Fipronil is a broad-spectrum insecticide widely used in agriculture and residential areas; its indiscriminate use leads to environmental pollution and poses health hazards. Early detection of ...fipronil is critical to prevent the deleterious effects. However, current insecticide analysis methods such as HPLC, LC/MS, and GC/MS are incompetent; they are costly, immobile, time-consuming, laborious, and need skilled technicians. Hence, a sensitive, specific, and cheap biosensor are essential to containing the contamination. Here, we designed two novel biosensors—the first design relied on fluorescent labeling/quenching, while the second sensor focused on label-free detection using Thioflavin T displacement. Altogether, we identified four candidate aptamers, predicted secondary structures, and performed 3D molecular modeling to predict the binding pocket of fipronil in FiPA6B aptamer. Furthermore, the aptameric sensors showed high sensitivity to fipronil of sub-ppb level LOD, attributed to stringent experimental design. The biosensors displayed high specificity against other phenylpyrazole insecticides and demonstrated robust sensitivity for fipronil in real samples like cabbage and cucumber. Notably, to the best of our knowledge, this is the first demonstration of noncanonical G4-quadruplex-like aptamer binding to fipronil, verified using CD spectroscopy. Such aptasensors possess considerable potential for real-time measurements of hazardous insecticides as point-of-care technology.
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•Ultrasensitive biosensors are crucial to detect and prevent toxic insecticide contamination.•Four unique ssDNA aptameric sensors are developed to detect fipronil.•Demonstrated two modes of detection: fluorescence labeling and label-free.•The aptamers displayed very high binding affinity (Kd ) and specificity.•Achieved detection sensitivity of the sub-ppb level.
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•Binding ability of eugenol with xanthine oxidase were analysed.•Multi spectroscopic and in silico docking studies were carried out.•Eugenol exerts remarkable binding capability with ...xanthine oxidase.
Eugenol, a major component in clove has various biological activities. The current study focused to the binding potential of eugenol with Xanthine oxidase (XO) were evaluated using multi spectroscopic techniques and in silico docking studies. Xanthine oxidase, a superoxide generating enzyme, catalyses hypoxanthine and xanthine to uric acid. An excessive uric acid and superoxide anion radical in our body causes many serious clinical complications. The activity and the structural alterations can be a significant method to reduce this kind of risk factors. The results obtained from the fluorescence titration exhibited the interactions initiated by a static quenching mechanism. The ultraviolet (UV), fourier-transform infrared (FTIR), circular dichroism (CD) spectroscopic analysis of eugenol bind with XO indicated the secondary structural alteration in XO. Docking studies showed molecular level interaction of eugenol with the amino acid residues of Thr 1010, Phe 914, Phe 1009, Leu 1014, Phe 1009, Val 1011, Arg 880, Ala 1078, Glu 802, Leu 648and Leu 873 which residing at the catalytic active site of the XO. These results inferred that the eugenol can interact with XO in a remarkable manner and these findings provide a supporting data for the XO inhibition studies to propose a new lead compound.
•Ag+ bound to central CC mismatched base pair in duplex DNA with 9 × 105 M–1.•Ag+ bound to central CC mismatched base pair in duplex DNA at 1:1 molar ratio.•Ag+ did not bind to terminal CC mismatched ...base pair in duplex DNA.•Ag+ did not bind to central C–A and C–T mismatched base pairs in duplex DNA.•Ag+ did not bind to terminal C–A and C–T mismatched base pairs in duplex DNA.
Metal ion-nucleic acid interactions are important for their contribution in structure formation and their potential applications in nanotechnology. Hg2+ and Ag+ bind to T–T and CC mismatched base pairs, respectively, at the center of duplex DNA to form T–Hg–T and C–Ag–C. Although primer-extension by DNA polymerases with Hg2+ incorporated thymidine 5′-triphosphate to form T–Hg–T, the same reaction with Ag+ did not incorporate deoxycytidine 5′-triphosphate to form C–Ag–C. Here, isothermal titration calorimetric analyses to examine the effect of CC position in duplex DNA on Ag+ binding demonstrated that Ag+ did not bind to the terminal CC base pair in duplex, but it bound to the central CC base pair in duplex at 1:1 molar ratio with 9 × 105 M–1 binding constant. Ag+ did not bind to the terminal and central C–A, C–G, and C–T base pairs in duplex. These findings are useful for developing efficient metal-mediated base pair formation in nanotechnology.
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This report describes purification strategies, biochemical properties and thermodynamic analysis of an alkaline serine protease from a marine actinomycete, Nocardiopsis dassonvillei strain OK-18. The ...solvent tolerance, broad thermal-pH stability, favourable kinetics and thermodynamics suggest stability of the enzymatic reaction. The enzyme was active in the range of pH 7–12 and 37–90 °C, optimally at pH 9 and 70 °C. The deactivation rate constant (Kd), half-life (t½), enthalpy (ΔH*), entropy (ΔS*), activation energy (E) and change in free energy (ΔG*) suggested stability and spontaneity of the reaction. β-Sheets as revealed by the Circular dichroism (CD) spectroscopy, were the major elements in the secondary structure of the enzyme, while Fourier-transform infrared spectroscopy (FTIR) indicated the presence of amide I and amide II. Based on the liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis, the amino acid sequence had only 38% similarity with other proteases of Nocardiopsis strains, suggesting its novelty. The Ramachandran Plot revealed the location of the amino acid residues in the most favored region. The blood de-staining, gelatin hydrolysis, silver recovery and deproteinization of crab shells established the biotechnological potential of the enzyme.
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•Cost-effective single-step purification•Thermodynamic, biochemical and structural elucidation of the protease•High enzyme stability in organic solvents, metal ions and other agents•Highly efficient in crab shell deproteinization, gelatin hydrolysis and silver recovery from used X-ray film•Highly compatible as a detergent supplement
Chirality is a fundamental molecular property that plays a crucial role in biophysics and drug design. Optical circular dichroism (OCD) is a well-established chiral spectroscopic probe in the ...UV–visible regime. Chirality is most commonly associated with a localized chiral center. However, some compounds such as helicenes (Figure 1) are chiral due to their screwlike global structure. In these highly conjugated systems, some electric and magnetic allowed transitions are distributed across the entire molecule, and OCD thus probes the global molecular chirality. Recent advances in X-ray sources, in particular the control of their polarization and spatial profiles, have enabled X-ray circular dichroism (XCD), which, in contrast to OCD, can exploit the localized and element-specific nature of X-ray electronic transitions. XCD therefore is more sensitive to local structures, and the chirality probed with it can be referred to as local. During the racemization of helicene, between opposite helical structures, the screw handedness can flip locally, making the molecule globally achiral while retaining a local handedness. Here, we use the racemization mechanism of 12helicene as a model to demonstrate the capabilities of OCD and XCD as time-dependent probes for global and local chiralities, respectively. Our simulations demonstrate that XCD provides an excellent spectroscopic probe for the time-dependent local chirality of molecules.
High pressure circular dichroism (HP-CD) spectroscopy below 200 MPa, especially in the near-ultraviolet region, has long been expected to probe the protein self-association phenomena. However, its ...experimental difficulties have hampered obtaining the data available for quantitative analyses. In this paper, near-ultraviolet CD spectra measurements under various temperatures and pressures were demonstrated on nitrilase from Rhodococcus rhodochrous J1 (J1-NTase), which thermally self-associates from inactive protomer to active oligomer at atmospheric pressure. The exploration of instrumental conditions and simple raw data correction enabled us to complete the spectra in a temperature-pressure plane. The quality of data was checked by multivariate curve resolution with the alternating least squares method (MCR-ALS), which gave model-free decomposition into component spectra and their concentration profiles. The resultant two components pointed out that the obtained data were self-consistent and appeared to reflect free and bound protomers.
The present study was aimed at investigating the binding between an important drug of Alzheimer's therapy, Rivastigmine tartrate (RT), with Bovine serum albumin (BSA). BSA is a model protein that is ...increasingly being used for studies related to drug-protein interaction owing to its structural similarity with human serum albumin (HSA) which is extremely abundant in the circulatory system comprising around 60% of the total plasma protein. Fluorescence spectroscopy implied that complex formation is taking place between BSA and RT; binding constant calculated was of the order of 104 M−1 implicative of the strength of this interaction. Fluorescence spectroscopy was carried out at three different temperatures in a bid to find out the operative mode of quenching; static quenching was taking place for RT-BSA interaction with a binding constant of 2.5 × 104 M−1 at 298 K. Further, changes in Far UV CD spectra clearly implied that RT induces structural transition in BSA suggestive of RT-BSA complex formation. The negative value of ∆G0 as obtained from fluorescence spectroscopy and isothermal titration calorimetry (ITC) suggests the reaction to be spontaneous and thermodynamically favorable. Additionally, molecular docking was employed to investigate different forces and critical residues involved in RT-BSA interaction. Furthermore, all-atom molecular dynamics simulation for 50 ns was performed on the BSA-RT complex to investigate its conformational behavior, stability and dynamics.
Characterization of oligomeric intermediate states populated at an early stage of misfolding and aggregation is essential to understanding molecular mechanism of pathogenic protein aggregation. ...Growing evidence also suggests that oligomeric species are more toxic than mature fibrillar counterparts. Here, we describe procedures for isolating oligomeric species of an aggregation-prone protein, transthyretin, associated with protein misfolding disorders, including cardiomyopathy and polyneuropathy. We also describe methods for structural studies of the oligomeric species using circular dichroism and solid-state NMR spectroscopy. These methods can be applied to structural characterization of oligomeric intermediates of other aggregation-prone proteins.
•Cu2+ and Al3+ increased the chlorogenic acid and caffeic acid binding affinities greatly with BSA.•Binding affinities were enhanced by esterification of quinic acid of caffeic acid.•Chlorogenic acid ...could form chlorogenic acid-Cu2 and chlorogenic acid-Al2 complex.•Binding with Al3+ prevented the changes in BSA conformation caused by chlorogenic acid.•Binding sites of both chlorogenic acid and caffeic acid were closed to Trp213 in BSA.
Despite the phenolic acids' health benefits, their interactions with proteins are still unclear. In this study, the interactions of Bovine Serum Albumin (BSA) with chlorogenic acid (CHA), caffeic acid (CA), and their Al3+, Cu2+ complexes were studied by using circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and UV/Vis spectroscopy. It was found that esterification of carboxyl group of CA with quinic acid increased the binding affinities for BSA. After chelating with Cu2+ and Al3+, both CHA and CA exhibited high binding affinities for BSA. CHA could form CHA-Cu2 and CHA-Al2 complex with Cu2+ and Al3+. The result of CD spectroscopy demonstrated that the binding of CHA and Al3+ with BSA contributed to the folding of BSA secondary structure. In addition, with the presence of CHA, binding with Al3+ could also induce changes in BSA conformation. The binding sites of both CHA and CA were closed to Trp213.
