant guanidinium chloride (GdmCl). It was shown that 8-ANS bound with protein yields a double-exponential fluorescence decay, where both decay times considerably exceed the decay time of free 8-ANS in water. This finding reflects the hydrophobic environment of the dye molecules attached to the proteins.

The fluorescence lifetime of the short-time component is affected by protein association and can be effectively quenched by acrylamide, indicating that 8-ANS molecules preferentially bind at the protein surface. The fluorescence lifetime of the long-time component is independent of the protein and acrylamide concentration and may be related to protein-embedded dye molecules.

Changes of the long lifetime component upon GdmCl-induced denaturation and unfolding of BCAB and -HLA correlate well with overall changes of the protein conformation. The transition from native protein to the molten globule state is accompanied by an increase of the number of protein-embedded 8-ANS molecules, while the number of dye molecules located at the protein surface decreases. For the transition from the molten globule to the unfolded state was the opposite behaviour observed.     

Thermal-unfolding Reaction of Triosephosphate Isomerase from <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Thermal denaturation of triosephosphate isomerase from Trypanosoma cruzi was studied by circular dicrhoism and fluorescence spectroscopies. The unfolding transition was found to be highly irreversible even at the very early stages of the reaction. Kinetic studies, allowed us to identify consecutive reactions. Firstly, only the tryptophan environment is altered. Next, changes on the secondary structure and hydrophobic surface exposure measured by 1-anilino-8-naphthalenesulfonate (ANS) binding were observed. Further conformational changes imply additional modifications on the secondary and tertiary structures and release of the hydrophobic dye leading to the formation of the unfolded state that is prone to aggregate. Edgar Mixcoha-Hernández and Liliana M. Moreno-Vargas contributed equally to this work     

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.     

Appropriateness of DSS and TSP as internal references for 1H NMR studies of molten globule proteins in aqueous media

Summary A marked dependence of the ¹H resonances of TSP and DSS (internal standards) on the concentration of proteins in the molten globule state has been found. This result indicates that TSP and DSS interact with these proteins. Therefore, when the chemical shift is used as an indicator of the residual structure of proteins in the molten globule state, great care must be taken in using TSP and DSS.     

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.     

Oriented immobilization of stem bromelain via the lone histidine on a metal affinity support

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from the stem of the pineapple plant (Ananas comosus) and is unique for it contains a single histidine residue (His-158) in the polypeptide. Based on the technology of protein separation with immobilized metal ion affinity chromatography (IMAC), a method for oriented immobilization of bromelain was selected. Bromelain was successfully immobilized on iminodiacetic acid carrier Sepharose 6B. Cu²⁺ complexed with iminodiacetate (IDA) was used as the chelating ligand to bind the lone histidine on bromelain. Simultaneously, preparation of a high affinity immobilized preparation was attempted using a soluble cross-linked preparation of bromelain on Cu-IDA-Sepharose. However this second method proved unsuccessful, possibly due to poor histidine accessibility in the cross-linked preparation. The immobilized preparation obtained using uncrosslinked bromelain was more resistant to thermal inactivation, as evidenced by retention of over enzyme 50% activity after incubation at 60 °C, as compared to 20% retained by the native enzyme. The immobilized preparation also exhibited a broader pH-activity profile in acidic range. The native, immobilized and soluble cross-linked bromelain showed apparent Michaelis constant (K_m) values of 1.08, 0.42, 1.56 mg/ml, respectively, using casein as the substrate. While the maximum velocity (V_max) values of the soluble and immobilized preparations were comparable, cross-linked preparation showed a 20% decrease, suggesting inactivation. The mild conditions used for predominantly oriented immobilization exploiting the unique property of single histidine, the high recovery of immobilized preparations, the stability, reusability and the regenerability of the matrix are the main features of the method reported here.     

1,1,1,3,3,3-hexafluoroisopropanol induced thermal unfolding and molten globule state of bovine alpha-lactalbumin: calorimetric and spectroscopic studies

The thermal denaturation of alpha-lactalbumin was studied at pH 7.0 and 9.0 in aqueous 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) by high-sensitivity differential scanning calorimetry. The conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. The most obvious effect of HFIP was lowering of the transition temperature with an increase in the concentration of the alcohol up to 0.30M, beyond which no calorimetric transition was observed. Up to 0.30M HFIP the calorimetric and van't Hoff enthalpy remained the same, indicating the validity of the two-state approximation for the thermal unfolding of alpha-lactalbumin. The quantitative thermodynamic parameters accompanying the thermal transitions have been evaluated. Spectroscopic observations confirm that alpha-lactalbumin is in the molten globule state in the presence of 0.50M HFIP at pH 7.0 and 0.75M HFIP at pH 9.0. The results also demonstrate that alpha-lactalbumin in the molten globule state undergoes a noncooperative thermal transition to the denatured state. It is observed that two of four tryptophans are exposed to the solvent in the HFIP induced molten globule state of alpha-lactalbumin compared to four in the 8.5M urea induced denatured state of the protein. It is also observed that the HFIP induced molten globule states at the two pH values are different from the acid induced molten globule state (A state) of alpha-lactalbumin.     

A single mutation induces molten globule formation and a drastic destabilization of wild-type cytochrome c at pH 6.0

Amino acid sequences of seven subfamilies of cytochromes c show that other than heme binding residues there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94, and Tyr/Trp/Phe97. These residues are 90% conserved in all sequences reported and are also considered to be involved in a common folding nucleus. To determine the importance of conserved interactions offered by the side chain of Leu94, we made an L94G mutant of horse cytochrome c. Characterization of this mutant by the far-UV, near-UV, and Soret circular dichroism, intrinsic and 1-Anilino-8-naphthalene sulfonate fluorescence, and dynamic light scattering measurements led to the conclusion that the L94G mutant has all the common structural characteristics of a molten globule at pH 6.0 and 25 °C. NaCl induces a cooperative transition between the acid-denatured state and a state of L94G having all the common structural characteristics of a pre-molten-globule state at pH 2 and 25 °C. Thermal denaturation studies showed that the midpoint of denaturation of the mutant is 28 °C less than that of the wild-type protein. Interestingly, the structure analysis using the coordinates given in the Protein Data Bank (1hrc) also suggested that the L94G mutant would be less stable than the wild-type protein.