Induced Molten Globule Research Articles

Sauromatum guttatum lectin: Spectral studies, lectin-carbohydrate interaction, molecular cloning and in silico analysis

Non-immune carbohydrate binding proteins are broadly defined as lectins. Having been reported from all kingdoms of life, phytolectins are the most widely studied group of lectins. Sauromatum guttatum agglutinin (SGA) was isolated from the plant tubers and characterized for structural variations due to solvent perturbation using polarimetry, fluorescence and light scattering. For the β-sheet rich SGA, a pH and temperature induced molten globule like intermediate was identified. In isothermal titration microcalorimetry, SGA demonstrated cooperative binding to a complex glycoprotein in enthalpically driven mechanism. Fine sugar specificity exploration identified core pentasaccharide as the most common and highest binding motif with complex N-glycans and fucosylated core N-glycans as additional motifs. Molecular cloning of SGA which has previously been demonstrated to have anti-cancer and anti-insect activities is being reported for the first time. Full length cDNA sequence was obtained with RACE-PCR based upon the conserved carbohydrate recognition site [QXDXNXVXY] present in all GNA-related lectins. Quaternary structure was proposed by homology modeling and an attempt was made to explain the structure-function relationship by in silico analysis.

Structural variations and molten globule state in Arisaema helliborifolium lectin under various treatments as monitored by spectroscopy.

Solvent perturbation was used to study variations in structure of Arisaema helliborifolium lectin (AHL) with the help of circular dichroism (CD), intrinsic fluorescence (IF), extrinsic fluorescence, quenching and dynamic light scattering (DLS). AHL was studied under acidic, alkaline and 6 M guanidine hydrochloride (GuHCl) equilibrium states. Three structural states were identified for AHL at different conditions, that are native (N; pH 7.0), molten globule (MG; pH 2.0) and unfolded (U; pH 12.0). CD analysis revealed that 50% of secondary structure of AHL was β-sheet component. A complete loss of secondary structure was observed at GuHCl treatment. The tertiary structural changes as studied by changes in microenvironment of trp residues also suggested a pH induced MG state as in case of CD. Parameter-A analysis pointed at the multi-step unfolding process of lectin under varying pH (pH 1-13). A comparision of CD and IF data further indicated that different pathways were followed for secondary and tertiary structure unfolding. Tryptophans of native AHL were only partially exposed to solvent belonging to Class II. Hydrodynamic diameter (Dh ) measurements of AHL via DLS also confirmed of a pH induced molten globule. A thermally induced molten globule was identified for AHL between 54-60 °C as monitored by DLS. An irreversible thermal denaturation was observed with the formation of a large aggregate. The Dh of AHL at neutral pH was confirmed by transmission electron microscopy (TEM).

Functional and conformational transitions of mevalonate diphosphate decarboxylase from Bacopa monniera

Functional and conformational transitions of mevalonate diphosphate decarboxylase (MDD), a key enzyme of mevalonate pathway in isoprenoid biosynthesis, from Bacopa monniera (BmMDD), cloned and overexpressed in Escherichia coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and Circular dichroism spectroscopy. Native BmMDD is a helix dominant structure with 45% helix and 11% sheets and possesses seven tryptophan residues with two residues exposed on surface, three residues partially exposed and two situated in the interior of the protein. Thermal denaturation of BmMDD causes rapid structural transitions at and above 40°C and transient exposure of hydrophobic residues at 50°C, leading to aggregation of the protein. An acid induced molten globule like structure was observed at pH 4, exhibiting altered but compact secondary structure, distorted tertiary structure and exposed hydrophobic residues. The molten globule displayed different response at higher temperature and similar response to chemical denaturation as compared to the native protein. The surface tryptophans have predominantly positively charged amino acids around them, as indicated by higher KSV for KI as compared to that for CsCl. The native enzyme displayed two different lifetimes, τ1 (1.203±0.036ns) and τ2 (3.473±0.12ns) indicating two populations of tryptophan.

Effect of galactose on acid induced molten globule state of Soybean Agglutinin: Biophysical approach

In the present study the formation of molten globule-like unfolding intermediate Soybean Agglutinin (SBA) in acidic pH range has been established with the help of acrylamide quenching, intrinsic fluorescence, ANS fluorescence measurement, far UV CD and dynamic light scattering measurement. A marked increase in ANS fluorescence was observed at pH 2.2. Ksv of acrylamide quenching was found to be higher at pH 2.2 than that of native SBA at pH 7. Far UV CD spectra of pH induced state suggest that SBA shows significant retention of secondary structure closure to native. Hydrodynamic radius of SBA at pH 2.2 was found be more as compared to native state and also in other pH induced states. Further we checked the effect of galactose on the molten globule state of SBA. This study suggests that SBA exist as molten globule at pH 2.2 and this study will help in acid induced molten globule state of other proteins.

Thermodynamic analysis of partially folded states of myoglobin in presence of 2,2,2-trifluoroethanol

The thermal denaturation of myoglobin was studied in the presence of 2,2,2-trifluoroethanol (TFE) at various pH values using differential scanning calorimetry and UV–visible spectroscopy. The most obvious effect of TFE was lowering the transition temperature up to 1.5mol·kg−1, beyond which no thermal transitions were observed. The protein conformation was analysed by fluorescence and circular dichroism measurements. Quantitative binding of ANS to the TFE induced molten globule state of myoglobin was studied by using isothermal titration calorimetry. The results enable quantitative estimation of the binding strength of ANS with the molten globule state of myoglobin along with the enthalpic and entropic contributions to the binding process. The results suggest occurrence of common structural features of the molten globule states of proteins offering two types of binding sites to ANS molecules which are a widely used fluorescence probe to characterise partially folded states of proteins.

Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography

Influence of acid concentration in the mobile phase on protein separation was studied in a wide concentration range using trifluoroacetic acid (TFA) and formic acid (FA). At low, 0.001–0.01 (v/v%) TFA concentration and appropriate solvent strength proteins elute before the column's dead time. This is explained by the proteins having a structured, but relatively extended conformation in the eluent; and are excluded from the pores of the stationary phase. Above ca. 0.01–0.05 (v/v%) TFA concentration proteins undergo further conformational change, leading to a compact, molten globule-like structure, likely stabilized by ion pairing. Proteins in this conformation enter the pores and are retained on the column. The results suggest a pore exclusion induced separation related to protein conformation. This effect is influenced by the pH and type of acid used, and is likely to involve ion-pair formation. The TFA concentration needed to result in protein folding (and therefore to observe retention on the column) depends on the protein; and therefore can be utilized to improve chromatographic performance. Conformation change was monitored by circular dichroism spectroscopy and mass spectrometry; and it was shown that not only TFA but FA can also induce molten globule formation.

FRET between a donor and an acceptor covalently bound to human serum albumin in native and non-native states

Fluctuation in the inter-domain distance of a protein, human serum albumin (HSA), in the native, molten globule and denatured states is studied by Förster resonance energy transfer (FRET). For this purpose, a donor (CPM) and an acceptor (Alexa Fluor 488) are covalently attached to HSA. Unfolding of the protein is induced by pH changes as well as by the addition of 6 M GdnHCl and addition of 1.5 M of a room temperature ionic liquid (RTIL, [pmim][Br]). The efficiency of FRET (εFRET) and hence donor (D) - acceptor (A) distances of protein molecules in the native and non-native states are determined using FRET. In the native state (N), there is only one value of εFRET and D-A distance. In the non-native states (molten globule and unfolded) there are multiple values of εFRET and D-A distances. This suggests the presence of multiple conformers in equilibrium in the non-native states. When the protein is unfolded (on addition of GdnHCl or RTIL), separation between the two domains (I and II) increases and as a result εFRET decreases. In the presence of both GdnHCl and RTIL, the protein undergoes compaction (to form N'). However, in spite of the decrease in the overall radius, the D-A distance in the compact state (N') is found to be larger than that in the native state (N) of the protein. In contrast, two acid induced molten globule states of HSA (formed at pH 2 and 4) exhibit high εFRET indicating short D-A distances. In summary, we show that under chemical denaturation HSA undergoes stepwise unfolding and different domains unfold independently.

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.

Multistate unfolding of α-mannosidase from Canavalia ensiformis (Jack Bean): Evidence for the thermostable molten globule

The relevance of partially ordered states of proteins (such as the molten-globule state) in cellular processes is beginning to be understood. We examined the conformational transitions in a multimeric and high molecular weight class II α-mannosidase from Canavalia ensiformis (Jack Bean) (Jbα-man) utilizing intrinsic fluorescence, solute quenching, hydrophobic dye binding, size exclusion chromatography and circular dichroism (CD) spectroscopy for the protein in presence of Guanidine hydrochloride (GdnHCl). The decomposition analysis of the protein spectra obtained during unfolding showed progressive appearance of class S, I, II and III trp. The parameter A and spectral center of mass showed multi state unfolding of the protein and phase diagram analysis revealed formation of an intermediate of Jbα-man in the vicinity of 1 M GdnHCl. The intermediate exhibited compact secondary and distorted tertiary structure with exposed hydrophobic amino acids on the surface, indicating the molten-globule nature. The dissociation, partial unfolding and aggregation of Jbα-man occurred simultaneously during chemical denaturation. The molten-globule possessed slightly higher hydrodynamic radius, perturbance in the structure up to 60 °C and stability of the structure up to 80 °C unlike the native Jack Bean α-mannosidase. The modes of chemical and thermal denaturation of the native protein were different. The solute quenching parameters confirmed the altered confirmation of the intermediate. Taken together, our results constitute one of the early reports of formation of GdnHCl induced molten globule in a class II α-mannosidase.

SDS induced molten globule state of heynein; a new thiol protease: Evidence of domains and their sequential unfolding

The molten globule state can be an intermediate in the protein-folding pathway and its detailed description can help understand the protein folding and an insight into the molecular structure of a protein. Sodium dodecyl sulfate (SDS), an anionic surfactant is known to induce molten globule sate in some proteins. SDS-induced changes in heynein were monitored by CD, fluorescence, 8-anilino-1-napthalenesulfonic acid (ANS) binding and proteolytic activity measurements. An enhancement in the α-helicity of protein with increasing concentration of SDS along with exposure of tryptophans is seen. At a concentration of SDS (∼2 mM) heynein loses activity and rigid tertiary structure but possesses considerable amount of secondary structure along with strong ANS binding, indicating the presence of an intermediate state, which is like molten globule state seen in the case heynein. Chemical and temperature induced unfolding of SDS-induced state of heynein is non-cooperative contrary to the protein in the absence of detergent. Further, the cooperative unfolding transition curve of heynein in the absence of SDS intersects at a point where the second transition of SDS-induced state starts suggesting that the molecule of heynein consist of at least two structural domains which are stabilized differentially and unfolds sequentially. Enhancement of α-helicity of heynein in the presence of SDS suggests the α-rich domain of the protein was stabilized and unfold later as compared with β-rich domain during temperature and chemical induced denaturation. Equilibrium unfolding pathway of heynein in SDS-induced state provides knowledge of the structure and stability of this plant cysteine protease at domain level.

Formation of conjugates between β-lactoglobulin and allyl isothiocyanate: Effect on protein heat aggregation, foaming and emulsifying properties

Whey proteins are widely used food ingredients due to their nutritional and functional properties (gelling, emulsifying, foaming). Owning to their structure (free thiol group, lysine residues, hydrophobic pocket), they can also be used as carriers for bioactives. In this study, conjugates between β-lactoglobulin (β-lg), and a bioactive metabolite from Brassicaceae vegetables, allyl isothiocyanate (AITC) were formed. Heat aggregation behavior (85 °C, 15 min), foaming and emulsifying properties of conjugates, at pH 4.0 and 7.1, were evaluated. Conjugates were formed by incubating β-lg (0.5 mM) with AITC (0.05–20 mM) in water at pH 8.5 and room temperature. AITC primarily reacted with β-lg’s free thiol group (K D = 0.2 ± 0.1 mM) and thereafter with its amino groups (K D = 10.8 ± 3.4 mM). AITC binding destabilized secondary and tertiary structure of β-lg at pH 7.1, whereas induced molten globule conformation at pH 4.0. Conjugation reduced the heat aggregation of β-lg at pH 7.1, while promoting it at pH 4.0. Conjugates adsorbed faster to air/water and oil/water interfaces at pH 4.0 than at pH 7.1. After 30 min, air/water surface tension was lower at pH 4.0 (47 mN m −1) than at pH 7.1 (57 mN m −1), while the surface tension of the oil/water interface was 8 mN m −1 at both pHs. Foams produced with β-lg–AITC conjugates at pH 4.0 exhibited higher volume and liquid stabilities compared to foams obtained at pH 7.1. Emulsions formed with conjugates at both pHs were destabilized by creaming due to flocculation, but coalescence was prevented. This study revealed that whey protein could potentially be used for the delivery of isothiocyanates in the form of foam or emulsion-based products.

PH Shifting Alters Solubility Characteristics and Thermal Stability of Soy Protein Isolate and Its Globulin Fractions in Different pH, Salt Concentration, and Temperature Conditions

Soy protein isolate (SPI), beta-conglycinin (7S), and glycinin (11S) were subjected to pH-shifting treatments, that is, unfolding at pH 1.5 or 12.0 followed by refolding at pH 7.0, to induce molten globule structures. Treated samples were analyzed for protein solubility, thermal stability, and aggregation in 0, 0.1, and 0.6 M NaCl solutions at pH 2.0-8.0. The pH(12) shifting resulted in drastic increases (up to 2.5-fold) in SPI solubility in the pH 6.0-7.0 range, especially at 0 M NaCl. The pH(1.5) shifting had a generally lesser effect on solubility. 11S exhibited a solubility pattern similar to that of SPI, but the solubility of 7S was unaffected by pH shifting except at 0.6 M NaCl. The pH shifting, notably at pH 12.0, produced soluble, disulfide-linked polymers from 11S and reduced (P < 0.05) its enthalpy but not its temperature of denaturation. Soy proteins structurally altered by pH shifting had a reduced sensitivity to thermal aggregation.

Thermodynamic insights into the binding of ANS with the salt induced molten globule states of cytochrome c

The molten globule (MG) state or the A-state of cytochrome c (cyt c) has been induced by addition of salts sodium perchlorate (NaClO 4), sodium thiocyanate (NaSCN), and sodium sulphate (Na 2SO 4) at pH 2.0. Isothermal titration calorimetry (ITC) has been used for determining the energetics of binding of 8-anilino naphthalene sulphonate (ANS) to the salt induced A-state of cyt c, and the accompanying thermodynamic parameters have been analyzed to elucidate the nature of the interactions between ANS and the A-state of cyt c. Temperature dependent studies of the binding process reveal that the binding is not a two state process and there are more than a single type of interactions involved. Addition of a bulky salt tetraethylammonium bromide (TEAB) increases the stoichiometry of binding significantly, with a reduction in the binding affinity at a higher concentration. The results provide quantitative information on the binding of ANS to the salt induced molten globule states of cyt c. It is further inferred that the binding involves a combination of hydrophobic and electrostatic interactions.

PH induced structural alterations in an aspartic protease from Vigna radiata indicating an alkali induced molten globule state

pH-dependent transitions in secondary and tertiary structure are described for a plant aspartic protease from Vigna radiata. The enzyme was pH stable with pH optima of 3.0. The Lineweaver Burk analysis at various pH yielded p K a values of 3.3 and 4.29 indicating acidic amino acids at the active site of the enzyme. The structural changes exemplified compact secondary structure collapsed tertiary structure and exposure of hydrophobic patches at pH 10. The changes at pH 10 are typical of a molten globule state. This alkali induced molten globule is novel since acid induced molten globule state is more reported.

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.

Elucidating the binding thermodynamics of 8‐anilino‐1‐naphthalene sulfonic acid with the A‐state of α‐lactalbumin: An isothermal titration calorimetric investigation

Isothermal titration calorimetry has been used to demonstrate that the heat profile associated with the binding of 8-anilino-1-naphthalene sulfonic acid (ANS) with the acid induced molten globule state (A-state) of alpha-lactalbumin (alpha-LA) is different from that with the native and denatured states of the protein. The results corroborate the spectroscopic observations that ANS binds more strongly to the partially folded states of the protein compared to that with the native and denatured states. ANS binds to the A-state of alpha-LA at two independent binding sites that remain nearly the same in the temperature range of 10-35 degrees C. The number of moles of ANS binding at site 1 at 10 degrees C is 14.0+/-0.2 and remains nearly the same with rise in temperature. However, the number of moles of ANS molecules binding at site 2 show an increase from 1.6+/-0.2 at 10 degrees C to 4.1+/-0.1 at 35 degrees C. The deviation of the slope of enthalpy-entropy compensation plot from unity and nonadherence to van't Hoff dictates implies that the binding sites on the A-state of alpha-LA for ANS are not well defined and specific; rather, these binding sites are formed due to greater exposure of hydrophobic clusters in the A-state of the protein. The results for the first time demonstrate the use of isothermal titration calorimetry in characterizing the A-state of alpha-LA both qualitatively and quantitatively.

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.

Effects of aspartate on rabbit muscle creatine kinase and the salt induced molten globule state

The aspartate (Asp)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, circular dichroism (CD) spectra, native polyacrylamide gel electrophoresis and ultraviolet difference spectra. The results showed that Asp caused inactivation and unfolding of CK, with no aggregation during CK denaturation. The kinetics of CK unfolding followed a one phase process. At higher concentrations of Asp (>2.5 mM), the CK dimers were partially dissociated. Inactivation occurred before noticeable conformational change during CK denaturation. Asp denatured CK was mostly reactivated and refolded by dilution. KCl induced the molten globule state with compact structure after CK was denatured with 10 mM Asp. These results suggest that the effect of Asp differed from that of other denaturants such as guanidine, HCl or urea during CK unfolding. Asp is a reversible protein denaturant and the molten globule state indicates that intermediates exist during CK folding.

Uncoupled analysis of secondary and tertiary protein structure by circular dichroism and electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) applied to protein conformational studies is a powerful new method that seems to provide specific information about protein tertiary structure. In this study, we analyzed the effect of trifluoroethanol (TFE) on a myoglobin peptide and cytochrome c (cyt c) at low pH by circular dichroism (CD) and ESI-MS. These experiments show that coil-to-helix transition per se does not affect ESI mass spectra, confirming that this technique is insensitive to the local conformation of the polypeptidic chain and, rather, reports on the tertiary contacts characterizing different protein conformations. This property makes ESI-MS an excellent method, complementary to CD, for the characterization of protein conformational changes. Fluorinated alcohols have been suggested to induce molten globule formation in acid-unfolded cyt c. The experiments described here show that TFE does not induce major changes in the ESI mass spectrum of cyt c at pH 2.2, indicating that no stabilization of compact, globular structures is detectable under the conditions employed. On the other hand, even low concentrations of TFE (2-5%) are shown to destabilize the folded state of the protein around the mid-point of its acid-induced unfolding transition.

Role of the disulfide cleavage induced molten globule state of type a botulinum neurotoxin in its endopeptidase activity.

Botulinum neurotoxins are produced by anaerobic Clostridium botulinum in an inactive form. The endopeptidase activity of type A botulinum neurotoxin (BoNT/A) is triggered by reduction of its disulfide bond between its heavy chain and light chain. By using circular dichroism spectroscopy, we show that, upon reduction of BoNT/A and under physiological temperature (37 degrees C), the BoNT/A loses most of its native tertiary structure, while retaining most of its secondary structure. This type of structure is characterized as a molten globule type conformation, which was further confirmed for BoNT/A by the characteristic binding of 1-anilinonaphthalene-8-sulfonic acid. Under nonreducing conditions where the interchain disulfide bond is intact, the enzymatically inactive BoNT/A did not show a molten globule type of structure. A temperature profile of the structure and enzyme activity of BoNT/A revealed that, under reducing conditions, there was a strong correlation in the existence of the molten globule structure and optimum endopeptidase activity at about 37 degrees C.