And 1-anilinonaphthalene-8-sulfonate Research Articles

Macrocyclic Diacetylene / Sulfonate Fluorophore Hierarchical Multifunctional Nanotoroids.

Functional supramolecular materials exhibit important features including structural versatility and versatile applications. Here, this study reports the construction of unique hierarchically organized nanotoroids exhibiting fluorescence, photocatalytic, and sensing properties. The nanotoroids comprise of macrocyclic diacetylenes (MCDA) and 8-anilino-1-naphthalene sulfonate (ANS), a negatively charged aromatic fluorescent dye. This study shows that the hierarchical structure of the nanotoroids consist of MCDA nanofibers formed by stacked diacetylene monomers as the basic units, which are further bent and aligned into toroidal organization by electrostatic and hydrophobic interactions with the ANS molecules. The amine moieties on the nanotoroids surface are employed for deposition of gold nanostructures - Au nanoparticles or Au nanosheets - which constitute effective platforms for photocatalysis and surface enhanced Raman scattering (SERS)-based sensing.

Clay-Polymer Nanocomposites Mediated Inhibition of Protein Aggregation: Possible Role in the Prevention of Proteinopathies.

The transformation of proteins from their native conformation into highly ordered fibrillar structures due to their misfolding and aggregation under particular conditions are described as beta-sheet enriched amyloid fibrils. The accumulation of these fibrils in different body parts is the major cause of several neurological and non-neurological conditions (proteinopathies). To prevent these proteinopathies, inhibition of protein aggregation is considered a promising strategy. Therefore, in this study, we synthesized montmorillonite (MMT) based poly- orthophenylenediamine (PoPD) nanocomposites (NCs) and characterized their size and morphology due to their remarkable biological properties. Further, the effect of these nanocomposites on inhibition of fibril formation was assessed. These nanocomposites were evaluated for their anti-amyloidogenic potential on two model proteins of amyloidopathies, i.e., human lysozyme and human serum albumin (HL & HSA), by using several biophysical methods, such as Thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) fluorescence, congo red dye binding assay (CR). Secondary structural content was evaluated by Circular dichroism (CD) spectroscopy. Results demonstrated that synthesized nanocomposites significantly inhibited fibril formation in dose-dependent manner that corresponds to their ability to arrest fibrillation. It is suggested that they may adsorb proteins to protect them against aggregation when they are subjected to aggregating conditions. This study offers an opportunity to understand the mechanism of inhibition of fibril formation by nanocomposites, showing that they inhibit amyloid formation and amyloid diseases. Thus, the study concludes that these nanocomposites are promising candidates as therapeutic molecules for proteinopathies and are envisaged to enrich the area of personalized medicine, augmenting the human healthcare system.

Investigation of the Effect of Pyrogallol on the Formation of Amylin Amyloid Fibrils as a Strategy for the Treatment of Type 2 Diabetic Patients: A Theoretical and Experimental Study.

In more than 50 to 90% of type 2 diabetic patients, under the influence of various factors, the production of islet amyloid polypeptide or amylin in pancreatic beta cells increases. Spontaneous accumulation of amylin peptide in the form of insoluble amyloid fibrils and soluble oligomers is one of the main causes of beta cell death in diabetic patients. The objective of the present study was to evaluate the effect of pyrogallol, as a phenolic compound, on inhibiting the formation of amylin protein amyloid fibrils. In this study, different techniques such as the thioflavin T (ThT) and 1-Anilino-8-naphthalene sulfonate (ANS) fluorescence intensity and the circular dichroism (CD) spectrum, will be used to investigate the effects of this compound on inhibiting the formation of amyloid fibrils. To investigate the interaction sites of pyrogallol with amylin, docking studies were performed. Our results that pyrogallol in a dose-dependent manner (0.5:1, 1:1, and 5:1, Pyr to Amylin) inhibits the amylin amyloid fibrils formation. Docking analysis revealed that pyrogallol forms hydrogen bonds with valine 17 and asparagine 21. In addition, this compound forms 2 more hydrogen bonds with asparagine 22. This compound also forms hydrophobic bonds with histidine 18. Considering this data and the direct relationship between oxidative stress and the formation of amylin amyloid accumulations in diabetes, the use of compounds with both antioxidant and anti-amyloid properties can be considered an important therapeutic strategy for type 2 diabetes.

Cryptotanshinone against vascular dementia through inhibition of Aβ aggregation and inflammatory responses in cerebrovascular endothelial cells

Abnormal aggregation of amyloid-β (Aβ) peptides and associated inflammation and apoptosis in cerebrovascular endothelial cells are prelude to inhibition of onset of vascular dementia (VaD). Although small molecules have been widely used to mitigate the cell damage induced by aggregated species of Aβ, its molecular mechanism based on anti-amyloid properties and corresponding mitigation of cytotoxicity against cerebrovascular endothelial cells have not been elucidated. Herein, we used cryptotanshinone as the major bioactive compound from the root of Salvia miltiorrhiza Bunge to effectively inhibit Aβ fibrillation and associated cytotoxicity.Thoflavin T (ThT) and 1-Anilino-8-naphthalene sulfonate (ANS) fluorescence, Congo red, and circular dichroism (CD) analyses indicted that cryptotanshinone potentially inhibit Aβ1-42 aggregation through elongation of nucleation phase, apparent decrease in the slope of the growth phase, and the final fluorescence intensity in a concentration-dependent manner. Also, cell viability, inflammation and capsae-3 assays showed that co-incubation of Aβ1-42 peptide with cryptotanshinone in the aggregation buffer not only mitigated their cytotoxicity, but also reduced the levels of TNF-α, IL-1β, IL-6 and caspase-3 activity in cerebrovascular endothelialcells induced by Aβ1-42. This study suggested that cryptotanshinone may show a great promise in the development of small molecule-based platforms for the treatment of VaD.

Exploring the inhibitory effects of liquiritigenin against tau fibrillation and related neurotoxicity as a model of preventive care in Alzheimer's disease

Aggregation of tau protein into the form of insoluble amyloid fibrils is linked with Alzheimer's disease. The identification of potential small molecules that can inhibit tau protein from undergoing aggregation has received a great deal of interest, recently. In the present study, the possible inhibitory effects of liquiritigenin as a member of chiral flavanone family on tau amyloid fibrils formation and their resulting neurotoxicity were assessed by different biophysical and cellular assays. The inhibitory effect of the liquiritigenin against tau amyloid formation was investigated using thioflavin T (ThT) and 1-Anilino-8-naphthalene sulfonate (ANS) fluorescence spectroscopy, Congo red (CR) binding assays, transmission electron microscopy (TEM) analysis, and circular dichroism (CD) spectroscopy. Neurotoxicity assays were also performed against neuron-like cells (SH-SY5Y) using 3-(4,5-Dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) reduction, reactive oxygen species (ROS), catalase (CAT) and caspase-3 activity measurements. We found that liquiritigenin served as an efficient inhibitor of tau amyloid fibrils formation through prevention of structural transition in tau structure, exposure of hydrophobic patches and their associated neurotoxicity mediated by decrease in the production of ROS and caspase-3 activity and elevation of CAT activity. These data may finally find applications in the development of promising inhibitors against amyloid fibril formation and treatment of Alzheimer's disease.

Molecular basis of inhibition of Schistosoma japonicum glutathione transferase by ellagic acid: Insights into biophysical and structural studies

Schistosoma japonicum glutathione transferase (Sj26GST), an enzyme central to detoxification of electrophilic compounds in the parasite, is upregulated in response to drug treatment. Therefore, Sj26GST may serve as a potential therapeutic target for the treatment of schistosomiasis. Herewith, we describe the structural basis of inhibition of Sj26GST by ellagic acid (EA). Using 1-chloro-2,4-dinitrobenzene and reduced glutathione (GSH) as Sj26GST substrates, EA was shown to inhibit Sj26GST activity by 66 % with an IC50 of 2.4 μM. Fluorescence spectroscopy showed that EA altered the polarity of the environment of intrinsic tryptophan and that EA decreased (in a dose-dependent manner) the interaction between Sj26GST and 8-Anilino-1-naphthalenesulfonate (ANS), which is a known GST H-site ligand. Thermodynamic studies indicated that the interaction between Sj26GST and EA is spontaneous (ΔG = -29.88 ± 0.07 kJ/mol), enthalpically-driven (ΔH = -9.48 ± 0.42 kJ/mol) with a favourable entropic change (ΔS = 20.40 ± 0.08 kJ/mol/K), and with a stoichiometry of four EA molecules bound per Sj26GST dimer. The 1.53 Å-resolution Sj26GST crystal structure (P 21 21 21 space group) complexed with GSH and EA shows that EA binds primarily at the dimer interface, stabilised largely by Van der Waal forces and H-bonding. Besides, EA bound near the H-site and less than 3.5 Å from the ε-NH2 of the γ-glutamyl moiety of GSH, in each subunit.

Effect of the fluorescent probes ThT and ANS on the mature amyloid fibrils

ABSTRACT Fluorescent probes thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) are widely used to study amyloid fibrils that accumulate in the body of patients with serious diseases, such as Alzheimer’s, Parkinson’s, prion diseases, etc. However, the possible effect of these probes on amyloid fibrils is not well understood. In this work, we investigated the photophysical characteristics, structure, and morphology of mature amyloid fibrils formed from two model proteins, insulin and lysozyme, in the presence of ThT and ANS. It turned out that ANS affects the secondary structure of amyloids (shown for fibrils formed from insulin and lysozyme) and their fibers clusterization (valid for lysozyme fibrils), while ThT has no such effects. These results confirm the differences in the mechanisms of these dyes interaction with amyloid fibrils. Observed effect of ANS was explained by the electrostatic interactions between the dye molecule and cationic groups of amyloid-forming proteins (unlike hydrophobic binding of ThT) that induce amyloids conformational changes. This interaction leads to weakening repulsion between positive charges of amyloid fibrils and can promote their clusterization. It was shown that when fibrillogenesis conditions and, consequently, fibrils structure is changing, as well as during defragmentation of amyloids by ultrasonication, the influence of ANS to amyloids does not change, which indicates the universality of the detected effects. Based on the obtained results, it was concluded that ANS should be used cautiously for the study of amyloid fibrils, since this fluorescence probe have a direct effect on the object of study.

Disassembling and Reassembling Complex Structure of Glutamate Dehydrogenase 1 (DGH-1), Monitored by Tryptophan and 1-Anilinonaphthalene-8-Sulfonate (ANS) Fluorimetry

GDH-1 which converts glutamate to alpha-ketoglutarate and ammonia while reducing NAD(P) is constitutively expressed in all nucleated cells of mammals. Encoded by a nuclear gene, its mitochondrial matrix concentration is high (5-10 mg /ml). Turnover is also high considering a mitochondrial t 1/2 of 1-2 days. The MW of GDH-1 subunits is 56K. Newly synthesized enzyme precursor subunits are transported into mitochondria, are rapidly cleaved and assemble there to the enzymatically active homo-hexamer of 336K MW. Furthermore, bovine liver GDH-1 does form complexes of about 1.5 Mill at concentration above 2-3 mg/ml. Activating point mutations (e.g H454Y) cause life threatening hyperinsulinism. To better understand the biology of this protein, essential for life and of high medical relevance, we are using tryptophan and ANS fluorescence to study (in model experiments) the process of disassembly caused by urea and reassembly after urea removal by dilution or dialysis. An extensive literature beginning with the report by Olson and Anfinsen in 1952 provides useful background information. We show by monitoring tryptophan and ANS fluorescence that the trimer of the protein, formed at 3M urea, can be readily reassembled to the original higher molecular forms but that smaller structures, formed when exposed to urea exceeding 3 M, cannot be reassembled to the original multimeric forms. At this juncture we focus on the reversible process between 0 and 3 M urea, determining the rates of interconversion of multimeric forms as influenced by pH, temperature, the enzyme's substrates and its known allosteric modifiers using tryptophan fluorescence. We are testing a hypothesis that interconversion rates between trimers, hexamers and poly-hexamers contribute to the regulation of GDH-1 which controls glutaminolysis in insulin secreting beta-cells and cancer cells.

Effect of Surface Ligand and Temperature on Lipid Vesicle-Gold Nanoparticle Interaction: A Spectroscopic Investigation.

We herein investigate the interactions of differently functionalized anionic and cationic gold nanoparticles (AuNPs) with zwitterionic phosphocholine (PC) as well as inverse phosphocholine (iPC) lipid bilayers via spectroscopic measures. In this study, we used PC lipids with varying phase-transition temperatures, i.e., DMPC( Tm = 24 °C), DOPC( Tm = -20 °C), and iPC lipid DOCP ( Tm = -20 °C) to study their interactions with AuNPs functionalized with anionic ligands citrate, 3-mercaptopropionic acid, glutathione, and cationic ligand cysteamine. We studied the interactions by steady-state and time-resolved spectroscopic studies using membrane-sensitive probes 6-propionyl-2-dimethylaminonaphthalene (PRODAN)and 8-anilino-1 naphthalenesulfonate(ANS), as well as by confocal laser scanning microscopy (CLSM) imaging and dynamic light scattering (DLS) measurements. We observe that AuNPs bring in stability to the lipid vesicle, and the extent of interaction differs with the different surface ligands on the AuNPs. We observe that AuNPs functionalized with citrate effectively increase the phase-transition temperature of the vesicles by interacting with them. Our study reveals that the extent of interaction depends on the bulkiness of the ligands attached to the AuNPs. The bulkier ligands exert less van der Waals force, resulting in a weaker interaction. Moreover, we find that the interactions are more strongly pronounced when the vesicles are near the phase-transition temperature of the lipid.The CLSMimaging and DLSmeasurements demonstrate the surface modifications in the vesicles as a result of these interactions.

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

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

The effect of tyrosine residues on α-synuclein fibrillation.

Aggregation of the intrinsically disordered protein α-synuclein into ordered amyloid fibrils is implicated in the pathogenesis of Parkinson's disease. To unravel the role of Tyr residues in α-synuclein fibrillation, we prepared recombinant N-terminal (Y39A) and C-terminal (Y(125,133,136)A) mutants of α-synuclein and examined their fibrillation propensities by thioflavin T and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescent probes, SDS-PAGE and atomic force microscopy. We demonstrate that in contrast to wild-type α-synuclein, both mutants show large, but comparable delays in the fibrillation process and exhibit enhanced hydrophobicity during fibril-like assembly. Both Tyr mutants form fibril-like structures after prolonged incubation periods, which are morphologically distinct from those of the wild-type protein. Our results suggest that the N-terminal and C-terminal Tyr residues of α-synuclein are important primarily for the initiation of the fibrillation process.

Electromagnetic fields with 217 Hz and 0.2 mT as hazardous factors for tubulin structure and assembly (in vitro study)

Nowadays exposure to extremely low frequency electromagnetic fields (ELF-EMFs) is an unavoidable fact in human societies. In spite of destructive effects of ELF-EMF such as impression on short-term memory and cognitive performances, neuroprotective and neurogeneration effects of ELF-EMF have been reported. On the other hand, microtubule (MT) proteins, the dynamic cytoskeleton proteins that are mostly located in axons, are able to produce local EMFs and possess key role in memory formation and consciousness. Therefore, MTs have strong potential to be affected by exogenous ELF-EMFs. In the present study, decline of MT polymerization was shown by transmission electron microscopy images and turbidimetry method after exposure to ELF-EMFs with 50, 100, and 217 Hz frequencies at 0.2 mT intensity. Circular dichroism and 8-anilino-1-naphthalenesulfonate (ANS) fluorometry were used to demonstrate the MT secondary and third structural changes. Decrement of MT polymerization through disruption of tubulin native structure, and increasing the β-sheets and random coils were observed in all exposure conditions. Therefore, ELF-EMFs with 50, 100, and specially 217 Hz frequencies at 0.2 mT could lead to disruption of MT functions in the neural cells.

Ribosylation of bovine serum albumin induces ROS accumulation and cell death in cancer line (MCF-7)

Formation of advanced glycation end products (AGE) is crucially involved in the several pathophysiologies associated with ageing and diabetes, for example arthritis, atherosclerosis, chronic renal insufficiency, Alzheimer's disease, nephropathy, neuropathy, and cataracts. Because of devastating effects of AGE and the significance of bovine serum albumin (BSA) as a transport protein, this study was designed to investigate glycation-induced structural modifications in BSA and their functional consequences in breast cancer cell line (MCF-7). We incubated D-ribose with BSA and monitored formation of D-ribose-glycated BSA by observing changes in the intensity of fluorescence at 410 nm. NBT (nitro blue tetrazolium) assay was performed to confirm formation of keto-amine during glycation. Absorbance at 540 nm (fructosamine) increased markedly with time. Furthermore, intrinsic protein and 8-anilino-1-naphthalenesulfonate (ANS) fluorescence revealed marked conformational changes in BSA upon ribosylation. In addition, a fluorescence assay with thioflavin T (ThT) revealed a remarkable increase in fluorescence at 485 nm in the presence of glycated BSA. This suggests that glycation with D-ribose induced aggregation of BSA into amyloid-like deposits. Circular dichroism (CD) study of native and ribosylated BSA revealed molten globule formation in the glycation pathway of BSA. Functional consequences of ribosylated BSA on cancer cell line, MCF-7 was studied by MTT assay and ROS estimation. The results revealed cytotoxicity of ribosylated BSA on MCF-7 cells.

Principal component analysis of chemical shift perturbation data of a multiple‐ligand‐binding system for elucidation of respective binding mechanism

Chemical shift perturbations (CSPs) in NMR spectra provide useful information about the interaction of a protein with its ligands. However, in a multiple-ligand-binding system, determining quantitative parameters such as a dissociation constant (K(d) ) is difficult. Here, we used a method we named CS-PCA, a principal component analysis (PCA) of chemical shift (CS) data, to analyze the interaction between bovine β-lactoglobulin (βLG) and 1-anilinonaphthalene-8-sulfonate (ANS), which is a multiple-ligand-binding system. The CSP on the binding of ANS involved contributions from two distinct binding sites. PCA of the titration data successfully separated the CSP pattern into contributions from each site. Docking simulations based on the separated CSP patterns provided the structures of βLG-ANS complexes for each binding site. In addition, we determined the K(d) values as 3.42 × 10⁻⁴ M² and 2.51 × 10⁻³ M for Sites 1 and 2, respectively. In contrast, it was difficult to obtain reliable K(d) values for respective sites from the isothermal titration calorimetry experiments. Two ANS molecules were found to bind at Site 1 simultaneously, suggesting that the binding occurs cooperatively with a partial unfolding of the βLG structure. On the other hand, the binding of ANS to Site 2 was a simple attachment without a significant conformational change. From the present results, CS-PCA was confirmed to provide not only the positions and the K(d) values of binding sites but also information about the binding mechanism. Thus, it is anticipated to be a general method to investigate protein-ligand interactions.

Kinetics and Thermodynamics of 1-anilino-8-naphthalene Sulfonate Interactions with Urinary Trypsin Inhibitor

The interactions between Urinary Trypsin Inhibitor (UTI) and 1-anilino-8-naphthalene sulfonate (ANS) were investigated by fluorescence spectra, isothermal titration calorimetry and molecular modeling. The results revealed the presence of four specific binding sites for ANS on UTI, with interactions driven mainly by electrostatic forces. The four specific binding sites indicated the involvement of four hydrophobic patches on UTI. Experimental data also confirmed the presence of a further five nonspecific binding sites that interacted mainly by the formation of salt bridges between the sulfonates of ANS and positive residues on the surface of UTI.

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

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

Optical Odor Imaging by Fluorescence Probes

Odor gas detection is important for the detection of explosives, environmental sensing, biometrics, foodstuffs and a comfortable life. Such odor-source localizations is an active research area for robotics. In this study, we tried to detect odor chemicals with an optical method that can be applied for the spatiotemporal detection of odor. We used four types of fluorescence dyes; tryptophan, quinine sulfate, acridine orange, and 1-anilinonaphthalene-8-sulfonate (ANS). As analyses, we measured the following four odor chemicals, 2-furaldehyde, vanillin, acetophenone, and benzaldehyde. The fluorescence-quenching mechanism of PET (Photoinduced Electron Transfer) or FRET (Fluorescence Resonance Electron Transfer), which occur between fluorescence dyes and odor compounds, could prevent unintended detection of various odorants that is caused by their unspecific adsorption onto the detecting materials. The fluorescence changes were then observed. Thus, we could detect the odor substances through fluorescent quenching by using the fluorescence dyes. Odor information could be obtained by response patterns across all the fluorescence dyes. Moreover, we captured odor images with a cooled CCD camera. Shapes of the targets that emitted odor could be roughly recognized by the odor-shape images. From the spatiotemporal images of odors, twodimensional odor expanse could be obtained.

Effects of Isorhamnetin on Tyrosinase: Inhibition Kinetics and Computational Simulation

We studied the inhibitory effects of isorhamnetin on mushroom tyrosinase by inhibition kinetics and computational simulation. Isorhamnetin reversibly inhibited tyrosinase in a mixed-type manner at Ki=0.235±0.013 mM. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate(ANS)-binding fluorescence showed that isorhamnetin did not induce significant changes in the tertiary structure of tyrosinase. To gain insight into the inactivation process, the kinetics were computed via time-interval measurements and continuous substrate reactions. The results indicated that inactivation induced by isorhamnetin was a first-order reaction with biphasic processes. To gain further insight, we simulated docking between tyrosinase and isorhamnetin. Simulation was successful (binding energies for Dock6.3: -32.58 kcal/mol, for AutoDock4.2: -5.66 kcal/mol, and for Fred2.2: -48.86 kcal/mol), suggesting that isorhamnetin interacts with several residues, such as HIS244 and MET280. This strategy of predicting tyrosinase interaction in combination with kinetics based on a flavanone compound might prove useful in screening for potential natural tyrosinase inhibitors.

A Two-Step Refolding of Acid-Denatured Microbial Transglutaminase Escaping from the Aggregation-Prone Intermediate

Microbial transglutaminase (MTG) is a monomeric globular enzyme made of 331 amino acid residues. The conformation of MTG was examined over the pH 2.0-6.0 region using circular dichroism (CD) and 1-anilino-8-naphthalenesulfonate (ANS). Under conditions of low ionic strength, a decrease of pH below 4 caused a stepwise unfolding with an intermediate exhibiting specific ANS-binding before full unfolding at pH 2.0. At high ionic strength, the decrease of pH led to only an intermediate without further unfolding. The intermediate corresponds to the molten globule state with a secondary structure similar to the native state but disordered tertiary structures. A pH- and NaCl concentration-dependent phase diagram showed that the fully unfolded state exists only under limited conditions of low pH and a low NaCl concentration. Although a refolding yield by the direct jump to pH 6.0 was low, a two-step refolding with incubation at pH 4.0, where MTG is marginally stable, and a subsequent jump to pH 6.0 improved the yield by suppressing the kinetic traps. We propose that the two-step refolding is useful for improving the yield of larger proteins with a high pI value.

Acid-Induced Formation of Molten Globule States in the Wild Type Escherichia coli 5-Enolpyruvylshikimate 3-Phosphate Synthase and its Three Mutated Forms: G96A, A183T and G96A/A183T

Recent advances in protein chemistry have led to progress in the understanding of protein folding and properties of possible intermediates during the folding of proteins. The molten globule (MG) state, a major intermediate of protein folding, has a denatured state with native-like secondary structure. In the present work, the acid-induced unfolding of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and its three different variants (G96A, A183T and G96A/A183T) were studied by far- and near-UV circular dichroism (CD), intrinsic fluorescent emission spectroscopy and 1-anilino naphthalene-8-sulfonate (ANS) binding. At pH<3.0, these EPSPS variants acquire partially folded state, which show the characteristics of the MG state, e.g., a drastic reduction of defined tertiary structure and almost no change in the secondary structure. ANS binding experiments show that hydrophobic surface of these variants is exposed to a greater extent in comparison to the native form, at acidic pH. Wild type, G96A, A183T and G96A/A183T acquire MG states at pH 2.0, 1.5, 3.0 and 3.0, respectively, which show that pH stability of MG state of G96A has increased in comparison to wild type; and pH stability of MG states of two other mutants is lower than that of the wild type. The results suggest that there is a direct relationship between stability of protein and pH stability of its folding intermediates.