Modulating protein unfolding and refolding via the synergistic association of an anionic and a nonionic surfactant.

HYPOTHESIS: Nonionic surfactants can counter the deleterious effect that anionic surfactants have on proteins, where the folded states are retrieved from a previously unfolded state. However, further studies are required to refine our understanding of the underlying mechanism of the refolding process. While interactions between nonionic surfactants and tightly folded proteins are not anticipated, we hypothesized that intermediate stages of surfactant-induced unfolding could define new interaction mechanisms by which nonionic surfactants can further alter protein conformation. EXPERIMENTS: In this work, the behavior of three model proteins (human growth hormone, bovine serum albumin, and ß-lactoglobulin) was investigated in the presence of the anionic surfactant sodium dodecylsulfate, the nonionic surfactant ß-dodecylmaltoside, and mixtures of both surfactants. The transitions occurring to the proteins were determined using intrinsic fluorescence spectroscopy and far-UV circular dichroism. Based on these results, we developed a detailed interaction model for human growth hormone. Using nuclear magnetic resonance and contrast-variation small-angle neutron scattering, we studied the amino acid environment and the conformational state of the protein. FINDINGS: The results demonstrate the key role of surfactant cooperation in defining the conformational state of the proteins, which can shift away or toward the folded state depending on the nonionic-to-ionic surfactant ratio. Dodecylmaltoside, initially a non-interacting surfactant, can unexpectedly associate with sodium dodecylsulfate-unfolded proteins to further impact their conformation at low nonionic-to-ionic surfactant ratio. When this ratio increases, the protein begins to retrieve the folded state. However, the native conformation cannot be fully recovered due to remnant surfactant molecules still adsorbed to the protein. This study demonstrates that the conformational landscape of the protein depends on a delicate interplay between the surfactants, ultimately controlled by the ratio between them, resulting in unpredictable changes in the protein conformation.

Changes in physicochemical, structural and functional properties, and lysinoalanine formation during the unfolding and refolding of pH-shifted black soldier fly larvae albumin.

The changes of physicochemical, structural and functional properties and the lysinoalanine (LAL) formation during the unfolding and refolding of black soldier fly larvae albumin (BSFLA) induced by acid/alkaline pH shift were explored. The results showed that acid/alkaline conditions induced unfolding of BSFLA structure, but also accompanied by the formation of some large aggregates due to the hydrophobic interactions, hydrogen bonds, and disulfide bonds. Compared with control or pH1.5 shift, pH12 shift treatment significantly increased the electrostatic repulsion, surface hydrophobicity, free sulfhydryl group, and deamidation reactions, but reduced the fluorescence intensity of BSFLA, and these change in protein conformation contributed to increase in solubility, emulsion activity, and emulsion stability. But the content of LAL in BSFLA was increased by 93.39 % by pH 12 shift treatment. In addition, pH1.5 shift modified BSFLA tended to form ß-sheet structure through unfolding and refolding, resulting in the formation of aggregates with larger particle sizes, and reducing the solubility and the LAL content by 7.93 % and 65.53 %, respectively. SDS-PAGE profile showed that pH12/1.5 shifting did not cause irreversible denaturation of protein molecules. Therefore, pH12-shift is good way to improve the functional properties of BSFLA, but the content of LAL should be reduced to make it better used in food.

A novel sulfamethoxazole derivative as an inhibitory agent against HSP70: A combination of computational with in vitro studies.

In the current study, a novel derivative of sulfamethoxazole (a sulfonamide containing anti-biotic) named ZM-093 (IUPAC name: (E)-4-((4-(bis(2-hydroxyethyl)amino)phenyl)diazenyl)-N-(5-methylisoxazole-3-yl)benzenesulfonamide) was synthesized via common diazotization-coupling reactions from sulfamethoxazole and subsequently characterized through NMR/FT-IR spectroscopy. After evaluation, the compound was geometrically optimized at the DFT level of theory with BL3YP method and 6/31++G (d,p) basis set and from the optimized structure, several molecular descriptors important in the biological reactivity of the compound, such as global reactivity parameters, molecular electrostatic potential, average local ionization energy, and drug-likeness features of the compound were computationally analyzed. The experimental in vitro investigations of the interaction between ZM-093 and heat shock protein 70 (HSP70), a protein that is highly expressed in several types of cancers, exhibited a significant inhibitory effect against the chaperone activity of HSP70 for the titled compound (P-value < 0.01) and the comparison between the experimental studies with the mentioned computational analysis, as well as molecular docking, illustrated that ZM-093 may inhibit HSP70 through binding to its substrate-binding domain. Finally, by taking all the previous results into account, a new method for assessing the inhibitory activity of ligand to HSP70 is introduced based on protonography, a recently developed method that is dependent on the catalytic activity of carbonic anhydrase on polyacrylamide gel electrophoresis.

Structural Refolding and Thermal Stability of Myoglobin in the Presence of Mixture of Crowders: Importance of Various Interactions for Protein Stabilization in Crowded Conditions.

The intracellular environment is overcrowded with a range of molecules (small and large), all of which influence protein conformation. As a result, understanding how proteins fold and stay functional in such crowded conditions is essential. Several in vitro experiments have looked into the effects of macromolecular crowding on different proteins. However, there are hardly any reports regarding small molecular crowders used alone and in mixtures to observe their effects on the structure and stability of the proteins, which mimics of the cellular conditions. Here we investigate the effect of different mixtures of crowders, ethylene glycol (EG) and its polymer polyethylene glycol (PEG 400 Da) on the structural and thermal stability of myoglobin (Mb). Our results show that monomer (EG) has no significant effect on the structure of Mb, while the polymer disrupts its structure and decreases its stability. Conversely, the additive effect of crowders showed structural refolding of the protein to some extent. Moreover, the calorimetric binding studies of the protein showed very weak interactions with the mixture of crowders. Usually, we can assume that soft interactions induce structural perturbations while exclusion volume effects stabilize the protein structure; therefore, we hypothesize that under in vivo crowded conditions, both phenomena occur and maintain the stability and function of proteins.

High levels of glucose alter Physcomitrella patens metabolism and trigger a differential proteomic response.

Sugars act not only as substrates for plant metabolism, but also have a pivotal role in signaling pathways. Glucose signaling has been widely studied in the vascular plant Arabidopsis thaliana, but it has remained unexplored in non-vascular species such as Physcomitrella patens. To investigate P. patens response to high glucose treatment, we explored the dynamic changes in metabolism and protein population by applying a metabolomic fingerprint analysis (DIESI-MS), carbohydrate and chlorophyll quantification, Fv/Fm determination and label-free untargeted proteomics. Glucose feeding causes specific changes in P. patens metabolomic fingerprint, carbohydrate contents and protein accumulation, which is clearly different from those of osmotically induced responses. The maximal rate of PSII was not affected although chlorophyll decreased in both treatments. The biological process, cellular component, and molecular function gene ontology (GO) classifications of the differentially expressed proteins indicate the translation process is the most represented category in response to glucose, followed by photosynthesis, cellular response to oxidative stress and protein refolding. Importantly, although several proteins have high fold changes, these proteins have no predicted identity. The most significant discovery of our study at the proteome level is that high glucose increase abundance of proteins related to the translation process, which was not previously evidenced in non-vascular plants, indicating that regulation by glucose at the translational level is a partially conserved response in both plant lineages. To our knowledge, this is the first time that metabolome fingerprint and proteomic analyses are performed after a high sugar treatment in non-vascular plants. These findings unravel evolutionarily shared and differential responses between vascular and non-vascular plants.

Comparative refolding of guanidinium hydrochloride denatured bovine serum albumin assisted by cationic and anionic surfactants via artificial chaperone protocol: Biophysical insight.

In the present study, we report the cooperative refolding/renaturation behaviour of guanidinium hydrochloride (GdnHCl) denatured bovine serum albumin (BSA) in the presence of cationic surfactant cetyltrimethylammonium bromide (CTAB), anionic surfactant sodium dodecyl sulphate (SDS) and their catanionic mixture in the solution of 60 mM sodium phosphate buffer of physiological pH 7.4, using artificial chaperone-assisted two-step method. Here, we have employed biophysical techniques to characterize the refolding mechanism of denatured BSA after 200 times of dilution in the presence of cationic, anionic surfactants and their catanionic mixture, separately. We have found that minimum refolding of diluted BSA in the presence of 1:1 rational mixture of CTAB and SDS (CTAB/SDS = 50/50), it may be due to the micelles formation which is responsible for the unordered microstructure aggregate formation. Other mixtures (CTAB/SDS = 20/80 and 80/20) slightly played an effective role during refolding process in the presence of methyl-ß-cyclodextrin. On other hand, CTAB and SDS are more effective and reflect a good renaturation tendency of denatured BSA solution separately and in existence of methyl-ß-cyclodextrin as compare to their mixture compositions. But overall, CTAB has the better renaturation tendency as compare to SDS in the existence of methyl-ß-cyclodextrin. These results ascribed the presence of charge head group and length of hydrophobic tail of CTAB surfactant that plays an important task during electrostatic and hydrophobic interactions at pH 7.4 at which BSA carries negative charge on their surface. These biophysical parameters suggest that, CTAB surfactant assisted artificial chaperone protocol may be utilized in the protein renaturation/refolding studies, which may address the associated problems of biotechnological industries for the development of efficient and inexpensive folding aides, which may also be used to produced genetically engineered cells related diseases, resulting from protein misfolding/aggregation.

Identification of vibsanin A analog as a novel HSP90 inhibitor.

Vibsanin A is the first natural product isolated from Viburnum awabuki and has several biological activities. We have reported that a vibsanin A analog, obtained from process of total synthesis of vibsanin A, has anti-proliferative activity against human cancer cell lines. In this study, we evaluated anti-proliferative effect of the vibsanin A analogs against various human cancer cell lines and examined molecular target of the analog in human cells. Among the vibsanin A analogs, vibsanin A analog C (VAC) showed anti-proliferative effect against various cancer cell lines, and the anti-proliferative activity was strongest among the vibsanin A analogs. Additionally, VAC fluctuated amounts of HSP90-related proteins in cells and inhibited HSP90-mediated protein refolding of luciferase in vitro. These results suggest that the anti-proliferative activity of VAC is due to HSP90 inhibition, and VAC has a potential as novel anti-cancer drug as HSP90 inhibitor.

Superior inhibition of influenza virus hemagglutinin-mediated fusion by indole-substituted spirothiazolidinones.

The influenza virus hemagglutinin (HA) mediates membrane fusion after viral entry by endocytosis. The fusion process requires drastic low pH-induced HA refolding and is prevented by arbidol and tert-butylhydroquinone (TBHQ). We here report a class of superior inhibitors with indole-substituted spirothiazolidinone structure. The most active analogue 5f has an EC50 value against influenza A/H3N2 virus of 1 nM and selectivity index of almost 2000. Resistance data and in silico modeling indicate that 5f combines optimized fitting in the TBHQ/arbidol HA binding pocket with a capability for endosomal accumulation. Both criteria appear relevant to achieve superior inhibitors of HA-mediated fusion.

Denaturation studies of Clarias gariepinus glutathione transferase in dilute and crowded solutions.

It is important to understand the effect of crowding conditions on the native structure and functional state of enzymes. Equilibrium denaturation studies of Clarius gariepinus GST (CgGST) by guanidine hydrochloride (GdHCl) under dilute conditions and in separate solutions of 0-100 g dm-3 Ficoll 70, polyethylene glycol 6000 (PEG 6000) and equal w/v mixtures of the two polymers at 25 °C and pH 7.4 were studied fluorometrically. The data were analyzed based on a two-state model assuming the native protein dimer separates into two monomers and then unfolds. The standard free energy of unfolding (ΔG°UN) increases with increasing concentration of each crowding agent in a manner suggesting that high concentrations of PEG 6000 and Ficoll 70 favour the native CgGST relative to the unfolded form. Ficoll 70 stabilizes the native CgGST better than PEG 6000 at low w/v concentration. A mixture of equal g/cm3 concentrations of both crowding agents, however, stabilizes the native form more effectively than either Ficoll 70 or PEG 6000 at equivalent w/v total concentration and is less sensitive to GdHCl. This is in strong agreement with the results of refolding studies, and suggests that a mixture of molecular crowders of widely different molecular weights might show enhanced excluded volume effects compared to a single crowder. Thus, mixed crowding agents more effectively protect the enzyme against denaturation and assist in renaturation better than a single crowder. This suggests a heterogeneous solution of crowders, as will be found within cells, enhances the beneficial effect of crowding on the folded protein stability.

Molecular cloning, expression, and functional characterization of 70-kDa heat shock protein, DnaK, from Bacillus halodurans.

In the present study, we report cloning, sequencing, and functional characterization dnaK gene of B. halodurans that is the central component in cellular network of molecular chaperones. The 3D structures of DnaK obtained by I-TASSER server showed that the overall structures of DnaK from B. halodurans and human HSP70 chaperone BiP are very similar with a homology of 88.8%. The purified recombinant DnaK consists of a His-tag at C-terminus and show a band on approximately 70-kDa region in SDS-PAGE. The resultant refolding assay revealed that the refolding rate was considerably improved by the addition of the novel DnaK chaperone for the refolding of heat-denatured carbonic anhydrase. Also, salt resistance experiments indicated that E. coli + DnaK survival had enhanced by 4.4-fold as compared with control cells in 0.4 M NaCl. The number of E. coli + DnaK colonies was 2.5-fold higher than control colonies in pH 9.5. We showed that DnaK refolding functions were decreased by increasing Cd2+ in nanomolar concentrations. Hg2+ had a biphasic effect on recombinant DnaK refolding function: inhibition at low and stimulation at high concentrations. It was concluded that the DnaK from B. halodurans can potentially be employed for improving functional properties of proteins in various applications.

Investigation of the role of central metal ion of Cyathus bulleri laccase 1 using guanidinium chloride-induced denaturation.

The structure and folding/unfolding kinetics of Cyathus bulleri laccase 1 (Lcc1), expressed in Pichia pastoris, were analyzed by spectroscopic methods to understand the role of central metal ion. Far UV CD structure analysis revealed major ß-sheet and minor α helical segments present in the Lcc1. A significant change in the spectrum of Lcc1, indicative of unfolding of secondary structures, was observed with increasing concentrations of guanidinium chloride (GdnHCl) during Trp fluorescence, absorption and CD measurements. A similar trend was also noticed for enzyme activity with respect to GdnHCl concentrations. To establish the role of copper ion in the catalytic activity of laccase, a complete removal of copper was carried out and addition of copper was found to restore the structure and activity during the refolding process. The apo form was also reconstituted by addition of zinc ion which restored nearly 84% of enzyme activity, indicating non-essential role of copper in maintaining conformation and activity. Structural studies and inductively coupled plasma mass spectrometry data supported these observations.

Spectroscopic probing of the refolding of an unfolded protein through the formation of mixed-micelles.

We report the unfolding of the globular protein, Bovine Serum Albumin (BSA) induced by anionic surfactant sodium dodecyl sulfate (SDS) and subsequently monitored the refolding of this denatured BSA using triblock copolymers F127 and P123 through the formation of mixed micelles. Our study exclusively represents the reversibility of this unfolding-refolding process using pluronic triblock copolymers F127/P123 as refolding agents. We confirm the recovery of its native state from its denatured state estimating the α-helical structure of the denatured protein from the CD data which support our steady state fluorescence spectra monitoring the fluorescence of the intrinsic Trp molecules present in BSA. Time resolved study also corroborates the stepwise recovery of the denatured BSA as well as the reversibility of the processes. Isothermal Titration Calorimetry (ITC) data explain the negligible interactions between the triblock copolymers and the native state of BSA. The high binding constant of SDS and triblock copolymers probably play the crucial role in the stepwise recovery of the unfolded BSA followed by reversibility of the refolding processes through the formation of the mixed micelles. The mechanism of mixed-micelle formation has been substantiated by the fact that the Guanidine Hydrochloride denatured BSA does not react with F127/P123 whereby no recovery of the protein was observed.

Chloride promotes refolding of active Vibrio alkaline phosphatase through an inactive dimeric intermediate with an altered interface.

Most enzymes are homodimers or higher order multimers. Cold-active alkaline phosphatase from Vibrio splendidus (VAP) transitions into a dimer with very low activity under mild denaturation conditions. The desire to understand why this dimer fails to efficiently catalyse phosphomonoester hydrolysis led us to investigate interfacial communication between subunits. Here, we studied in detail the unfolding mechanism at two pH values and in the presence or absence of sodium chloride. At pH 8.0, the denaturation model had to include an inactive dimer intermediate and follow the pathway: N2 → I2 → 2U. At pH 10.5, the model was of a two-state nature. Enzyme activity was not recovered under several examined refolding conditions. However, in the presence of 0.5 m NaCl, the enzyme was nearly fully reactivated after urea treatment. Thermal inactivation experiments were biphasic where the inactivation could be detected using CD spectroscopy at 190-200 nm. By incorporating a bimane fluorescence probe at the dimer interface, we could monitor inactivation/denaturation at two distinct sites at the dimer interface. A change in bimane fluorescence at both sites was observed during inactivation, but prior to the global unfolding event. Furthermore, the rate of change in bimane fluorescence correlated with inactivation rates at 40 °C. These results indicate and support the hypothesis that the subunits of VAP are only functional in the dimeric state due to the cooperative nature of the reaction mechanism when proper crosstalk between subunits is facilitated.

The heat shock protein amplifier arimoclomol improves refolding, maturation and lysosomal activity of glucocerebrosidase.

BACKGROUND: Gaucher Disease is caused by mutations of the GBA gene which encodes the lysosomal enzyme acid beta-glucosidase (GCase). GBA mutations commonly affect GCase function by perturbing its protein homeostasis rather than its catalytic activity. Heat shock proteins are well known cytoprotective molecules with functions in protein homeostasis and lysosomal function and their manipulation has been suggested as a potential therapeutic strategy for GD. The investigational drug arimoclomol, which is in phase II/III clinical trials, is a well-characterized HSP amplifier and has been extensively clinically tested. Importantly, arimoclomol efficiently crosses the blood-brain-barrier presenting an opportunity to target the neurological manifestations of GD, which remains without a disease-modifying therapy. METHODS: We used a range of biological and biochemical in vitro assays to assess the effect of arimoclomol on GCase activity in ex vivo systems of primary fibroblasts and neuronal-like cells from GD patients. FINDINGS: We found that arimoclomol induced relevant HSPs such as ER-resident HSP70 (BiP) and enhanced the folding, maturation, activity, and correct cellular localization of mutated GCase across several genotypes including the common L444P and N370S mutations in primary cells from GD patients. These effects where recapitulated in a human neuronal model of GD obtained by differentiation of multipotent adult stem cells. INTERPRETATION: These data demonstrate the potential of HSP-targeting therapies in GCase-deficiencies and strongly support the clinical development of arimoclomol as a potential therapeutic option for the neuronopathic forms of GD. FUNDING: The research was funded by Orphazyme A/S, Copenhagen, Denmark.

Role of Thiol Reactivity for Targeting Mutant p53.

Reactivation of mutant p53 has emerged as a promising approach for cancer therapy. Recent studies have identified several mutant p53-reactivating compounds that target thiol groups in mutant p53. Here we have investigated the relationship between thiol reactivity, p53 thermostabilization, mutant p53 refolding, mutant p53-dependent growth suppression, and induction of cell death. Analysis of the National Cancer Institute database revealed that Michael acceptors show the highest selectivity for mutant p53-expressing cells among analyzed thiol-reactive compounds. Further experimental testing demonstrated that Michael acceptors, aldehydes, imines, and primary alcohols can promote thermodynamic stabilization of mutant p53. Moreover, mild thiol reactivity, often coupled with combined chemical functional groups, such as in imines, aldehydes, and primary alcohols, can stimulate mutant p53 refolding. However, strong electrophile activity was associated with cellular toxicity. Our findings may open possibilities for rational design of novel potent and selective mutant p53-reactivating compounds.

Optimization of in vitro refolding conditions of recombinant Lepidium draba peroxidase using design of experiments.

The main objective of this study was to optimize the in vitro refolding conditions of the recombinant Lepidium draba peroxidase (LDP). Initially, the effects of various factors were investigated on LDP refolding yield using one-factor-at-a-time (OFAT) method. Based on the OFAT results, optimum concentrations for LDP refolding were 2 M urea, 2 mM CaCl2, 0.42 mM l-glutathione oxidized (GSSG), 0.20 mg/ml protein, and 12 µM hemin as well as pH 7. Secondly, according to the OFAT results, design of experiments (DOE) was applied for investigation of the interactions between factors including protein (P), urea (U), CaCl2 (C), and GSSG (G). The results showed the possible interaction between PC, PG, PU, and GU. Lastly, response surface methodology (RSM) was used for final refolding conditions optimization. The final optimized refolding conditions for LDP were conducted as 2 M urea, 1 mM CaCl2, 0.70 mM GSSG, 0.07 mM DTT, 0.15 mg/ml protein which they obtained from RSM results and 12 µM hemin, and pH 7 according to the results of OFAT method. Overall, under optimal conditions, 23.4 mg active refolded LDP per liter of expression medium was obtained. So, the refolding yield was calculated to be approximately 48%.

Triblock-Copolymer-Assisted Mixed-Micelle Formation Results in the Refolding of Unfolded Protein.

The present work reports a new strategy for triblock-copolymer-assisted refolding of sodium dodecyl sulfate (SDS)-induced unfolded serum protein human serum albumin (HSA) by mixed-micelle formation of SDS with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer EO20PO68EO20 (P123) under physiological conditions. The steady-state and time-resolve fluorescence results show that the unfolding of HSA induced by SDS occurs in a stepwise manner through three different phases of binding of SDS, which is followed by a saturation of interaction. Interestingly, the addition of polymeric surfactant P123 to the unfolded protein results in the recovery of ∼87% of its α-helical structure, which was lost during SDS-induced unfolding. This is further corroborated by the return of the steady-state and time-resolved fluorescence decay parameters of the intrinsic tryptophan (Trp214) residue of HSA to the initial nativelike condition. The isothermal titration calorimetry (ITC) data also substantiates that there is almost no interaction between P123 and the native state of the protein. However, the mixed-micelle formation, accompanied by substantial binding affinities, removes the bound SDS molecules from the scaffolds of the unfolded state of the protein. On the basis of our experiments, we conclude that the formation of mixed micelles between SDS and P123 plays a pivotal role in refolding the protein back to its nativelike state.

NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B.

Non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase that catalyzes replication of the hepatitis C virus (HCV) RNA genome and therefore is central for its life cycle. NS5B interacts with the intrinsically disordered domain 2 of NS5A (NS5A-D2), another essential multifunctional HCV protein that is required for RNA replication. As a result, these two proteins represent important targets for anti-HCV chemotherapies. Despite this importance and the existence of NS5B crystal structures, our understanding of the conformational and dynamic behavior of NS5B in solution and its relationship with NS5A-D2 remains incomplete. To address these points, we report the first detailed NMR spectroscopic study of HCV NS5B lacking its membrane anchor (NS5BΔ21). Analysis of constructs with selective isotope labeling of the δ1 methyl groups of isoleucine side chains demonstrates that, in solution, NS5BΔ21 is highly dynamic but predominantly adopts a closed conformation. The addition of NS5A-D2 leads to spectral changes indicative of binding to both allosteric thumb sites I and II of NS5BΔ21 and induces long-range perturbations that affect the RNA-binding properties of the polymerase. We compared these modifications with the short- and long-range effects triggered in NS5BΔ21 upon binding of filibuvir, an allosteric inhibitor. We demonstrate that filibuvir-bound NS5BΔ21 is strongly impaired in the binding of both NS5A-D2 and RNA. NS5A-D2 induces conformational and functional perturbations in NS5B similar to those triggered by filibuvir. Thus, our work highlights NS5A-D2 as an allosteric regulator of the HCV polymerase and provides new insight into the dynamics of NS5B in solution.

A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli.

Using anti-EGFR (epidermal growth factor receptor) nanobody is a good choice for diagnoses and therapeutics for high EGFR expression diseases. In the present study, the percentage composition of anti-EGFR nanobody attained 25% of the total cell protein expressed in Escherichia coli BL21 (DE3). However, almost all nanobodies were expressed as inclusion bodies. To acquire active nanobodies, a series of dilution refolding procedures were optimized after inclusion bodies were dissolved into 6 M urea and purified with immobilized metal affinity chromatography. The results showed the refolding rate of the anti-EGFR nanobodies attained to 73%, and about 100 mg nanobodies were refolded from 1 L cells under the conditions that the initial nanobody concentration was 0.3 mg/mL, the dilution speed was 2.5 mL/Min, the dilution buffer was Tris-HCl at pH 8.0, the additives were 0.2 M Arg, 5 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). Then the activity of the refolded nanobodies was confirmed. The results showed that the refolded anti-EGFR nanobodies, in a dose-dependent manner, bounded to the tumor cell surface of A431 and MCF-7 and significantly inhibited the proliferation of A431 caused by the epidermal growth factor. Our study provides a facile method to rapidly, efficiently, and massively prepare anti-EGFR antibodies and promotes anti-EGFR-based recognition in cancer diagnoses and therapeutics.

Dual Lifetimes for Complexes between Glutathione-S-transferase (hGSTA1-1) and Product-like Ligands Detected by Single-Molecule Fluorescence Imaging.

Single-molecule fluorescence techniques were used to characterize the binding of products and inhibitors to human glutathione S-transferase A1-1 (hGSTA1-1). The identification of at least two different bound states for the wild-type enzyme suggests that there are at least two conformations of the protein, consistent with the model that ligand binding promotes closure of the carboxy-terminal helix over the active site. Ligand induced changes in ensemble fluorescence energy transfer support this proposed structural change. The more predominant state in the ensemble of single molecules shows a significantly faster off-rate, suggesting that the carboxy-terminal helix is delocalized in this state, permitting faster exit of the bound ligand. A point mutation (I219A), which is known to interfere with the association of the carboxy-terminal helix with the enzyme, shows increased rates of interconversion between the open and closed state. Kinematic traces of fluorescence from single molecules show that a single molecule readily samples a number of different conformations, each with a characteristic off-rate.