Presence Of Guanidine Hydrochloride Research Articles

Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae

HtaA is a heme-binding protein that is part of the heme uptake system in Corynebacterium diphtheriae. HtaA contains two conserved regions (CR1 and CR2). It has been previously reported that both domains can bind heme; the CR2 domain binds hemoglobin more strongly than the CR1 domain. In this study, we report the biophysical characteristics of HtaA-CR2. UV–visible spectroscopy and resonance Raman experiments are consistent with this domain containing a single heme that is bound to the protein through an axial tyrosine ligand. Mutants of conserved tyrosine and histidine residues (Y361, H412, and Y490) have been studied. These mutants are isolated with very little heme (≤5%) in comparison to the wild-type protein (~20%). Reconstitution after removal of the heme with butanone gave an alternative form of the protein. The HtaA-CR2 fold is very stable; it was necessary to perform thermal denaturation experiments in the presence of guanidinium hydrochloride. HtaA-CR2 unfolds extremely slowly; even in 6.8M GdnHCl at 37°C, the half-life was 5h. In contrast, the apo forms of WT HtaA-CR2 and the aforementioned mutants unfolded at much lower concentrations of GdnHCl, indicating the role of heme in stabilizing the structure and implying that heme transfer is effected only to a partner protein in vivo.

Structural⿿functional insights and studies on saccharide binding of Sophora japonica seed lectin

Functional and conformational transitions of the Sophora japonica seed lectin (SJL) were studied in detail using bioinformatics and biophysical tools. Homology model of the lectin displayed all the characteristics of the legume lectin monomer and the experimental observations correlated well with the structural information. In silico studies were performed by protein-ligand docking, calculating the respective binding energies and the residues involved in the interactions were derived from LigPlot+ analysis. Fluorescence titrations showed three times higher affinity of T-antigen disaccharide than N-acetyl galactosamine (GalNAc) towards SJL indicating extended sugar binding site of the lectin. Thermodynamic parameters of T-antigen binding to SJL indicated the process to be endothermic and entropically driven while those of GalNAc showed biphasic process. SDS-PAGE showed post-translationally modified homotetrameric species of the lectin under native conditions. In presence of guanidine hydrochloride (0.5⿿5.0M), the tetramer first dissociated into dimers followed by unfolding of the protein as indicated by size exclusion chromatography, fluorescence and CD spectroscopy. Different structural rearrangements were observed during thermal denaturation of SJL at physiological pH 7.2, native pH 8.5 and molten globule inducing pH 1.0. Topological information revealed by solute quenching studies at respective pH indicated differential hydrophobic environment and charge density around tryptophan residues.

Residual structures in the unfolded state of starch-binding domain of glucoamylase revealed by near-UV circular dichroism and protein engineering techniques

Protein folding is a thermodynamic process driven by energy gaps between the native and unfolded states. Although a wealth of information is available on the structure of folded species, there is a paucity of data on unfolded species. Here, we analyzed the structural properties of the unfolded state of the starch-binding domain of glucoamylase from Aspergillus niger (SBD) formed in the presence of guanidinium hydrochloride (GuHCl). Although far-UV CD and intrinsic tryptophan fluorescence spectra as well as small angle X-ray scattering suggested that SBD assumes highly unfolded structures in the presence of GuHCl, near-UV circular dichroism of wild-type SBD suggested the presence of residual structures in the unfolded state. Analyses of the unfolded states of tryptophan mutants (W543L, W563A, W590A and W615L) using Similarity Parameter, a modified version of root mean square deviation as a measure of similarity between two spectra, suggested that W543 and W563 have preferences to form native-like residual structures in the GuHCl-unfolded state. In contrast, W615 was entirely unstructured, while W590 tended to form non-native ordered structures in the unfolded state. These data and the amino acid sequence of SBD suggest that local structural propensities in the unfolded state can be determined by the probability of the presence of hydrophobic or charged residues nearby tryptophan residues.

Liquid–liquid equilibria, electrical conductivity, and refractive indices of Poly(ethylene glycol) + sodium sulfate + guanidine hydrochloride aqueous two-phase systems: Correlation and thermodynamic modeling

The initial recovery steps for recombinant protein were carried out using aqueous two-phase systems in the presence of guanidine hydrochloride. To investigate the phase behavior of such systems, equilibrium data was obtained for molecular weight fractions of PEG (3000 and 6000) + sodium sulfate (pH 7) + guanidine hydrochloride (5% and 10% mass) + water at 25 °C. Displacement of the binodal toward higher concentrations through an increase in guanidine hydrochloride concentration was clearly observed. A slightly larger two-phase was obtained as the PEG molecular weight increased from PEG 3000 to 6000. The experimental binodal data of the system was successfully correlated with Bleasdale equations (high R2 and low SE). The effects of molecular weight, guanidine hydrochloride concentration, tie-line length, and slope of tie-line on the partition behaviors of guanidine hydrochloride were examined at constant pH. The compositions of each aqueous phase were calculated using two thermodynamic models for the activity coefficient. The calculated results showed that the extended Pitzer and extended UNIQUAC models can predict the data in the quaternary system well. The fitted binary interaction parameters of the models were reported.

Contribution of Electrostatics in the Fibril Stability of a Model Ionic-Complementary Peptide.

In this work we quantified the role of electrostatic interactions in the self-assembly of a model amphiphilic peptide (RADA 16-I) into fibrillar structures by a combination of size exclusion chromatography and molecular simulations. For the peptide under investigation, it is found that a net charge of +0.75 represents the ideal condition to promote the formation of regular amyloid fibrils. Lower net charges favor the formation of amorphous precipitates, while larger net charges destabilize the fibrillar aggregates and promote a reversible dissociation of monomers from the ends of the fibrils. By quantifying the dependence of the equilibrium constant of this reversible reaction on the pH value and the peptide net charge, we show that electrostatic interactions contribute largely to the free energy of fibril formation. The addition of both salt and a charged destabilizer (guanidinium hydrochloride) at moderate concentration (0.3-1 M) shifts the monomer-fibril equilibrium toward the fibrillar state. Whereas the first effect can be explained by charge screening of electrostatic repulsion only, the promotion of fibril formation in the presence of guanidinium hydrochloride is also attributed to modifications of the peptide conformation. The results of this work indicate that the global peptide net charge is a key property that correlates well with the fibril stability, although the peptide conformation and the surface charge distribution also contribute to the aggregation propensity.

Structure and functional investigation of ligand binding by a family 35 carbohydrate binding module (CtCBM35) of β-mannanase of family 26 glycoside hydrolase from Clostridium thermocellum.

Functional attributes of recombinant CtCBM35 (family 35 carbohydrate binding module) of β-mannanase of family 26 Glycoside Hydrolase from Clostridium thermocellum were deduced by biochemical and in silico approaches. Ligand-binding analysis of expressed CtCBM35 analyzed by affinity-gel electrophoresis and fluorescence spectroscopy exhibited association constants Ka ~ 1.2·10(5)and 3.0·10(5)M(-1) with locust bean galactomannan and mannotriose, respectively. However, CtCBM35 showed low ligand-binding affinity with insoluble ivory nut mannan with Ka of 5.0·10(-5)M(-1). Unfolding transition analysis by fluorescence spectroscopy explained the conformational changes of CtCBM35 in the presence of guanidine hydrochloride (5 M) and urea (6.25M). This explained that CtCBM35 has good conformational stability and requires higher free energy of denaturation to invoke unfolding. The three-dimensional (3-D) model of CtCBM35 from C. thermocellum generated by Modeller9v8 displayed predominance of β-sheets arranged as β-jelly-roll fold. The secondary structure of CtCBM35 by PredictProtein showed the presence of two α-helices (3%), 12 β-sheets (45%), and 15 random coils (52%). Secondary structural element analysis of cloned, expressed, and purified recombinant CtCBM35 by circular dichroism also corroborated the in silico predicted secondary structure. Multiple sequence alignment of CtCBM35 showed conserved residues (Tyr123, Gly124, and Phe125), which are commonly observed in mannan specific CBMs. Docking analysis of CtCBM35 with manno-oligosaccharide displayed the involvement of Tyr26, Gln29, Asn43, Trp66, Tyr68, Leu69, Arg76, and Leu127 residues, making polar contact with the ligand molecules. Ligand docking analysis of CtCBM35 exhibiting higher binding affinity with mannotriose and galactomannan (Man-Gal-Man moiety) substantiated the affinity binding and fluorescence results, displaying similar values of Ka.

The Quaternary Structure of the Recombinant Bovine Odorant-Binding Protein Is Modulated by Chemical Denaturants

A large group of odorant-binding proteins (OBPs) has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP) has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer’s β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl). Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M). This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

Selective Reduction of a,b-Unsaturated Steroidal Carbonyl Compounds by NaBH4 in Presence of Guanidine Hydrochloride in Dioxane

Selective Reduction of a,b-Unsaturated Steroidal Carbonyl Compounds by NaBH4 in Presence of Guanidine Hydrochloride in Dioxane

Quantitative Evaluation of Myoglobin Unfolding in the Presence of Guanidinium Hydrochloride and Ionic Liquids in Solution

The use of ionic liquids in biochemical and biophysical applications has increased dramatically in recent years due to their interesting properties. We report results of a thermodynamic characterization of the chaotrope-induced denaturation of equine myoglobin in two different ionic liquid aqueous environments using a combined absorption/fluorescence spectroscopic approach. Denaturation by guanidinium hydrochloride was monitored by loss of heme absorptivity and limited unfolding structural information was obtained from Förster resonance energy transfer experiments. Results show that myoglobin unfolding is generally unchanged in the presence of ethylmethylimidazolium acetate (EMIAc) in aqueous solution up to 150 mM concentration but is facilitated by butylmethylimidazolium boron tetrafluoride (BMIBF4) in solution. The presence of 150 mM BMIBF4 alone does not induce unfolding but destabilizes the structure as observed by a decrease in threshold denaturant concentration for unfolding and an 80% decrease in the magnitude of ΔGunfolding from 44 kJ/mol in the absence of BMIBF4 to 8 kJ/mol in the presence of 150 mM BMIBF4. Thus, the BMIBF4 significantly destabilizes the myoglobin structure while the EMIAc does not, likely due to differences in anion interaction capabilities. This is confirmed with control studies using NaAc and LiBF4 solutions. EMIAc may be chosen as cosolvent additive with minimal effects on protein structure while BMIBF4 may be used as a supplement in protein folding experiments, potentially allowing access to proteins which have been traditionally difficult to denature as well as designing ionic liquids to match protein characteristics.

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.

Comparison of the Thermal Denaturing of Human Serum Albumin in the Presence of Guanidine Hydrochloride and 1-Butyl-3-methylimidazolium Ionic Liquids

The interaction of proteins with aqueous solutions of ionic liquids (ILs) has attracted considerable recent attention owing to the challenges of finding biocompatible water-free ILs. These systems remain of great interest because of the potential for using ILs as designer solvents for biocatalytic processes. Increasing evidence demonstrates that aqueous solutions of water-miscible ILs, such as the well-studied 1-alkyl-3-methylimidazolium ILs, disrupt the native fold of proteins and can drive the formation of non-native aggregates that could negatively impact catalytic function. Here, we present a study comparing the thermal unfolding of human serum albumin (HSA) in a 1 M solution of the protein denaturant guanidine hydrochloride with two 1 M aqueous solutions of 1-butyl-3-methylimidazolium ILs, namely the chloride and the acetate. Small-angle neutron scattering (SANS) measurements found qualitative agreement between the thermally driven unfolding process for the three denaturants, as well as with a Tris buffer solution. HSA irreversibly aggregates and unfolds in the three denaturant solutions upon heating to temperatures below that required to drive the same process in a simple Tris buffer solution. The results reveal subtle differences in the interaction of the ILs and guanidine hydrochloride with the protein, although the final states of the protein were similar in all cases. The results indicate that the ions of water-miscible ILs and guanidine hydrochloride have specific roles in disrupting protein structure and driving aggregation. The experimental approach employed has the potential to provide new insights into protein interactions with ionic liquids that may aid in the search for more biocompatible ionic liquids.

Structural stability of commercial ficin under different denaturing conditions

Aim: To study the conformational stability of commercial ficin (CF) in the presence of guanidine hydrochloride (GdnHCl), urea, ethanol or at acidic pH and compare it with that reported for the major ficin fraction (MFF) obtained by purification of CF. Methods: Far-UV and near-UV CD spectral signals, intrinsic fluorescence, acrylamide quenching and enzymatic activity were used to study the effects of chemical denaturants and acidic pH on CF. The data were analyzed using two-state hypothesis, if required. Results: GdnHCl produced complete loss of secondary and tertiary structures of the protein. Loss of all enzymatic activity was observed at 4 M GdnHCl. CF showed structural resistance against 9 M urea and 50 % ethanol. Significant differences in emission maximum, acrylamide quenching, denaturation transition and enzymatic activity were noted between CF and MFF treated with different denaturants. CF showed greater stability at acidic pH than MFF. Conclusion: We conclude that CF is more structurally resistant than MFF against chemical and acid denaturations.

Functional and Structural Characterization of Helicobacter pylori ClpX: A Molecular Chaperone of Hsp100 Family

ClpX is a general stress protein which belongs to the heat shock protein, Clp/Hsp100 family of molecular chaperones. ClpX, in association with ClpP degrades proteins in an ATP dependent manner. Some members of the Clp family have been shown to be involved in the pathogenesis of many bacteria. The Helicobacter pylori genome demonstrates the presence of ClpX along with ClpA, ClpB and ClpP, the other members of the caseinolytic protease family. H. pylori ClpX is a 386 amino acid long protein. In this study, we have over-expressed H. pylori ClpX in E. coli, purified the recombinant protein to homogeneity and functionally characterized it. The recombinant H. pylori ClpX showed an inherent ATPase activity and prevented the heat induced aggregation of a model protein in vitro. The chaperonic activity of H. pylori ClpX was dependent on ATP hydrolysis and involved hydrophobic interaction with the substrate protein. Biophysical studies reveal the secondary structure tolerance of ClpX at various temperatures and in the presence of guanidine hydrochloride. The study demonstrates that H. pylori ClpX manifests chaperonic activity in the absence of any adaptor protein.

Length of polyglutamine tract affects secondary and tertiary structures of huntingtin protein

The role of polyglutamine (polyQ) tract on protein stability and disease pathology remains ambiguous. We monitored the unfolding/refolding patterns of huntingtin proteins with varying polyQ lengths. In the presence of urea, minor differences in unfolding and refolding efficiencies were observed. However, in the presence of guanidinium hydrochloride, the protein with a longer polyQ stretch was able to regain its secondary but not tertiary structure on step-wise removal of denaturant. Thus, in case of Huntington's disease, the higher aggregation propensity of the mutant protein is likely to be due to the lower stability of the protein due to elongated polyQ tract.

Four-State Folding of a SH3 Domain: Salt-Induced Modulation of the Stabilities of the Intermediates and Native State

Unstable intermediates on the folding pathways of proteins can be stabilized sufficiently so that they accumulate to detectable extents by the addition of a suitable cosolute. Here, the effect of sodium sulfate (Na(2)SO(4)) on the folding of the SH3 domain of PI3 kinase was investigated in the presence of guanidine hydrochloride (GdnHCl) using intrinsic tyrosine fluorescence and 1-anilinonaphthalene-8-sulfonate (ANS) binding. The free energy of unfolding in water of the native state (N) increases linearly with Na(2)SO(4) concentration, indicating stabilization via the Hofmeister effect. The addition of 0.5 M Na(2)SO(4) causes accumulation of an early intermediate L, which manifests itself as (1) a sub-millisecond change in tyrosine and ANS fluorescence and (2) a curvature in the chevron plot. It is shown that L is a specific structural component of the initially collapsed ensemble. An intermediate, M, also accumulates in unfolding studies conducted in the presence of 0.5 M Na(2)SO(4) and manifests itself by causing a curvature in the unfolding arm of the chevron. M is shown to be a wet molten globule that binds to ANS under unfolding conditions and is stabilized to the same extent as N in the presence of Na(2)SO(4). A four-state U ↔ L ↔ M ↔ N scheme satisfactorily modeled the kinetic data. Thus, the folding of the PI3K SH3 domain in the presence of salt commences via the formation of a structured intermediate ensemble L, which accumulates before the rate-limiting step of folding. L subsequently proceeds to N via the late intermediate M that forms after the rate-limiting transition of folding.

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.

Characterization of Membrane Protein Non-native States. 1. Extent of Unfolding and Aggregation of Rhodopsin in the Presence of Chemical Denaturants

Little is known about the general folding mechanisms of helical membrane proteins. Unfolded, i.e., non-native states, in particular, have not yet been characterized in detail. Here, we establish conditions under which denatured states of the mammalian membrane protein rhodopsin, a prototypic G protein coupled receptor with primary function in vision, can be studied. We investigated the effects of the chemical denaturants sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride (GuHCl), and trifluoroacetic acid (TFA) on rhodopsin's secondary structure and propensity for aggregation. Ellipticity at 222 nm decreases in the presence of maximum concentrations of denaturants in the order TFA > GuHCl > urea > SDS + urea > SDS. Interpretation of these changes in ellipticity in terms of helix loss is challenged because the addition of some denaturants leads to aggregation. Through a combination of SDS-PAGE, dependence of ellipticity on protein concentration, and 1D (1)H NMR we show that aggregates form in the presence of GuHCl, TFA, and urea but not in any concentration of SDS, added over a range of 0.05%-30%. Mixed denaturant conditions consisting of 3% SDS and 8 M urea, added in this order, also did not result in aggregation. We conclude that SDS is able to prevent the exposure of large hydrophobic regions present in membrane proteins which otherwise leads to aggregation. Thus, 30% SDS and 3% SDS + 8 M urea are the denaturing conditions of choice to study maximally unfolded rhodopsin without aggregation.

Crystallization of the Membrane-Associated Annexin B1: Roles of Additive Screen, Dynamic Light Scattering, and Bioactivity Assay

Annexin B1 (AnxB1) is a calcium-dependent phospholipid binding protein from Taenia solium cysticercus and has been reported to possess anticoagulant activity, to inhibit phospholipase A2, and to regulate membrane transport. Native AnxB1 and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. The results of dynamic light scattering analysis showed that Hepes buffer combined with low concentration salts (NaCl or CaCl2) was beneficial for preventing aggregation and for AnxB1 stabilization in the storage. After the additive screen, crystals have been yielded in the presence of guanidine hydrochloride (Gn-HCl). We determined that a low concentration of Gn-HCl significantly delayed clotting time and increased anticoagulant activity. Analysis of the crystal showed that in the presence of Gn-HCl, AnxB1 crystallizes in orthorhombic space group, which is modified from the cubic space group for crystals grown in the absence of Gn-HCl. A high quality data set (at 1.9 A) has been co...

An efficient and green approach for the synthesis of vinyl aryl ethers in the presence of guanidine hydrochloride

A stereoselective procedure for vinyl aryl ether bond formation by direct coupling of dialkyl acetylenedicarboxylates and substituted phenols or naphthols under mild reaction conditions has been developed. Using guanidine hydrochloride as a new catalyst and water-acetone as green solvent, dialkyl acetylenedicarboxylates were reacted with substituted phenols or naphthols in room temperature to produce vinyl aryl ethers in good to excellent yields.

A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon

Zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene) has long been known as an excellent colorimetric reagent for the detection of zinc and copper ions in aqueous solution. To extend the chelator’s versatility to the quantification of metal ions in metalloproteins, the spectral properties of Zincon and its complexes with Zn2+, Cu2+, and Co2+ were investigated in the presence of guanidine hydrochloride and urea, two common denaturants used to labilize metal ions in proteins. These studies revealed the detection of metals to be generally more sensitive with urea. In addition, pH profiles recorded for these metals indicated the optimal pH for complex formation and stability to be 9.0. As a consequence, an optimized method that allows the facile determination of Zn2+, Cu2+, and Co2+ with detection limits in the high nanomolar range is presented. Furthermore, a simple two-step procedure for the quantification of both Zn2+ and Cu2+ within the same sample is described. Using the prototypical Cu2+/Zn2+–protein superoxide dismutase as an example, the effectiveness of this method of dual metal quantification in metalloproteins is demonstrated. Thus, the spectrophotometric determination of metal ions with Zincon can be exploited as a rapid and inexpensive means of assessing the metal contents of zinc-, copper-, cobalt-, and zinc/copper-containing proteins.