Probing the binding of two fluoroquinolones to lysozyme: a combined spectroscopic and docking study

Ciprofloxacin (CPFX) and enrofloxacin (ENFX) are two of the most widely used fluoroquinolones (FQs) in human and veterinary medicines. Their occurrence in the environment has received much attention because of the potential adverse effects on humans and ecosystem functions. In this paper, we investigated the interaction mechanism between the two FQs and lysozyme by the spectroscopic and molecular docking methods. As shown by the fluorescence spectroscopy, additions of CPFX or ENFX effectively quenched the intrinsic fluorescence of lysozyme, which was attributed to the formation of a moderately strong complex. The enthalpy change (ΔH) and entropy change (ΔS) indicated that van der Waals forces and hydrogen bonds were the dominant intermolecular forces in the binding of two FQs to lysozyme. Furthermore, data obtained by UV-vis absorption, synchronous fluorescence and circular dichroism (CD) suggested that both CPFX and ENFX could lead to the conformational and some microenvironmental changes of lysozyme. Finally, the molecular docking illustrated that the two FQs had specific interactions with the residues of Trp62 and Trp63.     

A probe to study the toxic interaction of tartrazine with bovine hemoglobin at the molecular level

Tartrazine is an artificial azo dye commonly used in food products, but tartrazine in the environment is potentially harmful. The toxic interaction between tartrazine and bovine hemoglobin (BHb) was investigated using fluorescence, synchronous fluorescence, UV–vis absorption, circular dichroism (CD) and molecular modeling techniques under simulated physiological conditions. The fluorescence data showed that tartrazine can bind with BHb to form a complex. The binding process was a spontaneous molecular interaction, in which van der Waals' forces and hydrogen bonds played major roles. Molecular docking results showed that the hydrogen bonds exist between the oxygen atoms at position 31 of tartrazine and the nitrogen atom NZ7 on Lys99, and also between the oxygen atoms at position 15 of tartrazine and the nitrogen atom NZ7 on Lys104, Lys105. The results of UV–vis and CD spectra revealed that tartrazine led to conformational changes in BHb, including loosening of the skeleton structure and decreasing α helix in the secondary structure. The synchronous fluorescence experiment revealed that tartrazine binds into the hemoglobin central cavity, and this was verified using a molecular modeling study. Copyright © 2013 John Wiley & Sons, Ltd.     

The study on interactions between levofloxacin and model proteins by using multi-spectroscopic and molecular docking methods

The interactions of levofloxacin (LEV) with lysozyme (LYZ), trypsin and bovine hemoglobin (BHb) were investigated, respectively, by using multi-spectral techniques and molecular docking in vitro. Fluorescence studies showed that LEV quenched LYZ/trypsin fluorescence in a combined quenching ways and BHb fluorescence in a static quenching with binding constants of .14, .51 and .20 × 10⁵ L mol^?1 at 298 K, respectively. The thermodynamic parameters demonstrated that hydrophobic forces, hydrogen bonds, and van der Waals forces played the major role in the binding process. The binding distances between LEV and the inner tryptophan residues of LYZ, trypsin, and BHb were calculated to be 4.04, 3.38, and 4.52 nm, respectively. Furthermore, the results of circular dichroism spectra (CD), UV–vis, and three-dimensional fluorescence spectra indicated that the secondary structures of LYZ, trypsin, and BHb were partially changed by LEV with the α-helix percentage of LYZ-LEV system increased while that of BHb-LEV system was decreased, the β-sheet percentage of trypsin-LEV system increased from 41.3 to 42.9%. UV–vis spectral results showed that the binding interactions could cause conformational and some micro-environmental changes of LYZ, trypsin, and BHb. The results of molecular docking revealed that in LYZ and trypsin systems, LEV bound to the active sites residues GLU 35 and ASP 52 of LYZ and trypsin at the active site SER 195, and in BHb system, LEV was located in the central cavity, which was consistent with the results of synchronous fluorescence experiment. Besides, LEV made the activity of LYZ decrease while the activity of trypsin increased.     

The interaction of blood proteins with brucine

The features of brucine (BC) binding to two blood proteins, bovine hemoglobin (BHb), and bovine serum albumin (BSA), were investigated via fluorescence, circular dichroism and UV/Vis absorption spectroscopy. The results revealed that BC caused the fluorescence quenching of blood proteins by the formation of BC–protein complex. The corresponding thermodynamic parameters were measured at different temperatures. The process of binding BC molecule on protein was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased. Hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The molecular docking has been employed to explore the binding site of the BC in BHb and BSA on the Autodock 4.2. The distances r between BC and protein were calculated to be 4.93 and 5.08 nm for BHb, and BSA, respectively. The effect of BC on the conformation of blood proteins was analyzed using CD, synchronous fluorescence and three-dimensional fluorescence spectra.     

Spectroscopic characterization,calorimetric study and molecular docking to evaluate the bioconjugation of maltol with hemoglobin

Maltol, a food additive, is extensively used in our daily life. To date, its biological safety is still debated. In this article, binding interaction of maltol with bovine hemoglobin (BHb), an important functional protein, was studied by molecular docking research and spectroscopic and calorimetric measurements. We found that maltol could cause structural changes of BHb. By interacting with Glu 101 (1.27 Å) and Lys 104 (2.49 Å) residues, maltol changed the cavity structure and induced a microenvironment change around tryptophan (Trp) residue. Thermodynamic parameters obtained from isothermal titration calorimetry (ITC) measurement showed that hydrophobic forces were the main forces existing in this system. The association constant of K (8.0 ± 3.4 × 10⁴ M^?1) shows the mild ligand–protein binding for maltol with BHb. The α‐helix amount in BHb increased (59.6–62.6%) with different concentrations of maltol and the intrinsic fluorescence intensity was quenched by maltol, indicating the conformation changes and denaturation of BHb. This work presents the interactions of maltol with BHb at the molecular level and obtains evidence that maltol induces adverse effects to proteins in vitro.     

Affinity and Specificity of Ciprofloxacin-Bovine Serum Albumin Interactions: Spectroscopic Approach

Fluorescence spectroscopy in combination with UV–Vis absorption spectroscopy were employed to investigate the binding of an antibacterial drug Ciprofloxacin (CPFX) to bovine serum albumin (BSA) under the physiological conditions. In the discussion of the quenching mechanism, it was proved that the fluorescence quenching of BSA by CPFX is a result of the formation of CPFX-BSA complex. Binding parameters were determined using the modified Stern-Volmer equation and Scatchard equation to provide a measure of the binding affinity between CPFX and BSA. The results of thermodynamic parameters ΔG, ΔH, ΔS, at different temperatures indicate that the electrostatic interactions play a major role for CPFX-BSA association. Site marker competitive experiments indicated that the binding of CPFX to BSA primarily took place in site I. Furthermore, the effect of metal ions to CPFX-BSA system was studied, and the distance r between donor (BSA) and acceptor (CPFX) was obtained according to fluorescence resonance energy transfer (FRET). The conformation of BSA upon CPFX binding was evaluated by measuring synchronous fluorescence properties of the CPFX-BSA complex.     

Studies of the interaction between paraquat and bovine hemoglobin

The interaction between paraquat (PQ) and bovine hemoglobin (BHb) was investigated using fluorescence and UV/vis absorption spectroscopy. The reactivity of the heme centers with superoxide anions formed by PQ was judged on the basis of the decrease of the Soret band. The experimental results showed that the fluorescence quenching of BHb by PQ was a result of the formation of PQ-BHb complex; static quenching was confirmed to result in the fluorescence quenching. The binding site number n, apparent binding constant K(A) and corresponding thermodynamic parameters were measured at different temperatures. The process of binding PQ molecule on BHb was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased. Hydrophobic and electrostatic interactions played a major role in stabilizing the complex. The effect of PQ on the conformation of BHb was analyzed using synchronous fluorescence spectroscopy.     

Ultraviolet irradiation-dependent fluorescence enhancement of hemoglobin catalyzed by reactive oxygen species

Ultraviolet (UV) light has a potent effect on biological organisms. Hemoglobin, an oxygen-transport protein, plays an irreplaceable role in sustaining life of all vertebrates. In this study we scrutinize the effects of ultraviolet irradiation (UVI) as well as visible irradiation on the fluorescence characteristics of bovine hemoglobin (BHb) in vitro. Data show that UVI results in fluorescence enhancement of BHb in a dose-dependant manner. Furthermore, UVI-induced fluorescence enhancement is significantly increased when BHb is pretreated with hydrogen peroxide (H(2)O(2)), a type of reactive oxygen species (ROS). Meanwhile, The water-soluble antioxidant vitamin C suppresses this UVI-induced fluorescence enhancement. In contrast, green light irradiation does not lead to fluorescence enhancement of BHb no matter whether H(2)O(2) is acting on the BHb solution or not. Taken together, these results indicate that catalysis of ROS and UVI-dependent irradiation play two key roles in the process of UVI-induced fluorescence enhancement of BHb.     

Interactions of three bisphenol analogues with hemoglobin investigated by spectroscopy and molecular docking

Bisphenol F (BPF), bisphenol S (BPS), and bisphenol B (BPB) have been extensively used in food packaging, plasticizer, and paper products, causing more concern about their biosafety. The mechanism of these bisphenols' toxicity was investigated by determining diverse effects of them on common protein bovine hemoglobin (BHb). The effects at the molecular level were determined by ultraviolet‐visible, circular dichroism, resonance light scattering, fluorescence spectroscopy, and molecular docking. The irreversible cross‐linking results of bisphenols and BHb demonstrate that hydrogen (H) bonding and hydrophobic forces play major roles in the interaction. Both BPF and BPS decreased the amount of α‐helix, leading to the loosening of protein skeleton while BPB made little change. In the loose structure, BPF exposed the internal amino acids to a hydrophobic environment and BPS (above 10μM) obviously quenched characteristic fluorescence. The variant effects of BPF, BPS, and BPB may arise from different structural formula. Accordingly, BPB could be used as a better substitute for bisphenol A, and it is necessary to control the concentration of BPS and BPF below 10μM in application. This study provided important basis for application of BPB and safe use of bisphenol analogues in industry.     

Effects of N‐Acetyl‐L‐Cysteine‐Capped CdTe Quantum Dots on Bovine Serum Albumin and Bovine Hemoglobin: Isothermal Titration Calorimetry and Spectroscopic Investigations

The interactions of N‐acetyl‐L‐cysteine‐capped CdTe quantum dots (QDs) with bovine serum albumin (BSA) and bovine hemoglobin (BHb) were investigated by isothermal titration calorimetry (ITC), fluorescence, synchronous fluorescence, fluorescence lifetime, ultraviolet–visible absorption, and circular dichroism techniques. Fluorescence data of BSA–QDs and BHb–QDs revealed that the quenching was static in every system. While CdTe QDs changed the microenvironment of tryptophan in BHb, the microenvironment of BSA kept unchanged. Adding CdTe QDs affected the skeleton and secondary structure of the protein (BSA and BHb). The ITC results indicated that the interaction between the protein (BSA and BHb) and QDs‐612 was spontaneous and the predominant force was hydrophobic interaction. In addition, the binding constants were determined to be 1.19 × 10⁵ L mol^?1 (BSA–QDs) and 2.19 × 10⁵ L mol^?1 (BHb–QDs) at 298 K. From these results, we conclude that CdTe QDs have a larger impact on the structure of BHb than BSA.     

Studies on the binding of pepsin with three pyrethroid insecticides by multi‐spectroscopic approaches and molecular docking

In this study, the molecular interactions between pepsin and three pyrethroid insecticides, including fenvalerate, cyhalothrin and deltamethrin, were investigated by multi‐spectroscopic and molecular docking methods under mimic physiological pH conditions. The results indicated that all of these insecticides could interact with pepsin to form insecticide–pepsin complexes. The binding constants, number of binding sites and thermodynamic parameters measured at different temperatures indicated that these three pyrethroid insecticides could spontaneously bind with pepsin mainly through electrostatic forces and hydrophobic interactions with one binding site. According to the theory of Föster's non‐radioactive energy transfer, the distance (r) between pepsin and three pyrethroid insecticides were all found to be less than 7 nm, which implied that the energy transfer occurred between pepsin and these insecticides, leading to the quenching of pepsin fluorescence. Synchronous and three‐dimensional fluorescence, CD spectra and molecular docking results indicated that all tested pyrethroid insecticides bound directly into the enzyme cavity site and the binding of insecticides into the cavity influenced the microenvironment of the pepsin activity site which resulted in the extension of peptide strands of pepsin with loss of α‐helix structures.Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis,characterization, in silico ADMET prediction,and protein binding analysis of a novel zinc(II) Schiff-base complex: Application of multi-spectroscopic and computational techniques

By reaction of 1,2-diaminocyclohexane with the 2,3-butanedione monoxime in the presence of ZnCl₂, a new Schiff base complex was obtained. This complex was characterized by elemental analyses, FT-IR, ¹H NMR, UV–Vis, and conductivity measurements. The reactivity of this complex to human serum albumin (HSA) under simulative physiological conditions was studied by spectroscopic and molecular docking analysis. Experimental results at various temperatures indicated that the intrinsic fluorescence of protein was quenched through a static quenching mechanism. The negative value of enthalpy change and positive value of entropy change indicated that both hydrogen bonding and hydrophobic forces played a major role in the binding of Zn(II) complex to HSA. FT-IR, three-dimensional fluorescence, and UV–Vis absorption results showed that the secondary structure of HSA changed after Zn(II) complex bound to protein. The binding distance was calculated to be 4.96 nm, according to fluorescence resonance energy transfer. Molecular docking results confirmed the spectroscopic results and showed that above complex is embedded into subdomain IIA of protein. All these experimental and computational results clarified that Zn(II) complex could bind with HSA effectively, which could be a useful guideline for efficient Schiff-base drug design.     

Study on the interaction between pelargonidin‐3‐O‐glucoside and bovine serum albumin using spectroscopic,transmission electron microscopy and molecular modeling techniques

The aim of this study is to evaluate the binding behavior between pelargonidin‐3‐O‐glucoside (P3G) and bovine serum albumin (BSA) using multi‐spectroscopic, transmission electron microscopy (TEM) and molecular docking methods under physiological conditions. Fluorescence spectroscopy and time‐resolved fluorescence showed that the fluorescence of BSA could be quenched remarkably by P3G via a static quenching mechanism, and there is a single class of binding site on BSA. In addition, the thermodynamic functions ΔH and ΔS were –21.69 kJ/mol and 24.46 J/mol/K, indicating that an electrostatic interaction was a main acting force. The distance between BSA and P3G was 2.74 nm according to Förster's theory, illustrating that energy transfer occurred. In addition, the secondary structure of BSA changed with a decrease in the α‐helix content from 66.2% to 64.0% as seen using synchronous fluorescence, UV/vis, circular dichroism and Fourier transform infrared spectroscopies, whereas TEM images showed that P3G led to BSA aggregation and fibrillation. Furthermore, site marker competitive experiments and molecular docking indicated that P3G could bind with subdomain IIA of BSA. The calculated results of the equilibrium fraction showed that the concentration of free P3G in plasma was high enough to be stored and transported from the circulatory system to its target sites to provide therapeutic effects. Copyright © 2015 John Wiley & Sons, Ltd.     

Molecular interaction between 4-aminoantipyrine and catalase reveals a potentially toxic mechanism of the drug

4-Aminoantipyrine (AAP) is scarcely administered as an analgesic drug because of the potential side effects. The residue of AAP in the environment possesses a potential threat to human health. In this article, the binding mode of AAP with the important antioxidant enzyme catalase (CAT) was investigated using spectroscopic and molecular docking methods. AAP can interact with CAT to form an AAP-CAT complex. The binding constant, number of binding sites and thermodynamic parameters were measured, which indicated that AAP could spontaneously bind with CAT through electrostatic forces with one binding site. Molecular docking results revealed that AAP bound into the CAT central cavity. UV-visible absorption, synchronous fluorescence and circular dichroism (CD) results provide data concerning conformational and some microenvironmental changes of CAT. Furthermore, the binding of AAP can inhibit CAT activity in erythrocytes. The present study provides direct evidence at a molecular level to show that exposure to AAP could induce changes in the enzyme CAT structure and function. The estimated methods in this work can be applied to characterize interactions of enzyme systems and other pollutants and drugs.     

Characterisation of the interaction of the C-terminus of the dopamine D2 receptor with neuronal calcium sensor-1

NCS-1 is a member of the neuronal calcium sensor (NCS) family of EF-hand Ca(2+) binding proteins which has been implicated in several physiological functions including regulation of neurotransmitter release, membrane traffic, voltage gated Ca(2+) channels, neuronal development, synaptic plasticity, and learning. NCS-1 binds to the dopamine D2 receptor, potentially affecting its internalisation and controlling dopamine D2 receptor surface expression. The D2 receptor binds NCS-1 via a short 16-residue cytoplasmic C-terminal tail. We have used NMR and fluorescence spectroscopy to characterise the interactions between the NCS-1/Ca(2+) and D2 peptide. The data show that NCS-1 binds D2 peptide with a K(d) of ～14.3 μM and stoichiometry of peptide binding to NCS-1 of 2:1. NMR chemical shift mapping confirms that D2 peptide binds to the large, solvent-exposed hydrophobic groove, on one face of the NCS-1 molecule, with residues affected by the presence of the peptide spanning both the N and C-terminal portions of the protein. The NMR and mutagenesis data further show that movement of the C-terminal helix 11 of NCS-1 to fully expose the hydrophobic groove is important for D2 peptide binding. Molecular docking using restraints derived from the NMR chemical shift data, together with the experimentally-derived stoichiometry, produced a model of the complex between NCS-1 and the dopamine receptor, in which two molecules of the receptor are able to simultaneously bind to the NCS-1 monomer.     

Molecular interaction of human serum albumin with paracetamol: Spectroscopic and molecular modeling studies

The interaction between paracetamol and human serum albumin (HSA) under physiological conditions has been investigated by fluorescence, circular dichroism (CD) and docking. Fluorescence data revealed that the fluorescence quenching of HSA by paracetamol was the result of the formed complex of HSA–paracetamol, and the binding constant (K_a) and binding number obtained is 1.3 × 10⁴ at 298 K and 2, respectively for the primary binding site. Circular dichorism spectra showed the induced conformational changes in HSA by the binding of paracetamol. Moreover, protein–ligand docking study indicated that paracetamols (two paracetamols bind to HSA) bind to residues located in the subdomain IIIA.     

Anionic lipids in Ca(2+)-triggered fusion

Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca(2+)-triggered fusion pathway remains poorly defined. Here we utilize the tightly coupled quantitative molecular and functional analyses enabled by the cortical vesicle model system to elucidate the roles of specific anionic lipids in the docking, priming and fusion steps of regulated release. Studies with cholesterol sulfate established that effectively localized anionic lipids could contribute to Ca(2+)-sensing and even bind Ca(2+) directly as effectors of necessary membrane rearrangements. The data thus support a role for phosphatidylserine in Ca(2+) sensing. In contrast, phosphatidylinositol would appear to serve regulatory functions in the physiological fusion machine, contributing to priming and thus the modulation and tuning of the fusion process. We note the complexities associated with establishing the specific roles of (anionic) lipids in the native fusion mechanism, including their localization and interactions with other critical components that also remain to be more clearly and quantitatively defined.     

Study on the interactions of mapenterol with serum albumins using multi‐spectroscopy and molecular docking

The interactions of mapenterol with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated systematically using fluorescence spectroscopy, absorption spectroscopy, circular dichroism (CD) and molecular docking techniques. Mapenterol has a strong ability to quench the intrinsic fluorescence of BSA and HSA through static quenching procedures. At 291 K, the binding constants, K_a, were 1.93 × 10³ and 2.73 × 10³ L/mol for mapenterol–BSA and mapenterol–HAS, respectively. Electrostatic forces and hydrophobic interactions played important roles in stabilizing the mapenterol–BSA/has complex. Using site marker competitive studies, mapenterol was found to bind at Sudlow site I on BSA/HSA. There was little effect of K⁺, Ca²⁺, Cu²⁺, Zn²⁺ and Fe³⁺ on the binding. The conformation of BSA/HSA was changed by mapenterol, as seen from the synchronous fluorescence spectra. The CD spectra showed that the binding of mapenterol to BSA/HSA changed the secondary structure of BSA/HSA. Molecular docking further confirmed that mapenterol could bind to Sudlow site I of BSA/HSA. According to Förster non‐radiative energy transfer theory (FRET), the distances r₀ between the donor and acceptor were calculated as 3.18 and 2.75 nm for mapenterol–BSA and mapenterol–HAS, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Spectroscopic and molecular docking studies on interaction of two Schiff base complexes with bovine serum albumin

Abstract

This study was designed to examine interaction of two ternary copper (II) Schiff base complexes with bovine serum albumin (BSA), using spectroscopic and molecular docking techniques. The fluorescence quenching measurements revealed that the quenching mechanism was static and the binding site of both Schiff bases to BSA was singular. Förster energy transfer measurements, synchronous fluorescence spectroscopy, and docking study showed both Schiff bases bind to the Trp residues of BSA in short distances. Docking study showed that both Schiff base molecules bind with BSA by forming several hydrogen and van der Waals bonds. In addition, molecular docking study indicated that Schiff base A and Schiff base B were located within the binding pocket of subdomain IB and subdomain IIA of BSA, respectively. Results of Fourier transform-infrared spectroscopy demonstrated that bovine serum albumin interacts with both Schiff bases and the secondary structure of BSA was changed.

Communicated by Ramaswamy H. Sarma     

Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in determining binding affinity

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) proteins regulate a variety of cellular functions, including cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. Two docking sites, common docking (CD/ED) domain and F-site recruitment site (FRS), on ERK proteins have been identified. Specific interactions with the CD/ED domain and the FRS occur with substrates containing a docking site for ERK and JNK, LXL (DEJL) motif (D-domain) and a docking site for ERK, FXF (DEF) motif (F-site), respectively. However, the relative contributions of the ERK docking sites in mediating substrate interactions that allow efficient phosphate transfer are largely unknown. In these studies, we provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance to measure real time protein-protein interactions. ERK2 interacted with ELK-1 (DEF and DEJL motifs), RSK-1 (DEJL motif), and c-Fos (DEF motif) with K(D) values of 0.25, 0.15, and 0.97 μM, respectively. CD/ED domain mutations inhibited interactions with ELK-1 and RSK-1 by 6-fold but had no effect on interactions with c-Fos. Select mutations in FRS residues differentially inhibited ELK-1 or c-Fos interactions with ERK2 but had little effect on RSK-1 interactions. Mutations in both the ED and FRS docking sites completely inhibited ELK-1 interactions but had no effect on interactions with stathmin, an ERK substrate whose docking site is unknown. The phosphorylation status of ERK2 did not affect interactions with RSK-1 or c-Fos but did inhibit interactions with ELK-1 and stathmin. These studies provide a quantitative evaluation of specific docking domains involved in mediating interactions between ERK2 and protein substrates and define the contributions of these interactions to phosphate transfer.