Chemical- and Thermal-Induced Unfolding of Leishmania donovani Ribose-5-Phosphate Isomerase B: a Single-Tryptophan Protein

Ribose-5-phosphate isomerase B (RpiB), a crucial enzyme of pentose phosphate pathway, was proposed to be a potential drug target for visceral leishmaniasis. In this study, we have analyzed the biophysical properties of Leishmania donovani RpiB (LdRpiB) enzyme to gain insight into its unfolding pathway under various chemical and thermal denaturation conditions by using fluorescence and CD spectroscopy. LdRpiB inactivation precedes the structural transition at lower concentrations of both urea and guanidine hydrochloride (GdHCl). 8-Anilinonapthalene 1-sulfonic (ANS) binding experiments revealed the presence of molten globule intermediate at 1.5 M GdHCl and a nonnative intermediate state at 6-M urea concentration. Acrylamide quenching experiments further validated the above findings, as solvent accessibility of tryptophan residues increased with increase in GdHCl and urea concentration. The recombinant LdRpiB was completely unfolded at 6 M GdHCl, whereas the enzyme molecule was resistant to complete unfolding even at 8-M urea concentration. The GdHCl- and urea-mediated unfolding involves a three-state transition process. Thermal-induced denaturation revealed complete loss of enzyme activity at 65 °C with only 20 % secondary structure loss. The formation of the well-ordered β-sheet structures of amyloid fibrils was observed after 55 °C which increased linearly till 85 °C as detected by thioflavin T dye. This study depicts the stability of the enzyme in the presence of chemical and thermal denaturants and stability-activity relationship of the enzyme. The presence of the intermediate states may have major implications in the way the enzyme binds to its natural ligand under various conditions. Also, the present study provides insights into the properties of intermediate entities of this important enzyme.     

Ornithine Carbamoyltransferase Unfolding States in the Presence of Urea and Guanidine Hydrochloride

Ornithine carbamoyltransferase folding/unfolding is a complex and not completely understood process. Our experimental results suggest that ornithine carbamoyltransferase interacts in a completely different way with urea and guanidine hydrochloride. In fact, we noticed that, increasing concentration from 0.0 to 8.0 M of the two additives, the enzyme follows a simple one-step transition mechanism in the presence of guanidine hydrochloride, with two macroscopic states (the native and the denatured one) significantly populated, whereas in the presence of urea a lot of different protein states can be detected and analyzed. Circular dichroism and UV-visible spectroscopy reveal a similar mechanism of perturbation at high temperature, with opening of hydrophobic core and a significant loss in α-helix structure in the presence of guanidine hydrochloride that cannot be found in the presence of urea.     

Structural Stability and Unfolding Properties of Cutinases from Thermobifida fusca

A comparative analysis of the structural and functional aspects along with equilibrium unfolding of two homologous cutinases, Cut1 and Cut2, from Thermobifida fusca was carried out. The CD and fluorescence profile at different pH in the range of 6 to 9 showed no structural variations for both cutinases, indicating their stability to a wide range of pH. Tryptophan quenching studies suggested that all the four Trp residues in the protein are in inaccessible hydrophobic pockets. Further, near-UV CD analysis of tertiary structure revealed a dissimilar distribution of aromatic amino acid on the surface of these two enzymes. Denaturation profiles obtained in aqueous solutions of the guanidine hydrochloride revealed different tolerance levels for unfolding of the two cutinases, with Cut2 showing higher resistivity to unfolding in comparison to Cut1. Both cutinases retained all the structural parameters even in the presence of 8 M urea, indicating the protein to be highly resistant to urea-induced unfolding. Structural study by homology modeling revealed a high resemblance of secondary structure between the two cutinases; however, their tertiary structure, hydrophobicity, and surface electrostatic properties were very different, which contributed to the difference in the structural stability of these two cutinases.     

Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride

Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α?+?β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH?2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH?2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 °C; however, above this temperature, denaturation was observed.     

Molecular and Spectroscopic Characterization of A<Emphasis Type="Italic">spergillus flavipes</Emphasis> and <Emphasis Type="Italic">Pseudomonas putida</Emphasis> L-Methionine γ-Lyase in Vitro

Pseudomonas putida L-methionine γ-lyase (PpMGL) has been recognized as an efficient anticancer agent, however, its antigenicity and stability remain as critical challenges for its clinical use. From our studies, Aspergillus flavipes L-methionine γ-lyase (AfMGL) displayed more affordable biochemical properties than PpMGL. Thus, the objective of this work was to comparatively assess the functional properties of AfMGL and PpMGL via stability of their internal aldimine linkage, tautomerism of pyridoxal 5′-phosphate (PLP) and structural stability responsive to physicochemical factors. The internal Schiff base of AfMGL and PpMGL have the same stability to hydroxylamine and human serum albumin. Acidic pHs resulted in strong cleavage of the internal Schiff base, inducing the unfolding of MGLs, compared to neutral-alkaline pHs. At λ 280 nm excitation, both AfMGL and PpMGL have identical fluorescence emission spectra at λ 335 nm for the intrinsic tryptophan and λ 560 nm for the internal Schiff base. The maximum PLP tautomeric shift of ketoenamine to enolimine was detected at acidic pH causing complete enzyme unfolding, subunits dissociation and tautomeric shift of intrinsic PLP, rather than neutral-alkaline ones. The T _m of AfMGL and PpMGL in presence of thermal stabilizer/ destabilizer was assayed by DSF. The T _m of AfMGL and PpMGL was 73.1 °C and 74.4 °C, respectively, suggesting the higher proximity to the tertiary structure of both enzymes. The T _m of AfMGL and PpMGL was slightly increased by trehalose and EDTA in contrast to guanidine HCl and urea. The active site and PLP-binding domains are identically conserved in both AfMGL and PpMGL.     

Alpha-Glucosidase Folding During Urea Denaturation: Enzyme Kinetics and Computational Prediction

In this study, we investigated structural changes in alpha-glucosidase during urea denaturation. Alpha-glucosidase was inactivated by urea in a dose-dependent manner. The inactivation was a first-order reaction with a monophase process. Urea inhibited alpha-glucosidase in a mixed-type reaction. We found that an increase in the hydrophobic surface of this enzyme induced by urea resulted in aggregation caused by unstable folding intermediates. We also simulated the docking between alpha-glucosidase and urea. The docking simulation suggested that several residues, namely THR9, TRP14, LYS15, THR287, ALA289, ASP338, SER339, and TRP340, interact with urea. Our study provides insights into the alpha-glucosidase unfolding pathway and 3D structure of alpha-glucosidase.     

Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was studied through their intrinsic fluorescence emission spectra, fluorescence phase diagrams, fluorescence quenching, size‐exclusion chromatographies, native polyacrylamide gel electrophoreses and deactivation profiles. The results showed that during their unfolding in urea and guanidine hydrochloride solutions, bovine heart cytochrome c molecules existed only in a unimolecular form and their bi‐molecular and/or poly‐molecular aggregates and aggregate precipitates were not formed all along. When the urea and guanidine hydrochloride concentrations in denaturation solution were separately about 6.0 and 3.0 mol/L, they could be completely deactivated and almost all of the tryptophan residues originally embedded in the interior of their molecules were exposed to the surface of their molecules. Different from the unfolding of the most often used horse heart cytochrome c, that of bovine heart cytochrome c induced by urea and guanidine hydrochloride was separately a completely co‐operative procedure and followed a two‐state model.     

A study of the stability of 5-aza-2′-deoxycytidine in alkaline media in dependence on time

It has been determined that the first stage of the hydrolytic degradation of the cytostatically active substance, 5-aza-2′-deoxycytidine (AzDC), in alkaline solution yields N-amidinium-N′-(2-deoxy-β-d-erythropentofuranosyl) urea formate (AUF) due to the opening of the triazine ring between C6 and N1. In the second stage of the hydrolytic degradation, the -NHCO- bond in AUF is opened at an elevated temperature with formation of guanidine. The reaction mechanisms are given. The degradation was monitored using thin-layer chromatography and the standard substances of AUF and guanidine. HCl were prepared synthetically.     

蛋白质多态解折叠行为与稳定性Ⅱ:酶的多态解折叠行为

基于结构基元模型,进一步假设,由n个结构基元组成的蛋白酶,其活性中心由na(na?n)个结构基元组成,酶活性仅与组成活性中心的结构基元相关.由此,推导出适合于蛋白酶解折叠研究的变性曲线、解折叠结构基元平均自由能、物种分布等表达式.本文以盐酸胍诱导的卵清溶菌酶解折叠为例,通过荧光方法测定的溶菌酶解折叠曲线,得出卵清溶菌酶由2个结构基元组成,结构基元平均自由能?G0element(H2O)为48.47 kJ/mol.物种分布表明,酶活性随盐酸胍浓度的变化仅仅反映的是结构基元1(?-片结构域)的解折叠,而结构基元2(?-螺旋结构域)的解折叠反映在3.8~5.0 mol/L盐酸胍浓度范围内.结构基元模型既可描述蛋白酶多态解折叠的谱学行为,又可解释蛋白酶活性的两态性质.     

Identification of Folding Intermediates of Streblin,The Most Stable Serine Protease: Biophysical Analysis

Streblin, a serine proteinase from plant Streblus asper, has been used to investigate the conformational changes induced by pH, temperature, and chaotropes. The near/far UV circular dichroism activities under fluorescence emission spectroscopy and 8-aniline-1-naphthalene sulfonate (ANS) binding have been carried out to understand the unfolding of the protein in the presence of denaturants. Spectroscopic studies reveal that streblin belongs to the α+β class of proteins and exhibits stability towards chemical denaturants, guanidine hydrochloride (GuHCl). The pH-induced transition of this protein is noncooperative for transition phases between pH 0.5 and 2.5 (midpoint, 1.5) and pH 2.5 and 10.0 (midpoint, 6.5). At pH 1.0 or lower, the protein unfolds to form acid-unfolded state, and for pH 7.5 and above, protein turns into an alkaline denatured state characterized by the absence of ANS binding. At pH 2.0 (1 M GuHCl), streblin exists in a partially unfolded state with characteristics of a molten globule state. The protein is found to exhibit strong and predominant ANS binding. In total, six different intermediate states has been identified to show protein folding pathways.     

Effect of Chaotropes on Lipase Back Extraction Recovery in the Process of Reverse Micellar Extraction

Chaotropes could significantly enhance lipase activity recovery in reverse micellar back extraction. However, the mechanism of chaotropes promoting the release of enzyme from reverse micelles was not clear. In this study, chaotropes were added in the process of lipase reverse micellar extraction, and back extraction recovery was improved. In back extraction, at 0.6 M urea in stripping solution, 94.60 % total extraction recovery was obtained. Meanwhile at 0.3 M guanidine hydrochloride, nearly 65 % lipases were released into the stripping solution. DLS and Karl Fischer method results showed that the presence of urea in stripping solution could weaken the electrostatic interaction between lipase and cetyltrimethylammonium bromide molecules, promoting lipase release from reverse micelles, and guanidine hydrochloride could stimulate lipase and free water molecules enwrapped in reverse micelle release into the stripping solution. These experimental results provide a clue for understanding the mechanism of chaotropes influencing on protein recovery in reverse micelle back extraction.     

Probing protein unfolding through monitoring cysteine alkylation by matrix-assisted laser desorption/ionisation mass spectrometry

Matrix-assisted laser desorption/ionisation mass spectrometry was used to monitor interaction between three proteins and two basic Immobiline chemicals (pK 10.3 and pK >12) commonly used in immobilised pH gradients (IPG). For two of the investigated proteins, the observed alkylation channels of the cysteine residues exhibited unmistakable response to their gradual denaturation following treatment with different concentrations (0-8 M) of two commonly used denaturants, urea and guanidine hydrochloride. Our assessment for protein unfolding is based on the number and relative intensity of the alkylation channels, yet the present mass spectrometry data are in good agreement with data based on optical rotatory dispersion, in which another approach was used to assess protein unfolding. Whether the present simple, fast and specific mass spectrometry method can be developed as a probe for monitoring folding/unfolding of cysteine-containing proteins can only be demonstrated by generating similar data for a larger number of proteins.     

Direct probing of the folding/unfolding event of bovine hemoglobin at montmorillonite clay modified electrode by adsorptive-transfer voltammetry

An efficient way was proposed for probing the folding/unfolding event of bovine hemoglobin (Hb) through adsorptive-transfer voltammetry. Hb molecules in native and pre-unfolded in different urea conditions for 23 h were adsorbed onto the montmorillonite clay modified glassy carbon electrode (Hb/clay/GCE and uHb/clay/GCE, respectively). Cyclic voltammograms of Hb/clay/GCE and uHb/clay/GCE showed that the unfolding of Hb caused great change in the direct electron transfer between the heme irons within Hb and electrode surface, which was facilitated on clay film. From the amount of the electroactive Hb (W_Hbe) and the adsorbed Hb (Γ) on clay per unit mass, the minimal electroactive portion (MEP) of the adsorbed Hb was calculated to assess the unfolding state of Hb. With the increase of urea concentration, MEP showed a sigmoid curve. Thermodynamic parameters related to the unfolding event of Hb were also obtained based on the linear free energy model (LEM), including the free energy of folding in water (ΔG_U^water), the slope of the Santoro-Bolen equation (m), and the urea concentration required in for achieving half of the total change (S_m) in the unfolding curves. This work gave the first try for investigating protein unfolding at nano-materials modified electrode using adsorptive-transfer voltammetry, which improved the sensitivity of analysis and avoided the disadvantages involved in the existing electrochemical methods for protein unfolding. The proposed method will benefit the electrochemical studies of protein.     

Folding dynamics of cytochrome C using pulse radiolysis

Pulse radiolysis is a powerful method to realize real-time observation of various redox processes, which induces various structural and functional changes occurring in biological systems. However, its application has been mainly limited to studies of the redox reactions of rather smaller biological systems such as DNA because of an undesired reaction due to various free radicals generated by pulse radiolysis. For application of pulse radiolysis to generate plenty of redox reactions of biological systems, selective redox reactions induced by electron pulses have to be developed. In this study, we report that in the presence of the high concentration of the denaturant, guanidine HCl (GdHCl), the selective reduction of the oxidized cytochrome c (Cyt c) takes place in time scales of a few microseconds by the electron transfer from the guanidine radical that is formed by the fast reaction of e(aq)(-) with GdHCl, consequently leading to folding kinetics of Cyt c. By providing insight into the folding dynamics of Cyt c, we show that the pulse radiolysis technique can be used to track the folding dynamics of various biomolecules in the presence of a denaturant including GdHCl.     

Inhibitive effect by extract of Mentha rotundifolia leaves on the corrosion of steel in 1 M HCl solution

An extract of Mentha rotundifolia leaves (EMRL) was tested as a corrosion inhibitor of steel in 1 M HCl using electrochemical impedance spectroscopy, Tafel polarization methods, and weight loss measurements. The inhibition efficiency of the extract of Mentha rotundifolia leaves was calculated and compared. We note good agreement between these methods. The results obtained revealed that the inhibitor tested differently reduced the kinetics of the corrosion process of steel. Its efficiency increases with the concentration and attained 92.87 % at 35 %. The effect of temperature on the corrosion behavior of steel in 1 M HCl was also studied in the range 298 and 338 K. The thermodynamic data of activation were determined. Mentha rotundifolia extract is adsorbed on the steel surface according to a Langmuir adsorption model.     

Isomerization‐ and Temperature‐Jump‐Induced Dynamics of a Photoswitchable β‐Hairpin

Conformational changes in proteins and peptides can be initiated by diverse processes. This raises the question how the variation of initiation mechanisms is connected to differences in folding or unfolding processes. In this work structural dynamics of a photoswitchable β‐hairpin model peptide were initiated by two different mechanisms: temperature jump (T‐jump) and isomerization of a backbone element. In both experiments the structural changes were followed by time‐resolved IR spectroscopy in the nanosecond to microsecond range. When the photoisomerization of the azobenzene backbone switch initiated the folding reaction, pronounced absorption changes related to folding into the hairpin structure were found with a time constant of about 16 μs. In the T‐jump experiment kinetics with the same time constant were observed. For both initiation processes the reaction dynamics revealed the same strong dependence of the reaction time on temperature. The highly similar transients in the microsecond range show that the peptide dynamics induced by T‐jump and isomerization are both determined by the same mechanism and exclude a downhill‐folding process. Furthermore, the combination of the two techniques allows a detailed model for folding and unfolding to be presented: The isomerization‐induced folding process ends in a transition‐state reaction scheme, in which a high energetic barrier of 48 kJ mol^?1 separates unfolded and folded structures.     

Structural Analysis and Inhibitory Kinetics of Brain Type Creatine Kinase by Sodium Dodecyl Sulfate

The studies regarding the effect of sodium dodecyl sulfate (SDS) on enzyme activities and structures can provide a valuable insight into public health. We have predicted the 3D structure of the brain creatine kinase (CK-BB) with a high resolution and simulated the docking between CK-BB and SDS. The predicted structure had a root mean square deviation of 0.51 Å. The docking between CK-BB and SDS was successful with significant scores (?4.67 kcal/mol, AutoDock4 and ?48.32 kcal/mol, DOCK6). We have also investigated the inactivation by using SDS to study CK-BB’s folding behaviors. The two-phase rate constants as a first-order reaction were measured during inactivation. SDS strongly inhibited the CK-BB activity in a noncompetitive inhibition manner (K _i?=?1.22 mM). The tertiary structural change was induced by SDS binding with the exposure of hydrophobic surface. The methyl-β-cyclodextrin was used to strip SDS from the enzyme molecule to reactivate. The changes of thermodynamic parameters for the SDS ligand binding such as enthalpy, Gibbs free energy, and entropy were obtained as ?13?±?7.0 MJ/mol, 8.39 kJ/mol, and ?42.754 kJ/(K mol), respectively. Our study provides important structural information for CK-BB and its interaction with SDS with an insight on its folding and inhibition kinetics.     

Distribution and Transition of Native and Completely Unfolded Conformations in the Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was first studied through intrinsic fluorescence emission spectra and fluorescence phase diagram and the results showed that both of them separately followed a two‐state model. As the simplest sample of the unfolding of protein molecules induced by denaturants, an equation was presented to show the effect of the denaturant concentrations in denaturation solution on the residual activity ratios of bovine heart cytochrome c in their two‐state unfolding. There are two characteristic unfolding parameters K and m in this equation. The former is the thermodynamic equilibrium constant of the unfolding of bovine heart cytochrome c induced by denaturants, the latter is the number of denaturant molecules associated with a bovine heart cytochrome c molecule during the unfolding procedure, and through them the distribution and transition of native and completely unfolded bovine heart cytochrome c conformations under different concentrations of urea or guanidine hydrochloride in denaturation solution can be accurately described.     

Integration of Inhibition Kinetics and Molecular Dynamics Simulations: A Urea-Mediated Folding Study on Acetaldehyde Dehydrogenase 1

Understanding the mechanism of acetaldehyde dehydrogenase 1 (ALDH1) folding is important because this enzyme is directly involved in several types of cancers and other diseases. We investigated the urea-mediated unfolding of ALDH1 by integrating kinetic inhibition studies with computational molecular dynamics (MD) simulations. Conformational changes in the enzyme structure were also analyzed using intrinsic and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence measurements. Kinetic studies revealed that the direct binding of urea to ALDH1 induces inactivation of ALDH1 in a manner of mixed-type inhibition. Tertiary structural changes associated with regional hydrophobic exposure of the active site were observed. The urea binding regions on ALDH1 were predicted by docking simulations and were partly shared with active site residues of ALDH1 and with interface residues of the oligomerization domain for tetramer formation. The docking results suggest that urea prevents formation of the ALDH1 normal shape for the tetramer state as well as entrance of the substrate into the active site. Our study provides insight into the structural changes that accompany urea-mediated unfolding of ALDH1 and the catalytic role associated with conformational changes.     

Inactivation of Recombinant Human Brain-Type Creatine Kinase During Denaturation by Guanidine Hydrochloride in a Macromolecular Crowding System

In this study, we quantitatively examined the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000) and dextran 70, on guanidine hydrochloride (GdnHCl)-induced denaturation of recombinant human brain-type creatine kinase (rHBCK). Our results showed that both PEG 2000 and dextran 70 had a protective effect on the inactivation of rHBCK induced by 0.5 M GdnHCl at 25 °C. The presence of 200 g/L PEG 2000 resulted in the retention of 35.33 % of rHBCK activity after 4 h of inactivation, while no rHBCK activity was observed after denaturation in the absence of macromolecular crowding agents. The presence of PEG 2000 and dextran 70 at a concentration of 100 g/L could decelerate the k ₂ value of the slow track to 21 and 33 %, respectively, in comparison to values obtained in the absence of crowding agents. Interestingly, inactivation of rHBCK in the presence of 200 g/L PEG 2000 followed first-order monophasic kinetics, with an apparent rate constant of 8?×?10^?5?s^?1. The intrinsic fluorescence results showed that PEG 2000 was better than dextran 70 at stabilizing rHBCK conformation. In addition, the results of the phase diagram indicate that more intermediates may be captured when rHBCK is denatured in a macromolecular crowding system. Mixed crowding agents did not produce better results than single crowding agents, but the protective effects of PEG 2000 on the inactivation and unfolding of rHBCK tended to increase as the ratio of PEG 2000 increased in the mixed crowding agent solution. Though it is not clear which crowding agents more accurately simulated the intracellular environment, this study could lead to a better understanding of protein unfolding in the intracellular environment.