Photoinduced electron transfer between hen egg white lysozyme and anticancer drug menadione

The protein, hen egg white lysozyme, on photoexcitation undergoes electron transfer with menadione (2-methyl-1,4-naphthoquinone), a widely known anticancer drug. With the addition of menadione the fluorescence of lysozyme is quenched with the simultaneous formation of an excited state charge-transfer complex in the longer wavelength and a ground state complex. The former is further evident from laser flash photolysis studies, which indicate a tryptophan to menadione electron transfer. From fluorescence quenching studies the binding constant is found to be ∼1.7×10⁴ M⁻¹ with the corresponding changes in enthalpy (ΔH°) and entropy (ΔS°) as 12.24 kJ mol⁻¹ and 124.12 J mol⁻¹ K⁻¹, respectively, indicative of an entropy-driven process. The circular dichroism studies also show some structural changes with increase in α-helical content in the protein on interaction with menadione. Finally, docking studies give some insight into the role of Trp 108 of lysozyme in the interaction.     

光谱法结合分子模拟技术研究左氧氟沙星与胃蛋白酶的相互作用机理

在模拟生理条件下,采用荧光光谱法结合分子对接技术研究了左氧氟沙星与胃蛋白酶的相互作用。不同温度下的Stern-Volmer曲线结果表明,左氧氟沙星主要以静态猝灭的方式使胃蛋白酶的荧光产生猝灭。由热力学数据确定了两者之间存在的作用力主要为静电作用力。三维荧光光谱实验结果表明,在左氧氟沙星与胃蛋白酶发生作用过程中引起了胃蛋白酶中酪氨酸和色氨酸残基构象的变化,从而导致胃蛋白酶发生了猝灭作用。为了进一步探讨左氧氟沙星与胃蛋白酶相互作用的分子机理,利用分子对接技术对两者的相互作用进行模拟。结果表明两者发生作用的区域位于胃蛋白酶的催化活性中心,左氧氟沙星的进入导致酶催化中心氨基酸残基的微环境发生改变,从而对胃蛋白酶的活性造成影响,这可能也是左氧氟沙星引起消化不良的主要原因。     

Interaction of transferrin with cefuroxime sodium

ABSTRACT

The interaction between cefuroxime sodium and transferrin in physiological buffer (pH 7.4) was investigated by spectroscopy at 298, 310, and 318?K. The analysis of data indicated that quenching mechanism of the transferrin–cefuroxime sodium system was probably static. The electrostatic force played an important role in the conjugation reaction between transferrin and cefuroxime sodium. The order of magnitude of binding constants (K_a) was 10⁴, and the number of binding site (n) in the binary system was approximately equal to 1. Besides, the values of Hill’s coefficients were lager than 1, which indicated very weakly positive cooperativity in the transferrin–cefuroxime sodium system. The synchronous fluorescence spectra and circular dichroism spectra revealed that the microenvironment and the conformation of transferrin were changed during the binding reaction.     

硝基羟乙唑与溶菌酶反应机制的荧光光谱研究

分别在298,310,318 K温度下,利用荧光光谱法研究了pH=7.40生理条件下硝基羟乙唑(TRI)与溶菌酶(LYSO)的相互作用机理。结果表明,TRI与LYSO间通过静态猝灭方式相互作用。测定了LYSO与TRI反应的结合常数、结合位点数。利用反应过程的热力学参数,确定了LYSO-TRI体系的作用力类型;由Hill系数得出了LYSO或TRI的协同性;根据非辐射能量转移理论,确定了TRI到LYSO的结合距离,同时采用同步光谱法考察了TRI对LYSO构象的影响。     

计算机模拟和光谱法研究头孢他美酯与胃蛋白酶的结合机理

采用多种光谱法及计算机模拟技术研究了298,303,310 K温度下,头孢他美酯（CFP）与胃蛋白酶（PEP）之间的结合机理。结果表明,CFP主要以非辐射能量转移的静态猝灭方式猝灭PEP的荧光,两者主要通过静电作用力结合,其结合率在310 K为74.73%~92.13%。采用同步荧光法和圆二色谱法研究CFP对PEP的反应,结果表明两者的结合诱导了PEP的构象变化,使PEP的内源荧光猝灭。采用计算机模拟CFP与PEP的对接,结果表明CFP结合在PEP的催化活性位点处,该结论与光谱法所得结果一致。利用CFP对PEP的荧光猝灭反应,可以实现对实际药品中CFP含量的快速测定。     

Molecular interaction of silybin with hyaluronidase: A spectroscopic and molecular docking study

In this study, the molecular interaction of silybin with hyaluronidase was investigated by spectroscopic methods and molecular docking. It was found that silybin had strong ability to quench the intrinsic fluorescence of hyaluronidase by a static quenching procedure. The binding constants were obtained at three temperatures (293, 298, and 310 K). The results of synchronous fluorescence and three-dimensional fluorescence and molecular docking showed that silybin bound into the hyaluronidase cavity site and the binding of silybin to hyaluronidase could induce micro-environmental and conformational changes in hyaluronidase, which resulted in the reduced hyaluronidase activity. The thermodynamic parameter analysis and molecular docking experiments revealed that all types of non-covalent interaction, including hydrogen bonding interaction, van der Waals forces, hydrophobic interaction, and electrostatic interaction were present in the binding process of silybin with hyaluronidase. The results obtained here will provide direct evidence at a molecular level to understand the mechanism of the inhibitory effect of silybin against hyaluronidase.     

Deoxyribonucleic Acid and Bovine Serum Albumin Interaction with the Asymmetric Schiff Base Ligand and Its Molybdenum (VI) Complex: Multi Spectroscopic and Molecular Docking Studies

The interaction of an asymmetric Schiff base ligand derived from allylamine and 2,3-dihydroxybenzaldehyde and its molybdenum (VI) complex with deoxyribonucleic acid (DNA) and bovine serum albumin (BSA) were studied using spectroscopic and molecular docking methods. The spectroscopic results revealed that the DNA and BSA affinity for binding the Mo(VI) complex is greater than its ligand. Furthermore, the molecular docking calculations showed that H-bond, hydrophobic, π-π and π-cation interactions had the dominant roles in the stability of the compound-BSA complexes. The DNA interaction results suggested that the compounds interacted with DNA by the groove binding mechanism.     

The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

The crystallographic temperature factors(B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule.Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents.It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme.To test the theory of conformational flexibility of enzyme active site,crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions.It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M.It was also found that the B factors averaged over individually collected data sets were more accurate.Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets.Furthermore,we found that the correctly predicted active sites included not only the more flexible residues,but also some more rigid residues.Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network,covering the majority of the substrate binding residues.Therefore,this experimental prediction method may be useful for characterizing the binding site and the function of a protein,such as drug targeting.     

Fluorescence Investigation on the Interaction of a Prevalent Competitive Fluorescent Probe with Entomic Odorant Binding Protein

In recent years, one prevalent competitive fluorescent probe, N-phenyl-1-naphthylamine (1-NPN), was frequently utilized to measure the binding affinity of entomic odorant binding proteins (OBPs) with diverse plant volatiles or pheromones. Nevertheless, the details and model of the binding interaction are still largely unknown, although it is vital to investigate the physiological function of OBPs. Here we studied the binding interaction between 1-NPN and OBP2, a recombinant OBP from eastern honeybee, Apis cerana, by the combination of fluorescence quenching spectra, synchronous fluorescence spectra, ultraviolet spectra, circular dichroism spectra, and molecular docking. The Stern–Volmer curve of the fluorescence quenching of OBP2 by 1-NPN indicated it was a static quenching mechanism, and the binding constants and binding number were determined, respectively. Based on the Förster theory of nonradiation energy transfer (FRET), the binding distance was calculated, and the intrinsic fluorescent energy was predicted to transfer from the donor OBP2 to the acceptor 1-NPN. Synchronous fluorescence spectra and circular dichroism spectra were used to investigate the conformational change in binding progress. The thermodynamic parameters showed that the interaction was mainly driven by hydrophobic force, which was validated by the molecular docking; meanwhile, the binding mode was revealed and one hydrogen bond was found between the nitrogen atom of 1-NPN and Glu29 of OBP2.     

Interaction of chlorogenic acid with milk proteins analyzed by spectroscopic and modeling methods

Polyphenols are powerful antioxidants implicated in reducing the risk of human cancer and cardiovascular disease. Chlorogenic acid is a principal polyphenol in coffee, a beverage consumed worldwide on an immense scale. In many countries, coffee is consumed with milk, which has been shown to affect the bioavailability of polyphenols. Here we assessed the interactions of chlorogenic acid with five major milk proteins (α-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin) by spectroscopy and molecular docking calculations. The data showed that the number of binding sites in each chlorogenic acid–milk protein complex was close to 1. Fluorescence quenching of milk proteins by chlorogenic acid occurred through a static mechanism and the binding distance was smaller than 8 nm. Binding constant for chlorogenic acid–β-lactoglobulin was larger than those for chlorogenic acid-α-lactalbumin, chlorogenic acid–α-casein, chlorogenic acid–β-casein, and chlorogenic acid–κ-casein. Thermodynamic parameters revealed that Van der Waals forces and hydrogen bond interactions predominated in chlorogenic acid–α-casein and chlorogenic acid–α-lactalbumin complexes; hydrophobic interactions were predominant in chlorogenic acid–β-casein, chlorogenic acid–κ-casein, and chlorogenic acid–β-lactoglobulin. Molecular docking calculations identified chlorogenic acid was located near Pse66, Ile65, and Pro29 in the chlorogenic acid–α-casein adduct, Leu138, Thr126, Gln123, Ser124, Gln167, Ser166, Ser168 in the chlorogenic acid–β-casein adduct, Glu147, Asn123, Val143, Pse149, Pro120, Leu79, Ala148 in the chlorogenic acid–κ-casein adduct, Glu49, Tyr103, Gln54, His32, Trp104, Leu110 in the chlorogenic acid–α-lactalbumin adduct, and Cys66, Leu22, Lys60, Trp61, Ser21, Lys69, Gln59 in the chlorogenic acid–β-lactoglobulin adduct. Notably, the antioxidant activity of chlorogenic acid decreased significantly on interaction with each of the milk proteins (p < 0.05). This study reveals the binding behaviors of chlorogenic acid with five milk proteins and provides basic data that can help to clarify the binding mechanism.     

光谱法结合分子对接模拟技术研究头孢他啶与胰蛋白酶的相互作用机理

本文主要通过荧光光谱法与分子对接技术研究了在298,303,310 K温度下头孢他啶(CFD)与胰蛋白酶(TRP)之间的作用机制。研究结果表明,CFD与TRP之间是通过1∶1的静态猝灭方式相互作用。依照双对数方程处理荧光猝灭数据得到了CFD与TRP作用的结合常数Ka和结合位点数n。通过热力学方程求得了不同温度下CFD与TRP作用的热力学参数。实验数据表明,它们之间的作用力主要是疏水作用和氢键作用,这与分子对接技术所得的结果是一致的。     

Quinolinesulfonamides: Interaction between bovine serum albumin,molecular docking analysis,and antiproliferative activity against human breast carcinoma cells

The complex formation of bovine serum albumin (BSA) with library of all seven regioisomeric quinolinesulfonamides (QSAs) under physiological condition is studied in this paper. The fluorescence quenching mechanism of BSA by QSAs was discussed and the association constants, as well as the number of binding sites, were calculated. In addition, a molecular docking study of the tested sulfamoylquinolines on the active site of serum albumin is performed. The experimental data and molecular docking studies reveal that sulfamoylquinolines bind in the large hydrophobic cavity of subdomain IB, and in the hydrophobic pockets of BSA subdomains IIA and IIIA by hydrophobic interactions with tryptophanyl (Trp-213) and tyrosyl residues. Moreover, the antiproliferative activity of QSAs against two human breast cancer cell lines (human adenocarcinoma (MCF-7) and human ductal carcinoma (MDA-MB-231)) and a human normal fibroblast is also studied in this paper. The antiproliferative activity of the tested QSAs was comparable to those of cisplatin. The returned data indicate that some of the tested quinolinesulfamoyl derivatives display significant cytotoxic activity.     

Analysis of the binding mechanism between o-phenylenediamine and bovine hemoglobin by molecular spectroscopy and molecular modeling methods

Abstract

This study was aimed to reveal the binding mechanism between bovine hemoglobin and o-phenylenediamine by using molecular spectroscopy techniques and molecular modeling methods. The experimental results revealed that the fluorescence quenching mechanism was a combined dynamic and static quenching. The binding constant was (1.17?±?0.02)×10⁴ L/mol, and only a single binding site exists. The binding distance was 2.46?nm. The binding process was a spontaneous reaction, dominated by hydrophobic forces. The molecular docking simulations have also confirmed the results of the spectroscopic methods. The results reported here may significantly help understanding the interaction mechanism of o-phenylenediamine and hemoglobin.     

Improving the gel properties of salted egg white/cooked soybean protein isolate composite gels by ultrasound treatment: Study on the gelling properties and structure

In this study, the effects of ultrasound treatment on the texture, physicochemical properties and protein structure of composite gels prepared by salted egg white (SEW) and cooked soybean protein isolate (CSPI) at different ratios were investigated. With the increased SEW addition, the ζ-potential absolute values, soluble protein content, surface hydrophobicity and swelling ratio of composite gels showed overall declining trends (P < 0.05), while the free sulfhydryl (SH) contents and hardness of exhibited overall increasing trends (P < 0.05). Microstructural results revealed that composite gels exhibited denser structure with the increased SEW addition. After ultrasound treatment, the particle size of composite protein solutions significantly decreased (P < 0.05), and the free SH contents of ultrasound-treated composite gels were lower than that of untreated composite gels. Moreover, ultrasound treatment enhanced the hardness of composite gels, and promoted the conversion of free water into non-flowable water. However, when ultrasonic power exceeded 150 W, the hardness of composite gels could not be further enhanced. FTIR results indicated that ultrasound treatment facilitated the composite protein aggregates to form a more stable gel structure. The improvement of ultrasound treatment on the properties of composite gels was mainly by promoting the dissociation of protein aggregates, and the dissociated protein particles further interacted to form denser aggregates through disulfide bond, thus facilitating the crosslinking and reaggregation of protein aggregates to form denser gel structure. Overall, ultrasound treatment is an effective approach to improve the properties of SEW-CSPI composite gels, which can improve the potential utilization of SEW and SPI in food processing.     

Quenching interactions and nonexponential decay: tryptophan 138 of bacteriophage T4 lysozyme

Site-directed mutagenesis has been used to prepare variants of bacteriophage T4 lysozyme that contain only one tryptophan residue at position 138 and to change the residues in the immediate environment of this buried residue. Replacement of glutamine-105 by alanine results in a 2.7-fold increase in fluoresence quantum yield and converts the fluorescence decay from a highly nonexponential form to a single-exponential decay. This is atributed to electron transfer quenching of tryptophan-138 fluorescence by glutamine-105. Replacemeent of alanine-146 by threonine results in a 1.6-fold decrease in fluorescence intensity, indicating enhanced quenching by glutamine-105; replacement of glutamine-105 by alanine in this species results in a 5-fold in crease in fluorescence intensity. The interpretation of the nonexponential decay of the glutamine-105-containing species is discussed in terms of reversibility of the quenching process.     

头孢噻肟钠和氯霉素与牛血清白蛋白相互作用的荧光光谱分析

在pH=7.40 Tris-HCl缓冲溶液中,应用荧光光谱法分别研究了298,303,308 K时,头孢噻肟钠(CTX)、氯霉素(CHL)对牛血清白蛋白(BSA)荧光的猝灭反应.结果表明:药物与BSA的结合稳定常数Ka随温度增加而降低,两种药物对BSA的荧光猝灭皆为静态猝灭过程.标记竞争实验表明CTX、CHL在BSA中...     

Slow dynamics of supercooled hydration water in contact with lysozyme: examining the cage effect at different length scales

ABSTRACT

We study by computer simulation the dynamics of hydration water in solution with lysozyme upon approaching the glassy state of water. We calculate the self-density correlation function at different wavelengths to test the Mode Coupling Theory (MCT) of glassy dynamics at different length scales. The results show a strict and clear relation of the behaviour of the structural relaxation with the cage effect. We find a good agreement with the predictions of the MCT in the short and medium scale range, while at increasing length scales the interaction of water molecules with the protein's substrate induces deviations from the MCT behaviour, as found in previous studies. Besides at low temperatures the slow dynamics deviates from MCT due to hopping processes, similar to the bulk, as witnessed by a crossover from a fragile behaviour to a strong behaviour. We show that this deviation is evident at all length scales. Interestingly, we find that in the fragile region the confining cage decreases in radius with temperature while in the strong region it appears stable.     

The Interaction of a New Schiff Base Ligand with Human Serum Albumin: Molecular Docking and Molecular Dynamics Simulation Studies

The interaction of a new heterocyclic Schiff base bearing pyridine and pyrimidine cycles, with human serum albumin (HSA) using molecular docking and molecular dynamics simulation methods was examined. Molecular docking studies showed that the ligand was bonded to the IB domain of the protein. It was found that there was one hydrogen bond interaction between HSA and the ligand. The standard Gibbs free energy for binding of the ligand to HSA was calculated as ?9.63 kcal.mol^?1. The results of the molecular dynamics simulation showed that the root mean square deviation (RMSD) of the non-liganded HSA and the HSA–ligand complex reached equilibration after 1000 ps. The study of the radius of gyration revealed that there was a conformational change when the HSA–ligand complex was formed. Finally, analyzing the RMS fluctuations (RMSF) suggested that the structure of the ligand binding site remained approximately rigid during the simulation.     

Studies on the interaction of gliclazide with bovine serum albumin by fluorescence and synchronous fluorescence spectroscopy

The interaction between gliclazide and bovine serum albumin was investigated by fluorescence and synchronous fluorescence spectroscopy. From the experimental results, it was found that the quenching mechanism was static. The results of the synchronous fluorescence obtained indicated that the binding of gliclazide with bovine serum albumin could affect conformation in bovine serum albumin. In addition, the binding constants (K_a), binding sites (n), thermodynamic parameters, binding forces, Hill’s coefficient, and binding rate of gliclazide to protein calculated from two methods using the same equation were consistent at three different temperatures (298, 310, 318 K). This indicated that as a useful supplement to the conventional method, synchronous fluorescence spectroscopy could be used to study the mechanism of drugs and proteins. The conclusion was verified by UV/vis method.     

Spectroscopic study and application of the interaction mechanism of meloxicam with trypsin

Abstract

In order to explore the interaction between meloxicam and trypsin, the interaction mechanism between meloxicam and trypsin was studied by fluorescence spectrum, UV-vis absorption spectrum, circular dichroism spectrum, and molecular docking simulation under the experimental condition of pH = 7.40. The results of spectral experiments showed that meloxicam could effectively quench the internal fluorescence of trypsin in the form of static quenching, formed a stable complex at 1:1, and changed the conformation of trypsin. The results of thermodynamic constant showed that ΔG?H?S?>?0 indicates that the main force type of the binding system was hydrophobic interaction and hydrogen bonding. Molecular docking technique showed that the best binding site between meloxicam and trypsin was near the catalytic active center of trypsin, and the interaction between them changed the microenvironment of amino acid residues in the catalytic active center of trypsin. The mathematical model of drug and protein showed that when the concentration ratio of meloxicam to trypsin was 1:1, the protein binding rate of the binding system was 5.15%. The concentration ratio of meloxicam to trypsin was 30: 1, and the protein binding rate was 45.4%. The results showed that when the drug concentration was high, the binding effect of the system had a great influence on the concentration of free trypsin.