Spectroscopic studies on the interaction mechanisms of safranin T with herring sperm DNA using acridine orange as a fluorescence probe

Under the condition of physiological pH environment (pH = 7.40), the interactions of safranin T (ST) with herring sperm DNA were studied by means of spectral methods using acridine orange (AO) as a fluorescence probe. The spectroscopic characteristics of DNA–AO in the case of ST (along with the increase of concentration) were observed in an aqueous medium. The binding constants for ST stranded DNA and competitive bindings of ST interacting with DNA–AO systems were examined by fluorescence spectra, and the binding mechanism of ST with DNA was researched via viscosity measurements. All the testimony manifested that bonding modes between ST and DNA were evidenced to be intercalative binding and electrostatic binding, and the combining constant of ST with DNA was obtained. The binding of ST to DNA was driven by entropy and enthalpy through the calculated thermodynamic parameters (Δ_rH_m^?, Δ_rS_m and Δ_rG_m^?). Copyright © 2014 John Wiley & Sons, Ltd.     

Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

AIMS: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the beta-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. METHODS AND RESULTS: Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and beta-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 microg ml(-1) and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 microg ml(-1). The highest concentration that gave permissible growth rate was 50 microg ml(-1). The isolated survivals were screened on the bases of PGA and beta-lactamase activities. Among the retained mutants, the occurrence of beta-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). CONCLUSIONS: In seven of the mutants, PGA activity was enhanced with considerable decrease in beta-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of beta-lactamase activity and twofold increase in PGA activity [12.7 mg 6-amino-penicillanic acid (6-APA) h(-1) mg(-1) wet cells] compared with that in the wild-type strain (6.3 mg 6-APA h(-1) mg(-1) wet cells). SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of beta-lactamase. These mutants could be used for industrial production of PGA.     

Fluorescence enhancement effect of the morin-Al3+ -sodium dodecyl benzene sulphonate-protein system and the determination of proteins.

The fluorescence intensity of the morin-Al(3+) complex was greatly enhanced by proteins in the presence of sodium dodecyl benzene sulphonate (SDBS). Based on this, a new fluorimetric method for the determination of protein was developed. Under optimum conditions, the enhanced intensity of fluorescence was in proportion to the concentration of proteins in the range 1.0 x 10(-8)-1.3 x 10(-5) g/mL for bovine serum albumin (BSA), 4.0 x 10(-8)-1.2 x 10(-5) g/mL for egg albumin (EA) and 5.0 x 10(-8)-1.2 x 10(-5) g/mL for human serum albumin (HSA). Their detection limits (S:N = 3) were 5.0 x 10(-9), 1.8 x 10(-8) and 1.6 x 10(-8) g/mL, respectively. The interaction mechanism was also studied.     

Determination of trace carvedilol by solid substrate–room temperature phosphorimetry,based on its activating effect on hypochlorite‐oxidizing amaranth using sodium dodecyl benzene sulphonate as sensitizer

This work proposes a simple and sensitive solid substrate–room temperature phosphorimetry (SS–RTP) for the selective determination of carvedilol (CV). The method is based on the sensitizing effect of sodium dodecyl benzene sulphonate (SDBS) on CV to activate the oxidation between NaClO and amaranth, resulting in the intense quenching of room temperature phosphorescence (RTP) of the system. Compared with non‐SDBS system, the reduction of phosphorescence intensity (ΔI_p) with SDBS is 16.5 times higher and is directly proportional to the content of CV, covering a wide range 0.080–16.00 fg/spot. The regression equation of the working curve can be expressed as ΔI_p = 0.7780 + 7.057 m_CV (fg/spot) (correlation coefficient (r) = 0.9976, n = 8), with a detection limit (LD) of 0.020 fg/spot (corresponding concentration is 5.1 × 10⁻¹⁴ g/mL, sample volume is 0.40 μL/spot). This sensitive method has also been applied to determine trace CV in human plasma and the results agreed with synchronous fluorimetry (SF). The activation energy (E) and rate constant (k) of this activating reaction were 69.04 kJ/mol and 3.580 × 10⁻⁴ s⁻¹, respectively. The reaction mechanism is also discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Evaluation of the effect of Tb(IV)-NR complex on herring sperm DNA genetic information by mean of spectroscopic

Abstract

The interaction between Tb(IV)-NR complex and herring sperm DNA in buffer solution of Tris-HCl was investigated with the use of acridine orange(AO) as a spectral probe. The binding modes and other information were provided by the UV–spectrophotometry and fluorescence spectroscopy. The thermodynamic functions expressed that the binding constants of Tb(IV)-NR complex with DNA was K^θ_298.15K = 4.03?×?10⁵?L·mol^?1, K^θ_310.15K =1.30?×?10⁷?L·mol^?1, and the Δ_rGθ m _298.15?K＝?3.20?×?10⁴ J·mol^?1. The scatchard equation suggested that the interaction mode between Tb(IV)-NR complex and herring sperm DNA is electrostatic and weak intercalation bindings. FTIR spectroscopy results also indicate that there is a specific interaction between the Tb(IV)-NR complex and the A and G bases of DNA.     

Identification of interacting proteins for calcium-dependent protein kinase 8 by a novel screening system based on bimolecular fluorescence complementation

Protein kinases are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. We developed a novel assay system, based on the bimolecular fluorescence complementation (BiFC) technique in Escherichia coli, for detecting transient interactions such as those between kinases and their substrates. This system detected the interaction between OsMEK1 and its direct target OsMAP1. By contrast, BiFC fluorescence was not observed when OsMAP2 or OsMAP3, which are not substrates of OsMEK1, were used as prey proteins. We also screened for interacting proteins of calcium-dependent protein kinase 8 (OsCPK8), a regulator of plant immune responses, and identified three proteins as interacting molecules of OsCPK8. The interaction between OsCPK8 and two of these proteins (ARF-GEF and peptidyl prolyl isomerase) was confirmed in rice cells by means of BiFC technology. These results indicate that our new assay system has the potential to screen for protein kinase target molecules.     

Study on the fluorescent enhancement effect in terbium-gadolinium-protein-sodium dodecyl benzene sulfonate system and its application on sensitive detection of protein at nanogram level

The co-luminescence effect in a terbium-gadolinium-protein-sodium dodecyl benzene sulfonate (SDBS) system is reported here. Based on it, the sensitive quantitative analysis of protein at nanogram levels is established. The co-luminescence mechanism is studied using fluorescence, resonance light scattering (RLS), absorption spectroscopy and NMR measurement. It is considered that protein could be unfolded by SDBS, then a efficacious intramolecular fluorescent energy transfer occurs from unfolded protein to rare earth ions through SDBS acting as a "transfer bridge" to enhance the emission fluorescence of Tb3+ in this ternary complex of Tb-SDBS-BSA, where energy transfer from protein to SDBS by aromatic ring stacking is the most important step. Cooperating with the intramolecular energy transfer above is the intermolecular energy transfer between the simultaneous existing complexes of both Tb3+ and Gd3+. The fluorescence quantum yield is increased by an energy-insulating sheath, which is considered to be another reason for the resulting enhancement of the fluorescence. F?rster theory is used to calculate the distribution of enhancing factors and has led to a greater understanding of the mechanisms of energy transfer.     

Differential effect of brefeldin A on the palmitoylation of surfactant protein C proprotein mutants.

The surfactant protein C precursor (proSP-C) is palmitoylated on two cysteines adjacent to its transmembrane domain. We showed previously that palmitoylation of proSP-C occurs in a postendoplasmic reticulum compartment and is not affected by the Golgi-disturbing agent brefeldin A (BFA). In contrast, the investigations presented here showed that BFA almost completely abolished palmitoylation of proSP-C mutants that contained alterations in the region between the palmitoylated cysteines and the transmembrane domain, including a Pro 30 to Leu mutant associated with interstitial lung disease. This differential effect of BFA was not caused by differences in the palmitoylation kinetics between wild-type proSP-C and the mutants and was not mimicked by nocodazole and monensin. However, differences between the mutants and wild-type proSP-C in the relative degree of processing suggest that BFA may unmask a difference in routing. This would imply that the amino acids just N-terminal of the transmembrane domain may be important for a proper sorting of proSP-C.     

A bacterial two-hybrid system based on the twin-arginine transporter pathway of E. coli

We have developed a bacterial two-hybrid system for the detection of interacting proteins that capitalizes on the folding quality control mechanism of the Twin Arginine Transporter (Tat) pathway. The Tat export pathway is responsible for the membrane translocation of folded proteins, including proteins consisting of more than one polypeptide, only one of which contains a signal peptide ("hitchhiker export"). Here, one protein (bait) is expressed as a fusion to a Tat signal peptide, whereas the second protein (prey) is fused to a protein reporter that can confer a phenotype only after export into the bacterial periplasmic space. Since the prey-reporter fusion lacks a signal peptide, it can only be exported as a complex with the bait-signal peptide fusion that is capable of targeting the Tat translocon. Using maltose-binding protein as a reporter, clones expressing interacting proteins can be grown on maltose minimal media or on MacConkey plates. In addition, we introduce the use of the cysteine disulfide oxidase DsbA as a reporter. Export of a signal peptide-prey:bait-DsbA complex into the periplasm allows complementation of dsbA(-) mutants and restores the formation of active alkaline phosphatase, which in turn can be detected by a chromogenic assay.     

The effect of sodium dodecyl sulfate on the structure and function of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) has been shown to exist as a dimer of molecular weight of 23,000 in 25 mm phosphate buffer, at pH 7.0 (the ionic strength 0.1 m with NaCl), 25.0 °C in the concentration range of 0.01–10 mg/ml. In the present paper, the effects of an anionic detergent, sodium dodecyl sulfate (SDS), on the structure and function of SSI has been examined, [a]The molecular weight of SSI was measured in the SDS solution with the sedimentation equilibrium method of the multicomponent-polydisperse system under the conditions described above, and thereby it has been shown that SSI dissociates into monomers with SDS of 0.03–0.12% ($\text{w}\text{v}$) when the concentration of SSI is 1.00 mg/ml (87.0 μm as monomer), [b]As SSI dissociates into monomers, there were observed blue-shift troughs at 293 nm and 300 nm due to a tryptophyl residue and a red-shift of phenylalanyl residues in the absorption difference spectrum induced by the binding of SSI and SDS. [c] The inhibitory activity of SSI against subtilisin BPN′-catalyzed hydrolysis of p-nitrophenyl acetate was measured under the conditions that SSI is in monomer in the SDS solution. Unexpectedly half of the inhibitory activity of SSI against subtilisin BPN′ is lost in the SDS solution.     

Effect of sodium dodecyl sulphate on the high molecular weight protein fraction of mustard (Brassica juncea) and rapeseed (Brassica campestris)

The effect of sodium dodecyl sulphate on mustard and rapeseed 12S protein has been monitored by the techniques of ultracentrifugation, viscosity, difference spectra and fluorescence spectrophotometry. At low concentration of sodium dodecyl sulphate (<3.47 mM) mustard protein undergoes aggregation and at higher concentrations it dissociates to 1.8 S protein, the dissociation being complete at 17.3 mM sodium dodecyl sulphate. The rapeseed protein, on the other hand, undergoes dissociation at all the concentrations of sodium dodecyl sulphate. The reduced viscosity values of mustard protein in the presence of the denaturant are higher than those of rapeseed protein. Similarly in difference spectra change in absorbance values of mustard protein are higher.’ The relative fluorescence intensity of the mustard protein increases with sodium dodecyl sulphate concentration, upto 0.87 mM and this is followed by fluorescence quneching at higher denaturant concentrations. However, with the rapeseed protein fluorescence quenching was observed at all concentrations of sodium dodecyl sulphate.     

Precision of sodium dodecyl sulfate-polyacrylamide-gel electrophoresis for the molecular weight estimation of a membrane glycoprotein: studies on bovine rhodopsin.

Criteria for assessing the precision and accuracy of methods for estimation of molecular weight for proteins using sodium dodecyl sulfate-polyacrylamide-gel electrophoresis have been applied to rhodopsin from bovine visual cell outer segment membranes. Various methods of preparing this hydrophobic protein for electrophoresis differ in their ability to solubilize and disaggregate polypeptide constituents of the outer segment membrane, with resultant variations in the pattern of protein bands and the apparent molecular weight of rhodopsin. Even with optimal solubilization and disaggregation, the behavior of rhodopsin relative to a series of standard proteins is such that the apparent molecular weight decreases systematically from 40,400 to 34,500 as the acrylamide concentration increases from 4 to 10%. As demonstrated by Ferguson plots of logR_f vs gel concentration and split gel experiments, this discrepancy is explained by the fact that the extrapolated R_f for zero gel concentration (Y₀) for rhodopsin is significantly lower than the Y₀'s for the soluble proteins used as molecular weight standards. In such cases, a possibly more reliable molecular weight estimate is obtained by plotting the retardation coefficient (K_R) vs molecular weight. This method yields a value of 29,500 ± 1000 for bovine rhodopsin if only the errors in measurement of R_f are considered and a quadratic relationship between K_R and molecular weight is used. Using weighted linear regression for K_R vs molecular weight, we obtain a molecular weight estimate of 32,700 ± 5000 when the uncertainty in the calibration curve is considered. Because of uncertainties regarding the detergent-binding properties of rhodopsin and the relationship of its Stokes radius to its molecular weight by comparison with the soluble protein standards, these values must be viewed with caution.     

Development of a simple assay system for protein-stabilizing efficiency based on hemoglobin protection against denaturation and measurement of the cooperative effect of mixing protein stabilizers

We have elucidated the cooperative stabilization of proteins by sugars, amino acids, and other protein-stabilizing agents using a new and simple assay system. Our system determines the protein-stabilizing ability of various compounds by measuring their ability to protect hemoglobin from denaturation. Hemoglobin denaturation was readily measured by quantitative changes in its ultraviolet–visible absorption spectrum. The efficiency of our assay was confirmed using various sugars such as trehalose and sucrose that are known to be good protein stabilizers. We have also found that mixtures of two different types of protein stabilizers resulted in a cooperative stabilizing effect on protein.     

In silico two-hybrid system for the selection of physically interacting protein pairs

Deciphering the interaction links between proteins has become one of the main tasks of experimental and bioinformatic methodologies. Reconstruction of complex networks of interactions in simple cellular systems by integrating predicted interaction networks with available experimental data is becoming one of the most demanding needs in the postgenomic era. On the basis of the study of correlated mutations in multiple sequence alignments, we propose a new method (in silico two-hybrid, i2h) that directly addresses the detection of physically interacting protein pairs and identifies the most likely sequence regions involved in the interactions. We have applied the system to several test sets, showing that it can discriminate between true and false interactions in a significant number of cases. We have also analyzed a large collection of E. coli protein pairs as a first step toward the virtual reconstruction of its complete interaction network.     

Rational design of TNFα binding proteins based on the de novo designed protein DS119

De novo protein design offers templates for engineering tailor‐made protein functions and orthogonal protein interaction networks for synthetic biology research. Various computational methods have been developed to introduce functional sites in known protein structures. De novo designed protein scaffolds provide further opportunities for functional protein design. Here we demonstrate the rational design of novel tumor necrosis factor alpha (TNFα) binding proteins using a home‐made grafting program AutoMatch. We grafted three key residues from a virus 2L protein to a de novo designed small protein, DS119, with consideration of backbone flexibility. The designed proteins bind to TNFα with micromolar affinities. We further optimized the interface residues with RosettaDesign and significantly improved the binding capacity of one protein Tbab1‐4. These designed proteins inhibit the activity of TNFα in cellular luciferase assays. Our work illustrates the potential application of the de novo designed protein DS119 in protein engineering, biomedical research, and protein sequence‐structure‐function studies.     

Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

Promising insights into the kosmotropic effect of magnetic nanoparticles on proteins: The pivotal role of protein corona formation

Increasing concerns about biosafety of nanoparticles (NPs) has raised the need for detailed knowledge of NP interactions with biological molecules especially proteins. Herein, the concentration-dependent effect of magnetic NPs (MNPs) on bovine serum albumin and hen egg white lysozyme was explored. The X-ray diffraction patterns, zeta potential, and dynamic light scattering measurements together with scanning electron microscopy images were employed to characterize MNPs synthesized through coprecipitation method. Then, we studied the behavior of two model proteins with different surface charges and structural properties on interaction with Fe₃O₄. A thorough investigation of protein–MNP interaction by the help of intrinsic fluorescence at different experimental conditions revealed that affinity of proteins for MNPs is strongly affected by the similarity of protein and MNP surface charges. MNPs exerted structure-making kosmotropic effect on both proteins under a concentration threshold; however, binding strength was found to determine the extent of stabilizing effect as well as magnitude of the concentration threshold. Circular dichroism spectra showed that proteins with less resistance to conformational deformations are more prone to secondary structure changes upon adsorption on MNPs. By screening thermal aggregation of proteins in the presence of Fe₃O₄, it was also found that like chemical stability, thermal stability is influenced to a higher extent in more strongly bound proteins. Overall, this report not only provides an integrated picture of protein–MNP interaction but also sheds light on the molecular mechanism underling this process.     

A phage display system for studying the sequence determinants of protein folding.

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.