Expression, Purification and Biophysical Characterisation of Klebsiella Pneumoniae Protein Adenylyltransferase: A Systematic Integration of Empirical and Computational Modelling Approaches.

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.

Unveiling the potential of Muscadine grape Skin extract as an innovative therapeutic intervention in cancer treatment.

The use of muscadine grape extracts (MGSE). in cancer treatment has gained attention due to its distinctive composition of polyphenols and antioxidants. This review analyses the reported anti-cancer properties of MGSE. The study commences by reviewing the phytochemical composition of MGSE, highlighting the presence of resveratrol and ellagic acid. Furthermore, the review underscores the mechanism of action of these active compounds in MGSE in combating cancer cells. The anti-cancer potential of MGSE compared to other plant extracts is also discussed. In addition, it highlights MGSE's superiority and distinct phytochemical composition in preventing cancer growth by comparing its anti-cancer compounds with those of other anti-cancer medicinal plants. Lastly, the combinatory approaches of MGSE with traditional cancer therapies, its safety, and its possible side effects were highlighted. This work provides an understanding of the anti-cancer properties of MGSE, positioning it as a valuable and unique challenge within the field of cancer therapy.

Computational identification of potential inhibitors targeting cdk1 in colorectal cancer.

Introduction: Despite improved treatment options, colorectal cancer (CRC) remains a huge public health concern with a significant impact on affected individuals. Cell cycle dysregulation and overexpression of certain regulators and checkpoint activators are important recurring events in the progression of cancer. Cyclin-dependent kinase 1 (CDK1), a key regulator of the cell cycle component central to the uncontrolled proliferation of malignant cells, has been reportedly implicated in CRC. This study aimed to identify CDK1 inhibitors with potential for clinical drug research in CRC. Methods: Ten thousand (10,000) naturally occurring compounds were evaluated for their inhibitory efficacies against CDK1 through molecular docking studies. The stability of the lead compounds in complex with CDK1 was evaluated using molecular dynamics simulation for one thousand (1,000) nanoseconds. The top-scoring candidates' ADME characteristics and drug-likeness were profiled using SwissADME. Results: Four hit compounds, namely, spiraeoside, robinetin, 6-hydroxyluteolin, and quercetagetin were identified from molecular docking analysis to possess the least binding scores. Molecular dynamics simulation revealed that robinetin and 6-hydroxyluteolin complexes were stable within the binding pocket of the CDK1 protein. Discussion: The findings from this study provide insight into novel candidates with specific inhibitory CDK1 activities that can be further investigated through animal testing, clinical trials, and drug development research for CRC treatment.

Molecular dynamics-derived pharmacophores of Schistosoma glutathione transferase in complex with bromosulfophthalein: Screening and analysis of potential inhibitors.

Schistosoma glutathione transferases (GSTs) have been identified as attractive drug targets for the design of novel antischistosomals. Here, we used in silico methods to validate the discriminative inhibitory properties of bromosulfophthalein (BSP) against the 26-kDa GST from S. japonicum (Sj26GST), and the 28-kDa GST from S. haematobium (Sh28GST), versus human GST (hGST) isoforms alpha (hGSTA), mu (hGSTM) and pi (hGSTP). The use of BSP as an archetypal selective inhibitor was harnessed to produce molecular dynamics-derived pharmacophores of the two targets. Pharmacophore-based screening using a large dataset of experimental and approved drug compounds was performed to produce a shortlist of candidates. The top candidate for each target was prioritised via molecular docking, yielding guanosine-3'-monophosphate-5'-diphosphate (G3D) for Sj26GST, and quercetin-3'-O-phosphate (Q3P) for Sh28GST. Comparative molecular dynamics studies of both candidates compared to BSP showed similar characteristics of binding stability and strength, suggesting their potential to emulate the inhibitory effects of BSP.

Nuclear localisation sequences of chloride intracellular channels 1 and 4 facilitate nuclear import via interactions with import mediator importin-α: An empirical and theoretical perspective.

Chloride intracellular channel proteins (CLICs) display ubiquitous expression, with each member exhibiting specific subcellular localisation. While all CLICs, except CLIC3, exhibit a highly conserved putative nuclear localisation sequence (NLS), only CLIC1, CLIC3 and CLIC4 exist within the nucleus. The CLIC4 NLS, 199-KVVAKKYR-206, appears crucial for nuclear entry and interacts with mouse nuclear import mediator Impα isoform 1, omitting the IBB domain (mImpα1ΔIBB). The essential nature of the basic residues in the CLIC4 NLS has been established by the fact that mutating out these residues inhibits nuclear import, which in turn is linked to cutaneous squamous cell cancer. Given the conservation of the CLIC NLS, CLIC1 likely follows a similar import pathway to CLIC4. Peptides of the CLIC1 (Pep1; Pep1_S C/S mutant) and CLIC4 (Pep4) NLSs were designed to examine binding to human Impα isoform 1, omitting the IBB domain (hImpα1ΔIBB). Molecular docking indicated that the core CLIC NLS region (KKYR) forms a similar binding pattern to both mImpα1ΔIBB and hImpα1ΔIBB. Fluorescence quenching demonstrated that Pep1_S (Kd ≈ 237 µM) and Pep4 (Kd ≈ 317 µM) bind hImpα1ΔIBB weakly. Isothermal titration calorimetry confirmed the weak binding interaction between Pep4 and hImpα1ΔIBB (Kd ≈ 130 µM) and the presence of a proton-linked effect. This weak interaction may be due to regions distal from the CLIC NLS needed to stabilise and strengthen hImpα1ΔIBB binding. Additionally, this NLS may preferentially bind another hImpα isoform with different flexibility properties.

Biophysical description of Bromosulfophthalein interaction with the 28-kDa glutathione transferase from Schistosoma japonicum.

Glutathione transferases (GSTs) are major detoxification enzymes vital for the survival and reproduction of schistosomes during infection in humans. Schistosoma encode two GST isoenzymes, the 26- and 28-kDa isoforms, that show different substrate specificities and cellular localisations. Bromosulfophthalein (BSP) has been identified and characterised as a potent 26-kDa Schistosoma japonicum GST (Sj26GST) inhibitor with an anthelmintic potential. This study describes the structure, function, and ligandin properties of the 28-kDa Schistosoma japonicum GST (Sj28GST) towards BSP. Enzyme kinetics show that BSP is a potent enzyme inhibitor, with a specific activity decreases from 60.4 µmol/min/mg to 0.0742 µmol/min/mg and an IC50 in the micromolar range of 0.74 µM. Far-UV circular dichroism confirmed that purified Sj28GST follows a typical GST fold, which is predominantly alpha-helical. Fluorescence spectroscopy suggests that BSP binding occurs at a site distinct from the glutathione-binding site (G-site); however, the binding does not alter the local G-site environment. Isothermal titration calorimetry studies show that the binding of BSP to Sj28GST is exergonic (∆G°= -33 kJ/mol) and enthalpically-driven, with a stoichiometry of one BSP per dimer. The stability of Sj28GST (∆G(H2O) = 4.7 kcal/mol) is notably lower than Sj26GST, owing to differences in the enzyme's dimeric interfaces. We conclude that Sj28GST shares similar biophysical characteristics with Sj26GST based on its kinetic properties and susceptibility to low concentrations of BSP. The study supports the potential benefits of re-purposing BSP as a potential drug or prodrug to mitigate the scourge of schistosomiasis.

Caseinolytic Proteins (Clp) in the Genus Klebsiella: Special Focus on ClpK.

Caseinolytic proteins (Clp), which are present in both prokaryotes and eukaryotes, play a major role in cell protein quality control and survival of bacteria in harsh environmental conditions. Recently, a member of this protein family, ClpK was identified in a pathogenic strain of Klebsiella pneumoniae which was responsible for nosocomial infections. ClpK is linked to the thermal stress survival of this pathogen. The genome wide analysis of Clp proteins in Klebsiella spp. indicates that ClpK is present in only 34% of the investigated strains. This suggests that the uptake of the clpk gene is selective and may only be taken up by a pathogen that needs to survive harsh environmental conditions. In silico analyses and molecular dynamic simulations show that ClpK is mainly α-helical and is highly dynamic. ClpK was successfully expressed and purified to homogeneity using affinity and anion exchange chromatography. Biophysical characterization of ClpK showed that it is predominantly alpha-helical, and this is in agreement with in silico analysis of the protein structure. Furthermore, the purified protein is biologically active and hydrolyses ATP in a concentration- dependent manner.

Targeting the SARS-CoV-2 main protease using FDA-approved Isavuconazonium, a P2-P3 α-ketoamide derivative and Pentagastrin: An in-silico drug discovery approach.

The SARS-CoV-2 main protease (Mpro) is an attractive target towards discovery of drugs to treat COVID-19 because of its key role in virus replication. The atomic structure of Mpro in complex with an α-ketoamide inhibitor (Lig13b) is available (PDB ID:6Y2G). Using 6Y2G and the prior knowledge that protease inhibitors could eradicate COVID-19, we designed a computational study aimed at identifying FDA-approved drugs that could interact with Mpro. We searched the DrugBank and PubChem for analogs and built a virtual library containing ∼33,000 conformers. Using high-throughput virtual screening and ligand docking, we identified Isavuconazonium, a ketoamide inhibitor (α-KI) and Pentagastrin as the top three molecules (Lig13b as the benchmark) based on docking energy. The ΔGbind of Lig13b, Isavuconazonium, α-KI, Pentagastrin was -28.1, -45.7, -44.7, -34.8 kcal/mol, respectively. Molecular dynamics simulation revealed that these ligands are stable within the Mpro active site. Binding of these ligands is driven by a variety of non-bonded interaction, including polar bonds, H-bonds, van der Waals and salt bridges. The overall conformational dynamics of the complexed-Mpro was slightly altered relative to apo-Mpro. This study demonstrates that three distinct classes molecules, Isavuconazonium (triazole), α-KI (ketoamide) and Pentagastrin (peptide) could serve as potential drugs to treat patients with COVID-19.

Comparative Characterization of Plasmodium falciparum Hsp70-1 Relative to E. coli DnaK Reveals the Functional Specificity of the Parasite Chaperone.

Hsp70 is a conserved molecular chaperone. How Hsp70 exhibits specialized functions across species remains to be understood. Plasmodium falciparum Hsp70-1 (PfHsp70-1) and Escherichia coli DnaK are cytosol localized molecular chaperones that are important for the survival of these two organisms. In the current study, we investigated comparative structure-function features of PfHsp70-1 relative to DnaK and a chimeric protein, KPf, constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of the three Hsp70s exhibited similar secondary and tertiary structural folds. However, compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. In addition, PfHsp70-1 preferentially bound to asparagine rich peptide substrates, as opposed to DnaK. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. Co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. However, a combination of supplementary GroEL plus DnaK improved folding of PfAdoMetDC. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.

Double trouble? Gag in conjunction with double insert in HIV protease contributes to reduced DRV susceptibility.

HIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38↑N↑L) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naïve infant. Isothermal titration calorimetry showed that 10% less of L38↑N↑L protease was in the active conformation as compared with a reference strain. L38↑N↑L protease displayed a ±50% reduction in KM and kcat The catalytic efficiency (kcat/KM) of L38↑N↑L protease was not significantly different from that of wild type although there was a 42% reduction in specific activity for the variant. An in vitro phenotypic assay showed the L38↑N↑L protease to be susceptible to lopinavir (LPV), atazanavir (ATV) and darunavir in the context of an unrelated Gag. However, in the presence of the related Gag, L38↑N↑L showed reduced susceptibility to darunavir while remaining susceptible to LPV and ATV. Furthermore, a reduction in viral replication capacity (RC) was observed in combination with the related Gag. The reduced susceptibility to darunavir and decrease in RC may be due to PTAPP duplication in the related Gag. The present study shows the importance of considering the Gag region when looking at drug susceptibility of HIV-1 protease variants.

Structural and biochemical characterization of Plasmodium falciparum Hsp70-x reveals functional versatility of its C-terminal EEVN motif.

Plasmodium falciparum, the main agent of malaria expresses six members of the heat shock protein 70 (Hsp70) family. Hsp70s serve as protein folding facilitators in the cell. Amongst the six Hsp70 species that P. falciparum expresses, Hsp70-x (PfHsp70-x), is partially exported to the host red blood cell where it is implicated in host cell remodeling. Nearly 500 proteins of parasitic origin are exported to the parasite-infected red blood cell (RBC) along with PfHsp70-x. The role of PfHsp70-x in the infected human RBC remains largely unclear. One of the defining features of PfHsp70-x is the presence of EEVN residues at its C-terminus. In this regard, PfHsp70-x resembles canonical eukaryotic cytosol-localized Hsp70s which possess EEVD residues at their C-termini in place of the EEVN residues associated with PfHsp70-x. The EEVD residues of eukaryotic Hsp70s facilitate their interaction with co-chaperones. Characterization of the role of the EEVN residues of PfHsp70-x could provide insights into the function of this protein. In the current study, we expressed and purified recombinant PfHsp70-x (full length) and its EEVN minus form (PfHsp70-xT ). We then conducted structure- function assays towards establishing the role of the EEVN motif of PfHsp70-x. Our findings suggest that the EEVN residues of PfHsp70-x are important for its ATPase activity and chaperone function. Furthermore, the EEVN residues are crucial for the direct interaction between PfHsp70-x and human Hsp70-Hsp90 organizing protein (hHop) in vitro. Hop facilitates functional cooperation between Hsp70 and Hsp90. However, it remains to be established if PfHsp70-x and hHsp90 cooperate in vivo.

An update on the biophysical character of the human eukaryotic elongation factor 1 beta: Perspectives from interaction with elongation factor 1 gamma.

The ß-subunit of the human eukaryotic elongation factor 1 complex (heEF1ß) plays a central role in the elongation step in eukaryotic protein biosynthesis, which essentially involves interaction with the α- and γ-subunits (eEF1γ). To biophysically characterize heEF1ß, we constructed 3 Escherichia coli expression vector systems for recombinant expression of the full length (FL-heEF1ß), N-terminus (NT-heEF1ß), and the C-terminus (CT-heEF1ß) regions of the protein. Our results suggest that heEF1ß is predominantly alpha-helical and possesses an accessible hydrophobic cavity in the CT-heEF1ß. Both FL-heEF1ß and NT-heEF1ß form dimers of size 62 and 30 kDa, respectively, but the CT-heEF1ß is monomeric. FL-heEF1ß interacts with the N-terminus glutathione transferase-like domain of heEF1γ (NT-heEF1γ) to form a 195-kDa complex or a 230-kDa complex in the presence of oxidized glutathione. On the other hand, NT-heEF1ß forms a 170-kDa complex with NT-heEF1γ and a high molecular weight aggregate of size greater than 670 kDa. Surface plasmon resonance analysis confirmed that (by fitting the Langmuir 1:1 model) FL-heEF1ß associated with monomeric or dimeric NT-heEF1γ at a rapid rate and slowly dissociated, suggesting strong functional affinity (KD  = 9.6 nM for monomeric or 11.3 nM for dimeric NT-heEF1γ). We postulate that the N-terminus region of heEF1ß may be responsible for its dimerization and the C-terminus region of heEF1ß modulates the formation of an ordered heEF1ß-γ oligomer, a structure that may be essential in the elongation step of eukaryotic protein biosynthesis.

The effects of mutating Tyr9 and Arg15 on the structure, stability, conformational dynamics and mechanism of GSTA3-3.

Glutathione S-transferase A3-3 is the most catalytically efficient steroid isomerase enzyme known in humans, transforming Δ5-androstene-3-17-dione into Δ4-androstene-3-17-dione. GSTA3-3 catalyzes this reaction with ten-fold greater efficiency than GSTA1-1, its closest competitor in the Alpha class of GSTs. In order to examine the differences between Alpha class GSTs and to better elucidate the mechanism of GSTA3-3 the roles of Tyr9 and Arg15 were examined. Tyr9 is the major catalytic residue of Alpha class GSTs and Arg15 is proposed to be catalytically important to GSTA3-3 but never before experimentally examined. While the structure and stability of the Alpha class enzymes are highly comparable, subtle differences at the G-site of the enzymes account for GSTA3-3 having a ten-fold greater affinity for the substrate GSH. Y9F and R15L mutations, singly or together, have no effect on the structure and stability of GSTA3-3 (the same effect they have on GSTA1-1) despite the R15L mutation removing an interdomain salt-bridge at the active site. Hydrogen-deuterium exchange mass spectrometry also revealed that neither mutation had a significant effect on the conformational dynamics of GSTA3-3. The R15L and Y9F mutations are equally important to the specific activity of the steroid isomerase reaction; however, Arg15 is more important for lowering the pKa of GSH. Lowering the pKa of GSH being how GSTs catalyze their reactions. Additionally, there is evidence to suggest that Arg15 is integral to allowing GSTA3-3 to differentiate between Δ5-androstene-3-17-dione and Δ4-androstene-3-17-dione, indicating that Arg15 is a more important active-site residue than previously known.

Energetics of Glutathione Binding to Human Eukaryotic Elongation Factor 1 Gamma: Isothermal Titration Calorimetry and Molecular Dynamics Studies.

The energetics of ligand binding to human eukaryotic elongation factor 1 gamma (heEF1γ) was investigated using reduced glutathione (GSH), oxidised glutathione (GSSG), glutathione sulfonate and S-hexylglutathione as ligands. The experiments were conducted using isothermal titration calorimetry, and the findings were supported using computational studies. The data show that the binding of these ligands to heEF1γ is enthalpically favourable and entropically driven (except for the binding of GSSG). The full length heEF1γ binds GSSG with lower affinity (K d = 115 µM), with more hydrogen-bond contacts (ΔH = -73.8 kJ/mol) and unfavourable entropy (-TΔS = 51.7 kJ/mol) compared to the glutathione transferase-like N-terminus domain of heEF1γ, which did not show preference to any specific ligand. Computational free binding energy calculations from the 10 ligand poses show that GSSG and GSH consistently bind heEF1γ, and that both ligands bind at the same site with a folded bioactive conformation. This study reveals the possibility that heEF1γ is a glutathione-binding protein.

Plasmodium falciparum Hsp70-z, an Hsp110 homologue, exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion.

The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.

Overexpression, Purification and Characterisation of the Plasmodium falciparum Hsp70-z (PfHsp70-z) Protein.

Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur in the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.

Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation.

Many studies have characterized how changes to the stability and internal motions of a protein during activation can contribute to their catalytic function, even when structural changes cannot be observed. Here, unfolding studies and hydrogen-deuterium exchange (HX) mass spectrometry were used to investigate the changes to the stability and conformation/conformational dynamics of JNK1ß1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide binding on both inactive and active JNK1ß1 using the ATP analogue, 5'-adenylyl-imidodiphosphate (AMP-PNP). JNK1ß1 phosphorylation alters HX in regions involved in catalysis and substrate binding, changes that can be ascribed to functional modifications in either structure and/or backbone flexibility. Increased HX in the hinge between the N- and C-terminal domains implied that it acquires enhanced flexibility upon phosphorylation that may be a prerequisite for interdomain closure. In combination with the finding that nucleotide binding destabilizes the kinase, the patterns of solvent protection by AMP-PNP were consistent with a novel mode of nucleotide binding to the C-terminal domain of a destabilized and open domain conformation of inactive JNK1ß1. Solvent protection by AMP-PNP of both N- and C-terminal domains in active JNK1ß1 revealed that the domains close around nucleotide upon phosphorylation, concomitantly stabilizing the kinase. This suggests that phosphorylation activates JNK1ß1 in part by increasing hinge flexibility to facilitate interdomain closure and the creation of a functional active site. By uncovering the complex interplay that occurs between nucleotide binding and phosphorylation, we present new insight into the unique mechanisms by which JNK1ß1 is regulated.

Purification and characterisation of recombinant human eukaryotic elongation factor 1 gamma.

The eukaryotic elongation factor 1 gamma (eEF1γ) is a multi-domain protein, which consist of a glutathione transferase (GST)-like N-terminus domain. In association with α, ß and δ subunits, eEF1γ forms part of the eukaryotic elongation factor complex, which is mainly involved in protein biosynthesis. The N-terminus GST domain of eEF1γ interacts with the ß subunit. eEF1γ subunit is over-expressed in human carcinoma. The role of human eEF1γ (heEF1γ) is poorly understood. A successful purification of recombinant heEF1γ is the first step towards determining unknown properties of the protein, including putative GST-like activities and the structure of the protein. This paper describes the over-expression, purification and characterisation of recombinant full-length, and the N- and C-terminus domains of heEF1γ. All three recombinant heEF1γ constructs over-expressed in the soluble Escherichia coli cell fraction and were purified to homogeneity. Secondary structure analysis indicates that the heEF1γ constructs have high α-helical structural character. The full-length and N-terminus domain are dimeric, while the C-terminus is monomeric. Both full-length and N-terminus domain interact with 8-anilino-1-naphthalene sulfonate (ANS) with KD=70.0 (±5.7) µM and with reduced glutathione (GSH). Glutathione sulfonate displaced ANS bound to hydrophobic binding sites in the recombinant N-terminus domain. Using the standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay, the N-domain showed some enzyme activity (0.03µmolmin(-1) mg(-1) protein), while the full-length heEF1γ did not catalyse the GSH-CDNB conjugation. Consequently, we hypothesize the presence of a presumed GST-like active site structure in the heEF1γ, which comprises a glutathione binding site and a hydrophobic substrate binding site.

Stability of the domain interface contributes towards the catalytic function at the H-site of class alpha glutathione transferase A1-1.

Cytosolic glutathione transferases (GSTs) are major detoxification enzymes in aerobes. Each subunit has two distinct domains and an active site consisting of a G-site for binding GSH and an H-site for an electrophilic substrate. While the active site is located at the domain interface, the role of the stability of this interface in the catalytic function of GSTs is poorly understood. Domain 1 of class alpha GSTs has a conserved tryptophan (Trp21) in helix 1 that forms a major interdomain contact with helices 6 and 8 in domain 2. Replacing Trp21 with an alanine is structurally non-disruptive but creates a cavity between helices 1, 6 and 8 thus reducing the packing density and van der Waals contacts at the domain interface. This results in destabilization of the protein and a marked reduction in catalytic activity. While functionality at the G-site is not adversely affected by the W21A mutation, the H-site becomes more accessible to solvent and less favorable for the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Not only does the mutation result in a reduction in the energy for stabilizing the transition state formed in the S(N)Ar reaction between the substrates GSH and CDNB, it also compromises the ability of the enzyme to form and stabilize a transition state analogue (Meisenheimer complex) formed between GSH and 1,3,5-trinitrobenzene (TNB). The study demonstrates that the stability of the domain-domain interface plays a role in mediating the catalytic functionality of the active site, particularly the H-site, of class alpha GSTs.

Anti-peptide antibodies differentiate between plasmodial lactate dehydrogenases.

Malaria lactate dehydrogenase, a glycolytic enzyme, is a malaria diagnostic target in lateral flow immunochromatographic rapid diagnostic tests. Recombinant Plasmodium yoelii LDH was cloned into the pET-28a vector, expressed and the expressed protein purified from a Ni-NTA affinity matrix. A pan-malarial LDH antibody directed against a common malaria LDH peptide (APGKSDKEWNRDDLL) and two anti-peptide antibodies, each targeting a unique Plasmodium falciparum (LISDAELEAIFDC) and Plasmodium vivax (KITDEEVEGIFDC) LDH peptide were raised in chickens. The antibodies were affinity purified with the appropriate peptide affinity matrix. The affinity purified anti-peptide antibodies detected recombinant P. falciparum, P. vivax and P. yoelii LDH and native P. falciparum and P. yoelii LDH in western blots and immunofluorescence studies. The pan-malarial antibody detected LDH from the three malaria species in western blots. The species-specific anti-peptide antibodies differentiated between P. falciparum and P. vivax LDH. Affinity purified chicken antibodies against recombinant PfLDH, PvLDH and PyLDH proteins each detected the parent and orthologous proteins with similar titers in an ELISA. The study supports an anti-peptide antibody approach to the development of diagnostic reagents.