Energetics of MazG Unfolding in Correlation with Its Structural Features

MazG is a homodimeric α-helical protein that belongs to the superfamily of all-α NTP pyrophosphatases. Its function has been connected to the regulation of the toxin-antitoxin module mazEF, implicated in programmed growth arrest/cell death of Escherichia coli cells under conditions of amino acid starvation. The goal of the first detailed biophysical study of a member of the all-α NTP pyrophosphatase superfamily, presented here, is to improve molecular understanding of the unfolding of this type of proteins. Thermal unfolding of MazG monitored by differential scanning calorimetry, circular dichroism spectroscopy, and fluorimetry at neutral pH in the presence of a reducing agent (dithiothreitol) can be successfully described as a reversible four-state transition between a dimeric native state, two dimeric intermediate states, and a monomeric denatured state. The first intermediate state appears to have a structure similar to that of the native state while the final thermally denatured monomeric state is not fully unfolded and contains a significant fraction of residual α-helical structure. In the absence of dithiothreitol, disulfide cross-linking causes misfolding of MazG that appears to be responsible for the formation of multimeric aggregates. MazG is most stable at pH 7-8, while at pH < 6, it exists in a molten-globule-like state. The thermodynamic parameters characterizing each step of MazG denaturation transition obtained by global fitting of the four-state model to differential scanning calorimetry, circular dichroism, and fluorimetry temperature profiles are in agreement with the observed structural characteristics of the MazG conformational states and their assumed functional role.     

Low-cost equilibrium unfolding of heme proteins using 2 μl samples

Equilibrium unfolding experiments provide access to protein thermodynamic stability revealing basic aspects of protein structure–function relationships. A limitation of these experiments stands on the availability of large amounts of protein samples. Here we present the use of the NanoDrop for monitoring guanidinium chloride-induced unfolding by Soret absorbance of monomeric heme proteins. Unfolding experiments using 2 μl of reactant are validated by fluorescence and circular dichroism spectroscopy and supported with five heme proteins including neuroglobin, cytochrome b₅, and cyanoglobin. This work guarantees 2 orders of magnitude reduction in protein expense. Promising low-cost protein unfolding experiments following other chromophores and high-throughput screenings are discussed.     

Revisiting absorbance at 230 nm as a protein unfolding probe

Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230 nm (A₂₃₀) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A₂₃₀ as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A₂₃₀. Significant changes in A₂₃₀ were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A₂₃₀ in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A₂₃₀ under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A₂₃₀ allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A₂₃₀ is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications.     

Chemical and Thermal Unfolding of a Global Staphylococcal Virulence Regulator with a Flexible C-Terminal End

SarA, a Staphylococcus aureus-specific dimeric protein, modulates the expression of numerous proteins including various virulence factors. Interestingly, S. aureus synthesizes multiple SarA paralogs seemingly for optimizing the expression of its virulence factors. To understand the domain structure/flexibility and the folding/unfolding mechanism of the SarA protein family, we have studied a recombinant SarA (designated rSarA) using various in vitro probes. Limited proteolysis of rSarA and the subsequent analysis of the resulting protein fragments suggested it to be a single-domain protein with a long, flexible C-terminal end. rSarA was unfolded by different mechanisms in the presence of different chemical and physical denaturants. While urea-induced unfolding of rSarA occurred successively via the formation of a dimeric and a monomeric intermediate, GdnCl-induced unfolding of this protein proceeded through the production of two dimeric intermediates. The surface hydrophobicity and the structures of the intermediates were not identical and also differed significantly from those of native rSarA. Of the intermediates, the GdnCl-generated intermediates not only possessed a molten globule-like structure but also exhibited resistance to dissociation during their unfolding. Compared to the native rSarA, the intermediate that was originated at lower GdnCl concentration carried a compact shape, whereas, other intermediates owned a swelled shape. The chemical-induced unfolding, unlike thermal unfolding of rSarA, was completely reversible in nature.     

Identification of a dimeric intermediate in the unfolding pathway for the calcium-binding protein S100B

The S100 proteins comprise 25 calcium-signalling members of the EF-hand protein family. Unlike typical EF-hand signalling proteins such as calmodulin and troponin-C, the S100 proteins are dimeric, forming both homo- and heterodimers in vivo. One member of this family, S100B, is a homodimeric protein shown to control the assembly of several cytoskeletal proteins and regulate phosphorylation events in a calcium-sensitive manner. Calcium binding to S100B causes a conformational change involving movement of helix III in the second calcium-binding site (EF2) that exposes a hydrophobic surface enabling interactions with other proteins such as tubulin and Ndr kinase. In several S100 proteins, calcium binding also stabilizes dimerization compared to the calcium-free states. In this work, we have examined the guanidine hydrochloride (GuHCl)-induced unfolding of dimeric calcium-free S100B. A series of tryptophan substitutions near the dimer interface and the EF2 calcium-binding site were studied by fluorescence spectroscopy and showed biphasic unfolding curves. The presence of a plateau near 1.5 M GuHCl showed the presence of an intermediate that had a greater exposed hydrophobic surface area compared to the native dimer based on increased 4,4-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid fluorescence. Furthermore, ¹H-¹⁵N heteronuclear single quantum coherence analyses as a function of GuHCl showed significant chemical shift changes in regions near the EF1 calcium-binding loop and between the linker and C-terminus of helix IV. Together these observations show that calcium-free S100B unfolds via a dimeric intermediate.     

The dimerization of ferrihaems: I. The effect of buffer ions and specific cations on deuteroferrihaem dimerization

The dimerization of deuteroferrihaem in aqueous solution has been investigated using a parameter, named the dimerization index (R_obs). This is defined as the ratio of extinction coefficients at wavelengths corresponding to Soret band maxima for the monomeric and dimeric species, respectively. For solutions containing mainly monomeric species, R_obs > 2, whereas for solutions containing mainly dimeric species R_obs < 1.A computer programme has been applied to determine values of the dimerization constant, K, defined as: $\text{K =}\text{[}\text{dimer}\text{][}\text{H}{}^{\mathrm{+}}\text{]}\text{[}\text{monomer}\text{]}{}^2$.Phosphate buffer anions and Tris · HCl buffer enhanced dimerization. Monovalent and divalent cations also increased dimerization, but in a specific manner. The magnitudes of their effects increased in the order $\text{K}{}^{\mathrm{+}}\text{< Na}{}^{\mathrm{+}}\text{< Li}{}^{\mathrm{+}}\text{<}\text{Sr}{}^{\mathrm{2+}}\text{< Mg}{}^{\mathrm{2+}}\text{? Ca}{}^{\mathrm{2+}}$. Values of K were determined for several concentrations of Na⁺ and Sr²⁺. These data are interpreted in terms of a stabilization of the ferrihaem dimer by the formation of ion triplets with the added cation ‘sandwiched’ between carboxyl residues of the adjacent ferrihaem monomeric units. General guidelines are recommended for the choice of conditions which minimize dimerization.     

The tetrameric α-helical membrane protein GlpF unfolds via a dimeric folding intermediate

Many membrane proteins appear to be present and functional in higher-order oligomeric states. While few studies have analyzed the thermodynamic stability of α-helical transmembrane (TM) proteins under equilibrium conditions in the past, oligomerization of larger polytopic monomers has essentially not yet been studied. However, it is vital to study the folding of oligomeric membrane proteins to improve our understanding of the general mechanisms and pathways of TM protein folding. To investigate the folding and stability of the aquaglyceroporin GlpF from Escherichia coli, unfolding of the protein in mixed micelles was monitored by steady-state fluorescence and circular dichroism spectroscopy as well as by seminative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. On the basis of our results, it appears most likely that GlpF unfolds in a two-step process, involving the equilibrium of tetrameric, dimeric, and monomeric GlpF species. A kinetic analysis also indicates an intermediate along the kinetic GlpF unfolding pathway, and thus, two phases are involved in GlpF unfolding. While three-state unfolding pathways and a dimeric folding intermediate are not uncommon for water-soluble proteins, a stable (un)folding intermediate with a decreased oligomeric structure has not been detected or reported for any α-helical membrane protein.     

Dimeric procaspase-3 unfolds via a four-state equilibrium process.

We have examined the folding and assembly of a catalytically inactive mutant of procaspase-3, a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and 5 M urea, consistent with an apparent three-state unfolding process. However, the midpoint of the second transition as well as the amplitude of the plateau are dependent on the protein concentration. Overall, the data are well described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. By fitting the four-state model to the experimental data, we have determined the free energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mol. The free energy change for the dissociation of the dimeric folding intermediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third step in the unfolding mechanism represents the complete unfolding of the monomeric intermediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. These results show two important points. First, dimerization of procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantly to the conformational free energy of the protein (18.8 of 25.8 kcal/mol).     

Pressure and denaturants in the unfolding of triosephosphate isomerase: the monomeric intermediates of the enzymes from Saccharomyces cerevisiae and Entamoeba histolytica

Triosephosphate isomerase (TIM) is a dimeric enzyme formed by two identical (beta/alpha)8 barrels. In this work, we compare the unfolding and refolding of the TIMs from Entamoeba histolytica (EhTIM) and baker's yeast (yTIM). A monomeric intermediate was detected in the GdnHCl-induced unfolding of EhTIM. The thermodynamic, spectroscopic, catalytic, and hydrodynamic properties of this intermediate were found to be very similar to those previously described for a monomeric intermediate of yTIM observed in GdnHCl. Monomer unfolding was reversible for both TIMs; however, the dissociation step was reversible in yTIM and irreversible in EhTIM. Monomer unfolding induced by high pressure in the presence of GdnHCl was a reversible process. DeltaGUnf, DeltaVUnf, and P1/2 were obtained for the 0.7-1.2 M GdnHCl range. The linear extrapolation of these thermodynamic parameters to the absence of denaturant showed the same values for both intermediates. The DeltaVUnfH2O values calculated for EhTIM and yTIM monomeric intermediates are the same within experimental error (-57 +/- 10 and -76 +/- 14 mL/mol, respectively). These DeltaVUnf H2O values are smaller than those reported for the unfolding of monomeric proteins of similar size, suggesting that TIM intermediates are only partially hydrated. |DeltaVUnf| increased with denaturant concentration; this behavior is probably related to structural changes in the unfolded state induced by GdnHCl and pressure. From the thermodynamic parameters that were obtained, it is predicted that in the absence of denaturants, pressure would induce monomer unfolding (P1/2 approximately 140 MPa) prior to dimer dissociation (P1/2 approximately 580 MPa). Therefore, dimerization prevents the pressure unfolding of the monomer.     

Thermodynamic and structural analysis of homodimeric proteins: Model of β-lactoglobulin

The energetics of protein homo-oligomerization was analyzed in detail with the application of a general thermodynamic model. We have studied the thermodynamic aspects of protein-protein interaction employing β-lactoglobulin A from bovine milk at pH = 6.7 where the protein is mainly in its dimeric form. We performed differential calorimetric scans at different total protein concentration and the resulting thermograms were analyzed with the thermodynamic model for oligomeric proteins previously developed. The thermodynamic model employed, allowed the prediction of the sign of the enthalpy of dimerization, the analysis of complex calorimetric profiles without transitions baselines subtraction and the obtainment of the thermodynamic parameters from the unfolding and the association processes and the compared with association parameters obtained with Isothermal Titration Calorimetry performed at different temperatures. The dissociation and unfolding reactions were also monitored by Fourier-transform infrared spectroscopy and the results indicated that the dimer of β-lactoglobulin (N₂) reversibly dissociates into monomeric units (N) which are structurally distinguishable by changes in their infrared absorbance spectra upon heating. Hence, it is proposed that β-lactoglobulin follows the conformational path induced by temperature:N₂ ? 2N ? 2D. The general model was validated with these results indicating that it can be employed in the study of the thermodynamics of other homo-oligomeric protein systems.     

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2_hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2_hPg. However, the binding strength of VEK30 (K_D = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (K_D = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH₂ and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2_hPg with nearly the same affinity as PAM (K_D = 1–2 nm). However, addition of two PAM residues (Arg¹²⁶-His¹²⁷) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2_hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg¹¹³, His¹¹⁴, Glu¹¹⁶, Arg¹²⁶, His¹²⁷), mutation of which reduced PAM binding affinity for K2_hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.     

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

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

A comparison of the folding of two knotted proteins: YbeA and YibK

The extraordinary topology of proteins belonging to the alpha/beta-knot superfamily of proteins is unexpected, due to the apparent complexities involved in the formation of a deep trefoil knot in a polypeptide backbone. Despite this, an increasing number of knotted structures are being identified; how such proteins fold remains a mystery. Studies on the dimeric protein YibK from Haemophilus influenzae have led to the characterisation of its folding pathway in some detail. To complement research into the folding of YibK, and to address whether folding pathways are conserved for members of the alpha/beta-knot superfamily, the structurally similar knotted protein YbeA from Escherichia coli has been studied. A comprehensive thermodynamic and kinetic analysis of the folding of YbeA is presented here, and compared to that of YibK. Both fold via an intermediate state populated under equilibrium conditions that is monomeric and considerably structured. The unfolding/refolding kinetics of YbeA are simpler than those found for YibK and involve two phases attributed to the formation of a monomeric intermediate state and a dimerisation step. In contrast to YibK, a change in the rate-determining step on the unfolding pathway for YbeA is observed with a changing concentration of urea. Despite this difference, both proteins fold by a mechanism involving at least one sequential monomeric intermediate that has properties similar to that observed during the equilibrium unfolding. The rate of dimerisation observed for YbeA and YibK is very similar, as is the rate constant for formation of the kinetic monomeric intermediate that precedes dimerisation. The findings suggest that relatively slow folding and dimerisation may be common attributes of knotted proteins.     

Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C_H3-C_H3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.     

Structural and Mechanical Hierarchies in the α-Crystallin Domain Dimer of the Hyperthermophilic Small Heat Shock Protein Hsp16.5

In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric α-crystallin domain as the basic structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the α-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the α-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an “all-or-none” mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force spectroscopy, we found that the α-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the α-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer.     

Unfolding energetics and conformational stability of DLC8 monomer

To understand the rules governing the protein folding process it is essential to study the stability and unfolding of small monomeric proteins. Here, I present the pH dependent thermal unfolding energetics and conformational stability analysis of monomeric Dynein light chain protein (DLC8) in the pH range 3.5-2.0. DLC8 is the smallest and the most conserved light chain among the light chains of the dynein motor assembly. Thermal unfolding of DLC8 monomer is much complex with the presence of transient intermediates, which is in contrast to the notion that small proteins unfold via simple two-state process. The unfolding seems to be more cooperative at lower pH and the temperature of highest conformational stability (T(s)) is found to be maximum (295.7 K) at pH 2.76. Stability curves have been simulated to understand the thermodynamic parameters that govern the shapes of the experimentally obtained curves. Further, an effort has been made to correlate the observed differences in the denaturation energetics with the protein sequence in order to throw light on the structure-folding paradigm of the DLC8 monomer.     

Peroxidase-like activities of iron(III)-porphyrins: Kinetics of the reduction of a peroxidatically active derivative of deuteroferriheme by phenols

The kinetics of reduction of a peroxidatically active intermediate (formed by reaction of deuteroferriheme with hydrogen peroxide) by phenol and a series of substituted phenols have been studied by stopped-flow spectrophotometry. The complex effects of varying heme concentration have been interpreted in terms of the formation of a dimeric form of the intermediate by reaction of monomeric intermediate with unoxidized ferriheme monomer. Both the relative reactivity of phenols towards the two forms of the intermediate and the rate constants for reduction of intermediate are markedly influenced by phenol substituents. Effects of pH suggest that phenolate anions are the reactive forms of the reductants. Rate constants for the monomeric intermediate reactions (k_DFH) are related to those (k_HRP) for the corresponding reductions of horseradish peroxidase Compound I by the following relation: log k_DFH = 1.53 log k_HRP ? 3.74.     

Folding and aggregation of a multi-domain engineered immunotoxin

Engineered immunotoxins with specific targeting mechanisms have potential applications for the treatment of cancer and other diseases; however, their folding behavior is often poorly understood and this presents challenges during process development, manufacturing, and formulation. Folding thermodynamics of an antibody variable domain (V_H/V_L) genetically fused to a biological toxin payload were characterized at pH 6.0 and pH 8.0 in order to assess the relative domain stabilities, along with time scales on which they fold, and the competition between aggregation and folding. The toxin and V_H/V_L domains had considerably different unfolding free energies (ΔG_UNF), leading to a thermodynamically-distinct intermediate species, with the toxin domain unfolded and the V_H/V_L folded. The intermediate is the majority species over a range of denaturant concentrations (∼4–6 M urea; ∼2–4 M guanidine HCl). Thermal unfolding resulted in reversible unfolding of the toxin domain at pH 8, but at pH 6 thermal unfolding was convoluted with aggregation due to irreversible unfolding and aggregation for the V_H/V_L domain. Chemical unfolding of both domains was more easily reversible, provided that the refold was done stepwise, allowing the antibody domain to fold first at intermediate denaturant concentration, as folding of the V_H/V_L domain played a key role in aggregation of this antibody fusion protein.     

Thermal,Chemical and pH Induced Denaturation of a Multimeric β-Galactosidase Reveals Multiple Unfolding Pathways

Background

In this case study, we analysed the properties of unfolded states and pathways leading to complete denaturation of a multimeric chick pea β-galactosidase (CpGAL), as obtained from treatment with guanidium hydrochloride, urea, elevated temperature and extreme pH.

Methodology/Principal Findings

CpGAL, a heterodimeric protein with native molecular mass of 85 kDa, belongs to α+β class of protein. The conformational stability and thermodynamic parameters of CpGAL unfolding in different states were estimated and interpreted using circular dichroism and fluorescence spectroscopic measurements. The enzyme was found to be structurally and functionally stable in the entire pH range and upto 50°C temperature. Further increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were irreversible, non-coincidental and sigmoidal. Free energy of protein unfolding (ΔG₀) and unfolding constant (K_obs) were also calculated for chemically denatured CpGAL.

Significance

The protein seems to use different pathways for unfolding in different environments and is a classical example of how the environment dictates the path a protein might take to fold while its amino acid sequence only defines its final three-dimensional conformation. The knowledge accumulated could be of immense biotechnological significance as well.     

Biophysical characterization of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> phosphoserine aminotransferase (EhPSAT): role of cofactor and domains in stability and subunit assembly

We investigated the role of the cofactor PLP and its binding domain in stability and subunit assembly of phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica. Presence of cofactor influences the tertiary structure of EhPSAT because of the significant differences in the tryptophan microenvironment and proteolytic pattern of holo- and apo-enzyme. However, the cofactor does not influence the secondary structure of the enzyme. Stability of the protein is significantly affected by the cofactor as holo-enzyme shows higher T _m and C _m values for thermal and GdnHCl-induced denaturation, respectively, when compared to the apo-enzyme. The cofactor also influences the unfolding pathway of the enzyme. Although urea-dependent unfolding of both holo- and apo-EhPSAT is a three-state process, the intermediates stabilized during unfolding are significantly different. For holo-EhPSAT a dimeric holo-intermediate was stabilized, whereas for apo-EhPSAT, a monomeric intermediate was stabilized. This is the first report on stabilization of a holo-dimeric intermediate for any aminotransferase. The isolated PLP-binding domain is stabilized as a monomer, thus suggesting that either the N-terminal tail or the C-terminal domain of EhPSAT is required for stabilization of dimeric configuration of the wild-type enzyme. To the best of our knowledge, this is a first report investigating the role of PLP and various protein domains in structural and functional organization of a member of subgroup IV of the aminotransferases.