Probing the interactions between carboxylated multi‐walled carbon nanotubes and copper–zinc superoxide dismutase at a molecular level

In order to evaluate the toxicity of multi‐walled carbon nanotubes (MWCNTs‐COOH) at a molecular level, the effect of MWCNTs‐COOH on antioxidant enzyme copper–zinc superoxide dismutase (Cu/ZnSOD) was investigated using fluorescence spectroscopy, UV/vis absorption spectroscopy, circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC). By deducting the inner filter effect (IFE), the fluorescence emission spectra and synchronous fluorescence spectra indicated that there were interactions between MWCNTs‐COOH and Cu/ZnSOD. Moreover, the microenvironment of the amino acid residues in the enzyme was changed slightly. The UV/vis absorption and CD spectroscopic results showed appreciable conformational changes in Cu/ZnSOD. However, the results of a Cu/ZnSOD activity determination did not show any significant difference. In other words, MWCNTs‐COOH has no significant effect on enzyme activity. The ITC results showed that the binding of MWCNTs‐COOH to Cu/ZnSOD was a weak endothermic process, indicating that the predominant force of the binding was hydrophobic interaction. Moreover, it was essential to consider the IFE in fluorescence assays, which might affect the accuracy and precision of the results. The above results are helpful in evaluating the oxidative stress induced by MWCNTs‐COOH in vivo. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural Changes of Human Serum Albumin Induced by Cadmium Acetate

The structural changes of human serum albumin (HSA) induced by the addition of cadmium acetate were systematically investigated using UV–vis absorption, circular dichroism (CD), synchronous, and three‐dimentional (3D) fluorescence methods. The fluorescence spectra suggested the formation of cadmium acetate–HSA complex. UV absorption result indicated that the interaction between cadmium acetate and HSA could lead to the alteration of the protein skeleton. The structural analysis according to CD method showed that the cadmium acetate binding altered HSA conformation with a major reduction of α‐helix, inducing a partial protein unfolding. Synchronous fluorescence spectra suggested that cadmium acetate was situated closer to tryptophan residue compared to tyrosine residues, making tryptophan residue locate in a more hydrophobic environment. 3D fluorescence demonstrated that cadmium acetate could induce the HSA aggregation and cause a slight unfolding of the polypeptide backbone of the protein.     

Effect of 1-methyl-3-octyleimmidazolium chloride on the stability and activity of lysozyme: a spectroscopic and molecular dynamics studies

Herein, the binding of 1-methyl-3-octylimidazolium chloride [OMIM][Cl] ionic liquid with hen egg white lysozyme (HEWL) has been studied using fluorescence, time resolved fluorescence, UV–visible and circular dichroism (CD) spectroscopy, in combination with computational study. The fluorescence results revealed that [OMIM][Cl] quenches the fluorophore of HEWL through static quenching mechanism. The calculated thermodynamic parameters show that [OMIM][Cl] bind with HEWL through hydrophobic interactions. In addition, the negative value of Gibbs energy change (?G) indicates that the binding process was spontaneous. Furthermore, UV–vis and CD results indicate that [OMIM][Cl] induce the conformational change in HEWL and increase its enzymatic activity. Additionally, molecular docking results showed that [OMIM][Cl] binds at the active site of HEWL where both the fluorophore residues (Trp108 and Trp62) and the catalytic residues (Glu35 and Asp52) reside. Molecular dynamic simulation results show the reduction of intra-molecular hydrogen bond of HEWL when it binds with [OMIM][Cl].     

Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction

Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of L-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess L-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20A away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the L-lysine activation reaction. Furthermore, the microenvironmental conditions around the residue were made more polar upon the binding of L-lysine, though its contact with the solvent was still restricted. It was suggested that Trp314 was located in a less polar environment than was Trp332, after comparison of the wavelengths at the peaks of fluorescence emission and of the relative fluorescence quantum yields. Trp332 was thought, based on the fluorescence quenching by some perturbants and the chemical modification with N-bromosuccinimide, to be on the surface of the enzyme, whereas Trp314 was buried inside. The UV absorption difference spectra induced by the L-lysine binding indicated that the state of Trp314, including its electrostatic environment, changed during the process, but Trp332 did not change. The increased fluorescence from Trp314 at acidic pH compared with that at neutral pH suggests that carboxylate(s) are in close proximity to the Trp314 residue.     

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.     

白茯苓凝集素的荧光光谱研究

白茯苓凝集素（ＳＬＬ）分子中含有４个色氨酸（Ｔｒｐ）残基,ＮＢＳ修饰测得这４个Ｔｒｐ残基位于分子表面。ＳＬＬ在天然状态下荧光发射峰位于３３５ｎｍ处,离子强度和温度对其荧光光谱均无明显的影响。ＮＢＳ修饰后的ＳＬＬ失去凝血活性,相应荧光光谱的强度减弱,荧光发射峰发生蓝移,提示ＳＬＬ的构象发生改变。用ＫＩ·ＣｓＣｌ和丙烯酰胺淬灭剂研究ＳＬＬ分子中Ｔｒｐ残基的微环境,发现丙烯酰胺和ＣｓＣｌ能淬灭分子中１００％和５０％的Ｔｒｐ残基的荧光,而ＫＩ完全不能淬灭ＳＬＬ分子中Ｔｒｐ残基的荧光,因此Ｔｒｐ残基周围存在阴离子区,或者Ｔｒｐ残基处于分子表面的疏水环境中。     

Study on the interaction of taiwaniaquinoids with FTO by spectroscopy and molecular modeling

In this work, an attempt has been made to study the interaction of four taiwaniaquinoids with fat mass and obesity-associated protein (FTO) by UV–vis absorption, fluorescence spectroscopy, and molecular docking techniques. The results indicated that taiwaniaquinoids effectively quenched the intrinsic fluorescence of FTO via static quenching. According to the binding constants and thermodynamic parameters at three different temperatures, the hydrophobic force and electrostatic interactions appeared be the predominant intermolecular forces in stabilizing the complex. Results revealed that W-4 was the strongest quencher and W-3 was the weakest. The results of synchronous and three-dimensional fluorescence spectra showed that the conformation of FTO was changed. In addition, the influence of molecular structure on the quenching effect has been investigated.     

Dissection of binding of trypsin to its natural inhibitor Gensenoside-Rg1 using spectroscopic methods and molecular modeling

Abstract

The interaction of trypsin with Gensenoside-Rg1 (G-Rg1) was studied using fluorescence, ultraviolet–visible (UV–vis), and circular dichroism (CD) spectroscopies along with enzyme activity assay and molecular docking. The enzyme activity assays showed that G-Rg1 inhibited the activity of trypsin effectively. The fluorescence experiments indicated that a complex of G-Rg1–trypsin was formed and that the fluorescence of trypsin was quenched by G-Rg1 via a mixed-quenching mechanism (both static and dynamic quenching). The thermodynamic analysis suggested that hydrophobic interaction and hydrogen bond were the major forces between G-Rg1 and trypsin. According to the theory of Förster’s non-radiation energy transfer, the binding distance between trypsin and G-Rg1 was calculated to be 2.01?nm, which implies that energy transfer occurred within the complex. The experimental results obtained from UV–vis absorption spectra, synchronous fluorescence spectra, and CD spectra indicated that G-Rg1 was mainly located on tryptophan moiety and that the interaction between G-Rg1 and trypsin led to conformational changes of trypsin with some α-helix and unordered coil structures being transformed into β-sheet structures. In addition, docking results supported the above experimental findings and suggested the possible binding location of G-Rg1 on trypsin along with the possible hydrogen bonds and hydrophobic interactions between G-Rg1 and trypsin. The experimental results from this study should be useful to minimize the antinutritional effects and make full use of Genseng extracts in the food industry and also be helpful to the design of the drugs for the diseases related to overexpression of trypsin.

Communicated by Ramaswamy H. Sarma     

Interactions of phosphatidylinositol 3-kinase Src homology 3 domain with its ligand peptide studied by absorption, circular dichroism, and UV resonance raman spectroscopies

Absorption, circular dichroism (CD), and UV resonance Raman (UVRR) spectroscopies were applied to selectively examine the environmental and structural changes of Trp and Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain induced by ligand association. Comparison of the spectra of PI3K SH3 in the presence or absence of its ligand peptide RLP1 (RKLPPRPSK) indicated that RLP1 binding changed the environment of Trp55 of the SH3 to be more hydrophilic and its H bonding weaker and that of Tyr residues to be more hydrophobic. The D21N mutant (Asp21 --> Asn) of the SH3 yielded a UV CD distinct from that of the wild type, and its spectral changes induced by RLP1 binding were smaller and different from those of the wild type in absorption, CD, and UVRR spectra, suggesting that the mutation of conserved Asp21 affected the conformation of the ligand binding cleft and thus might lead to the decrease in the ligand affinity. These data provide direct evidence for the occurrence of environmental and structural changes of PI3K SH3 by the association of a ligand and the D21N mutation.     

Biophysical studies on the interactions of jatrorrhizine with bovine serum albumin by spectroscopic and molecular modeling methods

The interaction between jatrorrhizine (JAT) and bovine serum albumin (BSA) has been studied. The studies were carried out in a buffer medium at pH 7.4 using fluorescence spectroscopy, UV–vis spectroscopy, and molecular modeling methods. The results of fluorescence quenching and UV–vis absorption spectra experiments indicated the formation of the complex of BSA–JAT. Binding parameters were determined using the Stern–Volmer equation and Scatchard equation. The results of thermodynamic parameters ΔG, ΔH and ΔS at different temperatures indicate that the electrostatic interactions and hydrogen bonds play a major role for JAT–BSA association. Site marker competitive displacement experiments and molecular modeling calculation demonstrating that JAT is mainly located within the hydrophobic pocket of the subdomain IIIA of BSA. Furthermore, The distance between donor (BSA) and acceptor (JAT) was estimated according to fluorescence resonance energy transfer.     

Probing the Interaction of Mutiwalled Carbon Nanotubes and Catalase: Mutispectroscopic Approach

In this study, the mechanism of the interaction between multiwalled carbon nanotubes (MWCNTs) and catalase was investigated by fluorescence, UV–vis, and circular dichroism (CD) spectroscopy under physiological conditions. The fluorescence quenching mechanism of catalase by MWCNTs was shown to be a static quenching procedure and was a result of the formation of a catalase–MWCNT complex. The secondary structure and conformation of the catalase adsorbed on MWCNTs was determined by CD and UV‐vis spectroscopy, and the results indicate that the catalase in this complex is partially unfolded with its lost in α‐helical content and obtainment in β‐sheet content. Moreover, binding of MWCNTs to catalase inhibited the enzymatic activity, which may trigger some toxic effects and undesirable physiological consequences. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:493‐498, 2012;Viewthis article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21454     

Investigation on the interaction between isorhamnetin and bovine liver catalase by spectroscopic techniques under different pH conditions

The binding of isorhamnetin to bovine liver catalase (BLC) was first investigated at 302, 310 and 318 K at pH 7.4 using spectroscopic methods including fluorescence spectra, circular dichroism (CD) and UV–vis absorption. Spectrophotometric observations are rationalized mainly in terms of a static quenching process. The binding constants and binding sites were evaluated by fluorescence quenching methods. Enzymatic activity of BLC in the absence and presence of isorhamnetin was measured using a UV/vis spectrophotometer. The result revealed that the binding of isorhamnetin to BLC led to a reduction in the activity of BLC. The positive entropy change and enthalpy change indicated that the interaction of isorhamnetin with BLC was mainly driven by hydrophobic forces. The distance r between the donor (BLC) and acceptor (isorhamnetin) was estimated to be 2.99 nm according to fluorescence resonance energy transfer. Fluorescence, synchronous fluorescence, and CD spectra showed no obvious change in the conformation of BLC upon the binding of isorhamnetin. In addition, the influence of pH on the binding of isorhamnetin to BLC was investigated and the binding ability of the drug to BLC deceased under other pH conditions (pH 9.0, 6.5, 5.0, 3.5, or 2.0) as compared with that at pH 7.4. Copyright © 2016 John Wiley & Sons, Ltd.     

Fluorescence contributions of the individual Trp residues in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues. In order to obtain information on the fluorescence contribution of the individual Trp residues in native GLA, we recorded the fluorescence spectra of four GLA mutants, W26F, W60F, W104F, and W118F, in each of which a single Trp residue was replaced with phenylalanine (Phe). Comparison of the fluorescence spectra of the four mutants with that of wild-type GLA indicated that, in native GLA, three Trp residues (Trp60, Trp104, and Trp118) are strongly quenched and account for the partial indirect quenching of Trp26. As a consequence, the fluorescence of wild-type GLA and of the mutants W60F, W104F, and W118F mainly results from Trp26. An inspection of the crystal structure indicated that, in addition to the disulfide bonds that are in direct contact with the indole groups of Trp60 and Trp118, backbone peptide bonds that are in direct contact with the indole groups of Trp60, Trp104, and Trp118, contribute to the direct quenching effects. Interestingly, the lack of direct quenching of Trp26 explains why the cleavage of disulfide bonds by UV light is mediated more by the highly fluorescent Trp26 than by the less fluorescent Trp104 and Trp118.     

慈菇蛋白酶抑制剂A和B中Trp残基周围构象与酶抑制专一性的关系

通过定点诱变结合荧光光谱学方法研究了慈菇蛋白酶抑制剂A和B(APIA和APIB)Trp残基周围构象与酶抑制专一性之间的关系。研究表明APIB中的两个Trp残基 (93和 12 2位 )所处环境的疏水性要比APIA中的强。Trp定点诱变研究表明 ,在APIB中 ,Trp12 2 周围环境的疏水性要比Trp93 强。用Ser和Leu分别替代 82位Leu和 87位Arg ,使APIB中色氨酸荧光特性变得与APIA的基本相同 ,同时还发现其酶的抑制专一性也变得趋近APIA的 ,暗示Trp周围的构象与酶抑制剂的抑制专一性有关。     

纤维素酶在二甲基亚砜微扰条件下的动力学行为和光谱学变化

为了探索二甲基亚砜对纤维素酶催化活性的影响,以羧甲基纤维素钠(CMC)为底物来研究纤维素酶纯酶在二甲基亚砜中的动力学变化、紫外吸收光谱、紫外差示光谱和荧光发射光谱。实验表明:在3%的二甲基亚砜中,纤维素酶的催化活性下降了46.78%;其Km值从缓冲液中的2.500 mg/mL上升到二甲基亚砜中的3.922 mg/mL;在二甲基亚砜中,酶分子的肽键紫外吸收稍有改变,但其氨基酸基团的紫外吸收没有改变;其紫外差示光谱出现明显的正峰和负峰;其荧光发射光谱没有改变。研究结果证明:二甲基亚砜通过轻微改变酶分子的肽链结构,使分子构象改变,导致酶分子对底物的亲和力下降,从而降低其催化活性。     

Conformational dynamics of DnaB helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching

DnaB helicase of E. coli unwinds duplex DNA in the replication fork using the energy of ATP hydrolysis. We have analyzed structural and conformational changes in the DnaB protein in various nucleotides and DNA bound intermediate states by fluorescence quenching analysis of intrinsic fluorescence of native tryptophan (Trp) residues in DnaB. Fluorescence quenching analysis indicated that Trp48 in domain alpha is in a hydrophobic environment and resistant to fluorescence quenchers such as potassium iodide (KI). In domain beta, Trp294 was found to be in a partially hydrophobic environment, whereas Trp456 in domain gamma appeared to be in the least hydrophobic environment. Binding of oligonucleotides to DnaB helicase resulted in a significant attenuation of the fluorescence quenching profile, indicating a change in conformation. ATPgammaS or ATP binding appeared to lead to a conformation in which Trp residues had a higher degree of solvent exposure and fluorescence quenching. However, the most dramatic increase of Trp fluorescence quenching was observed with ADP binding with a possible conformational relaxation. Site-specific Trp --> Cys mutants of DnaB helicase demonstrated that conformational change upon ADP binding could be attributed exclusively to a conformational transition in the alpha domain leading to an increase in the solvent exposure of Trp48. However, formation of DnaB.ATPgammaS.DNA ternary complex led to a conformation with a fluorescence quenching profile similar to that observed with DnaB alone. The DnaB.ADP.DNA ternary complex produced a quenching curve similar to that of DnaB.ADP complex pointing to a change in conformation due to ATP hydrolysis. There are at least four identifiable structural/conformational states of DnaB helicase that are likely important in the helicase activity. The noncatalytic alpha domain in the N-terminus appeared to undergo the most significant conformational changes during nucleotide binding and hydrolysis. This is the first reported elucidation of the putative role of domain alpha, which is essential for DNA helicase action. We have correlated these results with partial structural models of alpha, beta, and gamma domains     

Fluorescence spectroscopic studies on the interaction of Gemini surfactant 14‐6‐14 with bovine serum albumin

The interaction of the cationic Gemini surfactant hexamethylene‐1,3‐bis (tetradecyldimethylammonium bromide) (14‐6‐14) with bovine serum albumin (BSA) has been investigated by fluorescence quenching spectra and three‐dimensional (3D) fluorescence spectra. The Stern–Volmer quenching constants K_SV and the corresponding thermodynamic parameters ΔH, ΔG and ΔS have been estimated by the fluorescence quenching method. The results indicated that hydrophobic forces were the predominant intermolecular forces between BSA and the surfactant. Competitive experiments and the number of binding sites calculation show that 14‐6‐14 can be inserted in site‐II (in subdomain IIIA) of BSA. The effect of 14‐6‐14 on the conformation of BSA was evaluated by synchronous fluorescence spectroscopy and 3D fluorescence spectral methods. The results show that the conformation of BSA was changed dramatically in the presence of 14‐6‐14, by binding to the Trp and Try residues of BSA. The investigation provides interaction between BSA and 14‐6‐14 as a model for molecular design and industrial research. Copyright © 2011 John Wiley & Sons, Ltd.     

Carbohydrate recognition of gramicidin S analogues in aqueous medium.

We have designed and synthesized of carbohydrate-binding peptides, gramicidin S analogues. Asn/Asp/Gln and Trp residues in the peptides were employed as the binding sites for carbohydrates by hydrogen-bonding interaction and the creation units for hydrophobic pocket to promote the interaction, respectively. The data of fluorescence spectroscopy and affinity column chromatography indicated that the peptides possessed the binding ability for some carbohydrates in aqueous medium. As a result of 1H NMR study, nuclear Overhauser effects between aromatic side chains of a peptide, [Gln(1,1'),Trp(3,3')]-gramisidin S and mannose were observed, indicating that the interaction of the peptide with the sugar occurred in the hydrophobic environment formed by Trp and Phe residues.     

Spectroscopic and molecular simulation studies on the interaction of di‐(2‐ethylhexyl) phthalate and human serum albumin

Di‐(2‐ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in industrial production, but may have a potential health risk. In this study, the binding characteristics of DEHP with human serum albumin (HSA) in aqueous solution at pH 7.4 were determined using UV/vis absorption, fluorescence, Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD), along with a molecular simulation technique. Analysis of the fluorescence titration data at different temperatures suggested that the fluorescence quenching mechanism of HSA by DEHP was static. The calculated thermodynamic parameters indicated that hydrophobic forces played a predominant role in formation of the DEHP–HSA complex, but hydrogen bonds could not be omitted. Site marker competitive experiments and denaturation studies showed that the binding of DEHP to HSA primarily took place in subdomain IIA of HSA, and molecular docking results further corroborated the binding sites. The synchronous fluorescence, UV/vis absorption, FTIR and CD spectra revealed that the addition of DEHP induced changes in the secondary structure of HSA. Protein surface hydrophobicity (PSH) tests indicated that DEHP binding to HSA caused an increase in the PSH. Moreover, the effects of some metal ions on the binding constant of DEHP − HSA interaction were also investigated. Copyright © 2014 John Wiley & Sons, Ltd.     

An insight into the binding of 6-hydroxyflavone with hen egg white lysozyme: a combined approach of multi-spectroscopic and computational studies

Abstract

The interaction of 6-hydroxyflavone (6HF) with hen egg white lysozyme (HEWL) has been executed using multi-spectroscopic and computational methods. Steady state fluorescence studies indicated that static quenching mechanism is involved in the binding of 6HF with HEWL, which was further supported by excited state lifetime and UV–vis absorption studies. The binding constant (K_b) of the HEWL–6HF complex was observed to be 6.44?±?0.09?×?10⁴ M^?1 at 293?K, which decreases with the increase in temperature. The calculation of the thermodynamic quantities showed that the binding is exothermic in nature with a negative enthalpy change (ΔH = ?11.91?±?1.02?kJ mol^?1) along with a positive entropy change (ΔS = +51.36?±?2.43 J K^?1 mol^?1), and the major forces responsible for the binding are hydrogen bonding and hydrophobic interactions. The possibility of energy transfer from tryptophan (Trp) residue to the 6HF ligand was observed from Fo¨rster’s theory. The inclusion of 6HF within the binding site of HEWL induces some micro-environmental changes around the Trp residues as indicated by synchronous and three-dimensional (3D) fluorescence studies. The changes in secondary structural components of HEWL are observed on binding with 6HF along with a reduction in % α-helical content. Computational studies correlate well with the experimental finding, and the ligand 6HF is found to bind near to Trp 62 and Trp 63 residues of HEWL. Altogether, the present study provides an insight into the interaction dynamics and energetics of the binding of 6HF to HEWL.

Communicated by Ramaswamy H. Sarma