Recombinant Cas9 protein production in an endotoxin-free system and evaluation with editing the BCL11A gene in human cells.

Many therapeutic proteins are expressed in Escherichia coli bacteria for the low cost and high yield obtained. However, these gram-negative bacteria also generate undesirable endotoxin byproducts such as lipopolysaccharides (LPS). These endotoxins can induce a human immune response and cause severe inflammation. To mitigate this problem, we have employed the ClearColi BL21 (DE3) endotoxin-free cells as an expression host for Cas9 protein production. Cas9 is an endonuclease enzyme that plays a key role in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (CRISPR/Cas9) genome editing technique. This technology is very promising for use in diagnostics as well as treatment of diseases, especially for genetic diseases such as thalassemia. The potential uses for this technology thus generate a considerable interest for Cas9 utilization as a therapeutic protein in clinical treatment. Therefore, special care in protein production should be a major concern. Accordingly, we expressed the Cas9 protein in endotoxin-free bacterial cells achieving 99% purity with activity comparable to commercially available Cas9. Our protocol therefore yields a cost-effective product suitable for invitro experiments with stem cells.

Characterization of endotoxin free protein production of brain-derived neurotrophic factor (BDNF) for the study of Parkinson model in SH-SY5Y differentiated cells.

Human neuronal cells are a more appropriate cell model for neurological disease studies such as Alzheimer and Parkinson's disease. SH-SY5Y neuroblastoma cells have been widely used for differentiation into a mature neuronal cell phenotype. The cellular differentiation process begins with retinoic acid incubation, followed by incubation with brain-derived neurotrophic factor (BDNF), a recombinant protein produced in E. coli cells. Endotoxin or lipopolysaccharide (LPS) is the major component of the outer membrane of bacterial cells that triggers the activation of pro-inflammatory cytokines and ultimately cell death. Consequently, any endotoxin contamination of the recombinant BDNF used for cell culture experiments would impact on data interpretation. Therefore, in this study, we expressed the BDNF recombinant protein in bacterial endotoxin-free cells that were engineered to modify the oligosaccharide chain of LPS rendering the LPS unable to trigger the immune response of human cells. The expression of DCX and MAP-2 in differentiated cells indicate that in-house and commercial BDNF are equally effective in inducing differentiation. This suggests that our in-house BDNF protein can be used to differentiate SH-SY5Y neuroblastoma cells without the need for an endotoxin removal step.

Neuroprotective effects of Withania somnifera in the SH-SY5Y Parkinson cell model.

Parkinson's disease is the most frequent neurodegenerative motor disorder. The clinical syndrome and pathology involve motor disturbance and the degeneration of dopaminergic neurons in the substantia nigra. Root extracts of Withania. somnifera, commonly called Ashwagandha, contain several major chemical constituents known as withanolides. Studies have shown that W. somnifera extracts exhibit numerous therapeutic effects including inflammation and oxidative stress reduction, memory and cognitive function improvement. This study aimed to evaluate the protective effects of KSM-66, W. somnifera root extract, on 6-hydroxydopamine (6-OHDA)-induced toxicity in the human neuroblastoma SH-SY5Y cell line, as well as the associated oxidative response protein expression and redox regulation activity focused on S-glutathionylation. SH-SY5Y cells were treated with 6-OHDA preceded or followed by treatment with the KSM-66 extract. Using KSM-66 concentrations ranging from 0.25 to 1 mg/ml before and after treatment of the cells with 6-OHDA has resulted in an increased viability of SH-SY5Y cells. Interestingly, the extract significantly increased glutathione peroxidase activity and thioltransferase activity upon pre- or post- 6-OHDA treatment. KSM-66 also modulated oxidative response proteins: peroxiredoxin-I, VGF and vimentin proteins upon 6-OHDA pre/post treatments. In addition, the extract controlled redox regulation via S-glutathionylation. Pre-treatment of SH-SY5Y cells with KSM-66 decreased protein-glutathionylation levels in the cells treated with 6-OHDA. The rescue of mitochondria with 0.5 mg/ml KSM-66 extract showed an increase in ATP levels. These findings suggest that W. somnifera root extract acts as a neuroprotectant, thereby introducing a potential agent for the treatment or prevention of neurodegenerative diseases.

Anticancer effects of the combined Thai noni juice ethanolic extracts and 5-fluorouracil against cholangiocarcinoma cells in vitro and in vivo.

Application of 5-fluorouracil (5-FU) in cholangiocarcinoma (CCA) is limited by adverse side effects and chemoresistance. Therefore, the combination therapy of 5-FU with other substances, especially natural products may provide a new strategy for CCA treatment. The aim of this study was to evaluate the combination effects of 5-FU and two ethanolic extracts of Thai noni juice (TNJ) products on CCA cell lines and nude mice xenografts. The results of antiproliferative assay showed the combination treatment of 5-FU and each TNJ ethanolic extract exerted more cytotoxicity on CCA cells than either single agent treatment. Synergistic effects of drug combinations can enable the dose reduction of 5-FU. The mechanism underlying a combination treatment was apoptosis induction through an activation of p53 and Bax proteins. In the nude mouse xenograft model, combination treatments of 5-FU with each TNJ ethanolic extract suppressed the growth of CCA cells implanted mice more than single agent treatments with no effects on mouse body weight, kidney, and spleen. Moreover, low doses of TNJ ethanolic extracts reduced the hepatotoxicity of 5-FU in nude mice. Taken together, these data suggested that the ethanolic extracts of TNJ products can enhance the anti-CCA effect and reduce toxicity of 5-FU.

Phytochemical andrographolide modulates NF-κB and JNK in human neuroblastoma SH-SY5Y cells, a cell model for Parkinson's disease.

Andrographis paniculata has been an important plant for traditional medicine in Asia for centuries. Andrographolide is the primary bioactive phytochemical from the plant and is known to exhibit many different protective effects through modulation of various proteins and signaling pathways. Andrographolide has been reported to exert anti-inflammatory and neuroprotective effects as well as being an antioxidant itself. We therefore studied whether andrographolide could provide protective effects to the SH-SY5Y neuroblastoma cell model for Parkinson's disease. In this study, we observed andrographolide inhibiting activation of NF-κB p65 (nuclear factor kappa-light-chain-enhancer of activated B cells) and JNK MAPK (c-Jun N-terminal Kinase Mitogen-Activated Protein Kinase) pathways, however, it did not provide any protective effect against induced stress in the SH-SY5Y cells. We propose the sustained low-level activation of JNK and the inhibition of NF-κB promoted ROS (Reactive Oxygen Species) production that yielded the observed cell death. Therefore, the protective effects observed with andrographolide appear to be cell/tissue specific responses.

Glutathione transferase Omega 1-1 (GSTO1-1) modulates Akt and MEK1/2 signaling in human neuroblastoma cell SH-SY5Y.

In the human neuroblastoma SH-SY5Y cell line, the glutathione transferase Omega 1-1 (GSTO1-1) appears to modulate Akt and MEK1/2 kinase activation. We observed a glutathionylation modification was involved in the activation of Akt but not MEK1/2. With the specific GSTO1-1 inhibitor ML175, we show the enzyme activity of GSTO1-1 is important for modulation as the inhibited GSTO1-1 allowed activation of both Akt and MEK1/2. The inhibition of GSTO1-1 showed a similar extent of activation of Akt and MEK1/2 as treatment by the endotoxin lipopolysaccharide. The GSTO1-1 also either directly interacts with Akt and MEK1/2 or interacts with a protein complexed with Akt and MEK1/2 as both kinases coimmunoprecipitated with GSTO1-1. The results suggest that GSTO1-1 enzyme activity inhibits the activation of these two kinases to maintain basal levels. The possible regulation by GSTO1-1 is of interest as both kinases have hundreds of potential downstream targets that are known to have contributions to various cellular processes including survival, growth, proliferation, and metabolism.

Proteomic analysis of human glutathione transferase omega (hGSTO1) stable transfection in a 6-hydroxydopamine-induced neuronal cells.

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. The disease is associated with dopaminergic neuron losses in the substantia nigra area of the brain and the formation of cytoplasmic inclusion bodies. Human glutathione transferase omega 1 (hGSTO1) appears to have a role in modulating stress response. The study was aimed to elucidate differentially expressed proteins caused by oxidative stress induced by 6-hydroxydopamine (6-OHDA). Human neuronal cells SH-SY5Y overexpressing hGSTO1 were used to investigate protein glutathionylation and the modulation of cellular protein expression. Therefore SH-SY5Y/hGSTO1 and SH-SY5Y/control lysate proteins were separated by 2D-gel electrophoresis compared with untreated conditions in both standard and non-reducing conditions. In standard conditions, the analysis of protein profiles demonstrated 25 differentially expressed spots and 10 spots were chosen for further protein identification by LC-MS analysis. Several proteins were later identified as vimentin, galectin-1, high mobility group protein B2, clathrin, tropomyosin, heterogenous nuclear ribonucleoprotein and peroxiredoxin-2. Search Tool for Interactions of Chemicals (STITCH) analysis suggested that oxidative stress induced by 6-OHDA involved carbohydrate metabolism in SH-SY5Y via a lactose metabolic pathway. Our results raise the possibility that hGSTO1 modulates the functions of many proteins that play a role in the degenerative cell response of a Parkinson's model.

Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities.

It has been estimated for dengue infection that the global population at risk is 3.5 billion people, which makes dengue an important public health problem. The causative agents of dengue are dengue viruses. For dengue virus replication, the dengue virus NS5 protein is of special importance as it has several enzyme activities important for viral replication. Previous reports of phosphorylation and SUMOylation of dengue NS5 have shown these protein modifications have important consequences for NS5 functions. In this report we identify glutathionylation, another reversible post translation modification that impacts on NS5 enzyme activity. Using dengue virus infected cells we employed specific antibodies and mass spectrometry to identify 3 cysteine residues of NS5 protein as being glutathionylated. Glutathionylation is a post translational protein modification where glutathione is covalently attached to a cysteine residue. We showed glutathionylation occurs on 3 conserved cysteine residues of dengue NS5. Then we generated two flavivirus recombinant full length proteins, dengue NS5 and Zika NS5, to characterize two of the NS5 enzyme activities, namely, guanylyltransferase and RNA-dependent RNA polymerase activities. We show glutathionylation of dengue and Zika NS5 affects enzyme activities of the two flavivirus proteins. The data suggests that glutathionylation is a general feature of the flavivirus NS5 protein and the modification has the potential to modulate several of the NS5 enzyme functions.

Ubiquitin-Conjugating Enzyme E2 L3 is Downregulated by the Chikungunya Virus nsP2 Protease.

PURPOSE: Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that causes chikungunya fever in humans. The CHIKV non-structural protein 2 (nsP2) is a multifunctional protein that additionally modulates the host cell to dampen the innate immune response and inhibit other cellular processes. EXPERIMENTAL DESIGN: To further investigate the interactions of nsP2 with host cells, the protease domain of CHIKV nsP2 (nsP2-pro) is transfected into Hela cells, and differential protein expression is detected by 2D polyacrylamide gel electrophoresis. RESULTS: A total of 21 differentially regulated (six upregulated, 15 downregulated) spots are observed, of which five are identified by mass spectrometry. The downregulation of one of the identified proteins, ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is confirmed by western blotting of both nsP2-pro transfection and CHIKV natural infection, and the downregulation of UBE2L3 is additionally shown to require an enzymatically active nsP2 protease domain. Transfection of full length UBE2L3 into HEK293T/17 cells prior to CHIKV infection reduce levels of infection and E protein expression but do not alter RNA genome levels. CONCLUSION: These results suggest that UBE2L3 is a cellular target of the CHIKV nsP2 protease, and this possibly mediates the pathogenesis of chikungunya fever.

Glutathionylation of chikungunya nsP2 protein affects protease activity.

BACKGROUND: Chikungunya fever is an emerging disease caused by the chikungunya virus and is now being spread worldwide by the mosquito Aedes albopictus. The infection can cause a persistent severe joint pain and recent reports link high levels of viremia to neuropathologies and fatalities. The viral protein nsP2 is a multifunctional enzyme that plays several critical roles in virus replication. Virus infection induces oxidative stress in host cells which the virus utilizes to aid viral propagation. Cellular oxidative stress also triggers glutathionylation which is a post-translational protein modification that can modulate physiological roles of affected proteins. METHODS: The nsP2 protease is necessary for processing of the virus nonstructural polyprotein generated during replication. We use the recombinant nsP2 protein to measure protease activity before and after glutathionylation. Mass spectrometry allowed the identification of the glutathione-modified cysteines. Using immunoblots, we show that the glutathionylation of nsP2 occurs in virus-infected cells. RESULTS: We show that in virus-infected cells, the chikungunya nsP2 can be glutathionylated and we show this modification can impact on the protease activity. We also identify 6 cysteine residues that are glutathionylated of the 20 cysteines in the protein. CONCLUSIONS: The virus-induced oxidative stress causes modification of viral proteins which appears to modulate virus protein function. GENERAL SIGNIFICANCE: Viruses generate oxidative stress to regulate and hijack host cell systems and this environment also appears to modulate virus protein function. This may be a general target for intervention in viral pathogenesis.

Comparative proteome analysis of rubber latex serum from pathogenic fungi tolerant and susceptible rubber tree (Hevea brasiliensis).

Many cultivated rubber trees (Hevea brasiliensis) are invaded by various Phytophthora species fungi, especially in tropical regions which result in crop yield losses. Comparative proteome analysis coupled with liquid chromatography electrospray/ionization (LC-ESI) mass spectrometry identification was employed to investigate the relative abundance of defense related proteins in Phytophthora sp. susceptible (RRIM600) and tolerant (BPM24) clones of rubber tree. Proteome maps of non-rubber constituent of these two model clones show similar protein counts, although some proteins show significant alterations in their abundance. Most of the differentially abundant proteins found in the serum of BPM24 illustrate the accumulation of defense related proteins that participate in plant defense mechanisms such as beta-1,3-glucanase, chitinase, and lectin. SDS-PAGE and 2-D Western blot analysis showed greater level of accumulation of beta-1,3-glucanase and chitinase in latex serum of BPM24 when compared to RRIM600. A functional study of these two enzymes showed that BPM24 serum had greater beta-1,3-glucanase and chitinase activities than that of RRIM600. These up-regulated proteins are constitutively expressed and would serve to protect the rubber tree BPM24 from any fungal invader. The information obtained from this work is valuable for understanding of defense mechanisms and plantation improvement of H. brasiliensis. BIOLOGICAL SIGNIFICANCE: Non-rubber constituents (latex serum) have almost no value and are treated as waste in the rubber agricultural industry. However, the serum of natural rubber latex contains biochemical substances. The comparative proteomics analysis of latex serum between tolerant and susceptible clones reveals that the tolerant BPM24 clone contained a high abundance of several classes of fungal pathogen-responsive proteins, such as glucanase and chitinase. Moreover, other proteins identified highlighted the accumulation of defensive-associated proteins participating in plant fungal immunity. The isolation of beta-1,3-glucanase, chitinase, and lectin from latex serum should be further investigated and may provide a therapeutic application. This investigation will lead to possible use of latex serum as a great biotechnological resource due to the large quantity of serum produced and the biochemicals contained therein.

Yellow head virus infection in black tiger shrimp reveals specific interaction with granule-containing hemocytes and crustinPm1 as a responsive protein.

Yellow head virus (YHV) causes acute infections and mass mortality in black tiger shrimp culture. Our study aims to investigate molecular interaction between YHV and circulating hemocytes of Penaeus monodon at early infection. Total shrimp hemocytes were isolated by Percoll gradient centrifugation and identified by flow cytometric analysis. At least three types of hemocyte cells were identified as hyaline, semi-granular, and granular hemocytes. Experimental infection of YHV in shrimp culture demonstrated drastic changes in total and each hemocyte cell counts. Immunohistochemistry analysis demonstrated interaction and replication of YHV mainly with the granule-containing hemocytes and little to none in hyaline cell. These granule-containing hemocytes are proposed to be YHV targets providing the first line of defense to viral infection. Protein expression profiling of granule-containing hemocytes revealed several immune-responsive proteins including antimicrobial protein crustins (crustinPm1 and crustinPm4), alpha-2-macroglobulin, and kazal-type serine proteinase inhibitor. During an early phase of YHV infection at 6 hpi crustinPm1 illustrated a significant increase of mRNA and protein expression level in plasma. The results suggest that an antimicrobial crustinPm1 may participate in shrimp defense mechanism against YHV, especially on the granule-containing hemocytes.

Chikungunya nsP2 protease is not a papain-like cysteine protease and the catalytic dyad cysteine is interchangeable with a proximal serine.

Chikungunya virus is the pathogenic alphavirus that causes chikungunya fever in humans. In the last decade millions of cases have been reported around the world from Africa to Asia to the Americas. The alphavirus nsP2 protein is multifunctional and is considered to be pivotal to viral replication, as the nsP2 protease activity is critical for proteolytic processing of the viral polyprotein during replication. Classically the alphavirus nsP2 protease is thought to be papain-like with the enzyme reaction proceeding through a cysteine/histidine catalytic dyad. We performed structure-function studies on the chikungunya nsP2 protease and show that the enzyme is not papain-like. Characterization of the catalytic dyad cysteine residue enabled us to identify a nearby serine that is catalytically interchangeable with the dyad cysteine residue. The enzyme retains activity upon alanine replacement of either residue but a replacement of both cysteine and serine residues results in no detectable activity. Protein dynamics appears to allow the use of either the cysteine or the serine residue in catalysis. This switchable dyad residue has not been previously reported for alphavirus nsP2 proteases and would have a major impact on the nsP2 protease as an anti-viral target.

Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site.

Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme.

The crystal structure of JNK from Drosophila melanogaster reveals an evolutionarily conserved topology with that of mammalian JNK proteins.

BACKGROUND: The c-Jun N-terminal kinases (JNKs), members of the mitogen-activated protein kinase (MAPK) family, engage in diverse cellular responses to signals produced under normal development and stress conditions. In Drosophila, only one JNK member is present, whereas ten isoforms from three JNK genes (JNK1, 2, and 3) are present in mammalian cells. To date, several mammalian JNK structures have been determined, however, there has been no report of any insect JNK structure. RESULTS: We report the first structure of JNK from Drosophila melanogaster (DJNK). The crystal structure of the unphosphorylated form of DJNK complexed with adenylyl imidodiphosphate (AMP-PNP) has been solved at 1.79 Å resolution. The fold and topology of DJNK are similar to those of mammalian JNK isoforms, demonstrating their evolutionarily conserved structures and functions. Structural comparisons of DJNK and the closely related mammalian JNKs also allow identification of putative catalytic residues, substrate-binding sites and conformational alterations upon docking interaction with Drosophila scaffold proteins. CONCLUSIONS: The DJNK structure reveals common features with those of the mammalian JNK isoforms, thereby allowing the mapping of putative catalytic and substrate binding sites. Additionally, structural changes upon peptide binding could be predicted based on the comparison with the closely-related JNK3 structure in complex with pepJIP1. This is the first structure of insect JNK reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of insect JNK.

Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates.

Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. Enzyme characterization shows that the protease domain alone has different properties compared with the full length nsP2 protease. We also show chikungunya nsP2 protease possesses different substrate specificity to the canonical alphavirus nsP2 polyprotein cleavage specificity. Moreover, the chikungunya nsP2 also appears to differ from other alphavirus nsP2 in its distinctive ability to recognize small peptide substrates.

Micro-plasticity of genomes as illustrated by the evolution of glutathione transferases in 12 Drosophila species.

Glutathione transferases (GST) are an ancient superfamily comprising a large number of paralogous proteins in a single organism. This multiplicity of GSTs has allowed the copies to diverge for neofunctionalization with proposed roles ranging from detoxication and oxidative stress response to involvement in signal transduction cascades. We performed a comparative genomic analysis using FlyBase annotations and Drosophila melanogaster GST sequences as templates to further annotate the GST orthologs in the 12 Drosophila sequenced genomes. We found that GST genes in the Drosophila subgenera have undergone repeated local duplications followed by transposition, inversion, and micro-rearrangements of these copies. The colinearity and orientations of the orthologous GST genes appear to be unique in many of the species which suggests that genomic rearrangement events have occurred multiple times during speciation. The high micro-plasticity of the genomes appears to have a functional contribution utilized for evolution of this gene family.

Yellow head virus binding to cell surface proteins from Penaeus monodon hemocytes.

Our previous data revealed that viral particles of yellow head virus (YHV) specifically interacted with granule-containing hemocytes. After isolation of targeted hemocytes, biotinylation was performed using Biotin-NSH-LC. Biotinylated protein was extracted and separated by 2-D PAGE. Electro-transferred proteins on a nitrocellulose membrane were probed with streptavidin-HRP complex to detect biotinylated proteins. The data from 2-D PAGE combined with affinity pull down purification revealed 8 and 6 biotinylated proteins specific to hyaline and granule containing hemocytes, respectively. Four proteins were found in common for both two hemocytes. The majority of proteins detected in granular hemocytes are membrane-associated proteins and immune-related proteins such as alpha-2-macroglobulin (A2M), kazal-type serine protease inhibitor (SPI) and crustin. CrustinPm1 was found to bind to YHV as shown with biotinylation pull-down assay and confirmed with two-dimensional virus overlay protein binding assay (2-D VOPBA). The expression of crustinPm1 was observed in semigranular and granular hemocytes whereas very low or no expression occurred in hyaline hemocytes. CrustinPm1 appears to either be directly involved in cellular binding or mediating virus internalization into permissive hemocytes.

The impact of nitric oxide toxicity on the evolution of the glutathione transferase superfamily: a proposal for an evolutionary driving force.

Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10(-9) M) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 10(2)-10(4) times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO.

Structural evidence for conformational changes of Delta class glutathione transferases after ligand binding.

We report four new crystal structures for Delta class glutathione transferases from insects. We compare these new structures as well as several previously reported structures to determine that structural transitions can be observed with ligand binding. These transitions occurred in the regions around the active site entrance, including alpha helix 2, C-terminus of alpha helix 4 including the loop to helix 5 and the C-terminus of helix 8. These structural movements have been reported or postulated to occur for several other glutathione transferase classes; however, this is the first report showing structural evidence of all these movements occurring, in this case in Delta class glutathione transferases. These fluctuations also can be observed occurring within a single structure as there is ligand bound in only one subunit and each subunit is undergoing different conformational transitions. The structural comparisons show reorganizations occur both pre- and post-GSH ligand binding communicated through the subunit interface of the quaternary assembly. Movements of these positions would allow 'breathing' of the active site for substrate entrance, topological rearrangement for varying substrate specificity and final product release.