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.     

MCR-ALS结合分子对接研究隐色孔雀石绿与BSA的作用过程

孔雀石绿(LMG)在治理鱼卵中霉菌和杀灭鱼体寄生虫等方面的效果显著, 广泛应用于水产运输和养殖。孔雀石绿进入动物机体后, 通过生物转化代谢为脂溶性的隐色孔雀石绿(LMG), LMG的毒性超过MG; LMG能快速在组织中蓄积, 具有致癌、致畸、致突变等毒副作用。白蛋白可与多种内源和外源化合物结合, 是血浆中含量最丰富的载体蛋白, 也是药物发挥作用的重要载体和靶标。模拟pH 7.4的生理条件, 通过荧光光谱和圆二色谱法(CD)采集两种不同滴加方式的LMG与牛血清白蛋白(BSA)动态作用过程中的多维数据, 并应用化学计量学多元曲线分辨-交替最小二乘法(MCR-ALS)对多维波谱数据进行解析和描述, 从重叠严重的光谱数据中同时得到作用体系的定量和定性信息。从解析得到的浓度趋势图中, 说明体系在LMG∶BSA=2∶1时达到动态平衡, 并可确认复合物LMG₂-BSA的生成; 解析得到的与所测量的BSA荧光和CD图符合, 印证由MCR-ALS获得的浓度趋势图的可靠性和正确性; 通常由重叠光谱中无法辨别的LMG₂-BSA复合物荧光光谱和CD谱图也可由数学解析获得, 进一步印证了复合物的存在。原子力显微镜(AFM)测量结果表明BSA与LMG结合后, BSA的形貌发生改变, 表面粗糙度(RMS)由(1.24±0.28) nm增至(13.47±0.53) nm; 同时由CD实验结果可知LMG与BSA作用达到平衡时, α-螺旋结构的含量从46.5%降低到42.3%, 推测是BSA所处微环境和构象发生变化所致。荧光探针实验发现经典site Ⅰ标记物华法林加入后, LMG-BSA的猝灭常数由2.65×106 L·mol-1降低为1.88×106 L·mol-1, 但加入site Ⅱ标记物布洛芬后, LMG-BSA的猝灭常数变化不明显, 由此推断LMG可能结合在蛋白质的亚域ⅡA, 即site Ⅰ位。分子对接证实BSA的Ⅰ位有足够的空间容纳LMG, 且LMG与BSA之间的主要作用是疏水作用力。该研究从分子水平了解LMG与生物大分子的作用机制, 并为LMG的毒副作用研究提供重要的信息。     

同步荧光光谱法结合分子对接研究金雀花碱与牛血清白蛋白间相互作用

金雀花碱(Cy)是一种生物碱,主要存在于豆科毒豆属植物种子中。Cy具有较强的生物活性,特别是作为戒烟药物已得到广泛应用。在模拟生理条件下,应用荧光光谱法研究了Cy同牛血清白蛋白(BSA)之间的相互作用以及Cy猝灭BSA荧光发射的机理。详细考查了水浴温度、水浴时间以及溶液pH等因素对荧光猝灭的影响,并且通过Stem-Volmer方程计算了Cy与BSA间的结合类型、结合位点数目以及结合常数。结果表明,Cy与BSA可形成摩尔比为1∶1的非共价复合物,其结合常数为5.6×103,其猝灭类型为静态猝灭。同步荧光光谱研究结果表明,Cy的结合主要影响BSA 的Trp残基的荧光发射。进一步应用分子对接研究表明,氢键与疏水作用是Cy与BSA形成复合物的主要推动力。Cy与BSA中Trp213及其周围的氨基酸残基间存在氢键与疏水作用,这种作用将改变Trp213所处微环境的疏水情况,从而导致BSA的荧光发生猝灭。     

荧光法研究牛血清白蛋白与三羟基苯基荧光酮-钼(Ⅵ) 配合物探针的作用机理

用荧光光谱法研究了三羟基苯基荧光酮(TH-PF)-钼(Ⅵ)配合物与牛血清白蛋白的结合反应。探讨了TH-PF-Mo(Ⅵ)配合物对蛋白质内源荧光的猝灭机理,并测定了不同温度下的结合常数,温度为25 ℃时,荧光猝灭法测得该反应的结合常数为K=4.78×104 L·mol-1,温度为40 ℃时,荧光猝灭法测得该反应的结合常数为K=3.72×104 L·mol-1。根据Frster非辐射能量转移理论,确定了给体-受体之间的作用距离和能量转移效率(E=0.314),并根据热力学参数确定了TH-PF-Mo(Ⅵ)配合物与牛血清白蛋白之间的作用力类型,以静电引力为主。     

Interaction of BSA with proflavin: A spectroscopic approach

The interaction of bovine serum albumin (BSA) with proflavin was investigated by spectroscopic tools like absorption and fluorescence spectroscopy as well as laser flash photolysis. Absorption spectroscopy proved the formation of ground-state BSA-proflavin complex. Proflavin was found to quench the intrinsic fluorescence of BSA via static quenching. High value of quenching constant suggested that energy transfer occurred from BSA to proflavin. Distance between the fluorophore in the protein and the ligand (proflavin) was evaluated. Binding constant and number of binding site were determined for proflavin-BSA interaction both in phosphate buffer (pH∼6.8) and in sodium dodecylsulphate media. The values of the thermodynamic parameters suggested that the key interacting forces are van der Waal's interaction and hydrogen bonding. Laser flash photolysis study reconfirmed the formation of complex between BSA and proflavin.     

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。     

多光谱法和分子对接模拟法研究黄腐植酸和牛血清白蛋白的相互作用

在模拟生理环境中,使用荧光光谱法、紫外光谱法、圆二色谱法、同步荧光光谱法、三维荧光光谱法与分子对接模拟法研究黄腐植酸和牛血清白蛋白(BSA)之间相互作用。在荧光光谱法研究中,经Stern-Volmer方程计算得到298,303和308 K温度下的动态荧光猝灭速率常数K_q和猝灭常数,证明BSA与黄腐殖酸(FA)相互作用的猝灭过程为静态猝灭;同时根据计算得出的结合位点数n都在1附近,FA与BSA体系相互作用比为1∶1;利用静态猝灭双对数方程计算三个温度下的热力学参数,焓变ΔH<0,熵变ΔS＜0,得出结论,FA与BSA之间的主要作用力为氢键和范德华力;ΔG＜0,说明作用过程为自发过程。采用Förster’s偶极-偶极非辐射能量转移理论,计算出结合距离r=6.340 nm,表明BSA与FA之间存在非辐射能量转移。分子对接模拟结果表明FA与BSA残基的结合作用力具有氢键和范德华力,同时二者之间还存在疏水作用力,多种力共同作用使FA与BSA能够稳定结合。通过对FA与BSA相互作用的紫外-可见吸收光谱分析,发现BSA最大吸收峰发生了较为明显的红移,表明FA使BSA的二级结构发生改变。通过研究FA与BSA相互作用的同步荧光光谱,得到FA使BSA中的色氨酸（Trp）残基周围的微环境极性增强,疏水性减弱,亲水性增强,使BSA的蛋白质构象发生了一定程度的改变。通过研究FA与BSA相互作用的三维荧光光谱,峰1（peak 1）与峰2（peak 2）的最大发射波长峰都发生了红移,证明FA与BSA发生了相互作用,FA使BSA周围环境的极性增大,疏水性减小,亲水性增加,BSA蛋白质构象发生变化。最后采用圆二色谱法进行分析,利用软件计算得出该实验相互作用体系下α-螺旋（α-Helix）减少2.3％、β-折叠（β-sheet）增加7.7％、β-转角（β-Turn）增加0.6％和无规则结构（Random coil）含量减少1.2％,β-折叠（β-sheet）含量增加最为明显, 强有力地说明了FA使BSA结构发生了改变。     

多光谱法与分子对接法研究盐酸四环素与牛血清白蛋白的相互作用

盐酸四环素属于抗生素类, 目前有关盐酸四环素和牛血清白蛋白二级结构的影响及作用机理报道较少。在模拟生理条件下,采用荧光光谱法、三维荧光光谱法、紫外-可见光谱法、圆二色谱法和傅里叶红外光谱法以及分子对接模拟法,研究了盐酸四环素与牛血清白蛋白(BSA)之间的相互作用。荧光光谱表明,盐酸四环素能有效猝灭BSA的内源荧光,猝灭机制属静态猝灭,通过Stern-Volmer方程计算结合常数Ka为2.813×105 L·mol-1(298 K)。根据Vant’s Hoff方程确定结合过程中的热力学参数ΔS=-151.1 J·mol-1·K-1、ΔH=-76.09 kJ·mol-1_, 两者之间作用为氢键和范德华力。同步荧光光谱、紫外光谱、三维荧光光谱、红外光谱、圆二色谱结果证明盐酸四环素能够改变BSA的二级结构和微环境。根据Föster’s非辐射能量转移理论,盐酸四环素与BSA结合距离为0.49 nm。希尔系数(n_H)值小于1,表明盐酸四环素与BSA结合后存在药物间协同作用。圆二色谱(CD)定量测定了盐酸四环素与BSA作用前后的二级结构含量：α-螺旋含量增加了9.16%(1:1)。分子对接模拟表明盐酸四环素通过氢键、疏水作用和范德华力等多种作用力结合在BSA的site Ⅰ(亚域ⅡA)。本研究有助于了解盐酸四环素与BSA的作用机制,也有助于理解盐酸四环素对蛋白质在储运过程中功能的影响。     

Multiple Spectroscopic,Docking and Cytotoxic Study of a Synthesized 2,2′ Bipyridin Phenyl Isopentylglycin Pt(II) Nitrate Complex: Human Serum Albumin and Breast Cancer Cell Line of MDA-MB231 as Targets

In the present study, the biological activities of a new synthesized Pt(II)-complex, 2,2′ bipyridinphenyl isopentylglycin Pt(II) nitrate was investigated via its interaction with the most important blood carrier protein of human serum albumin (HSA), using fluorescence and Far-UV circular dichroism (CD) spectroscopic techniques and also molecular docking. Moreover, cytotoxicity activity of the complex was studied against breast cancer cell line of MDA MB231 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The Pt(II)-complex has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. According fluorescence quenching data, the binding parameters of the interaction were calculated and showed that hydrophobic interaction has an important role. The molecular docking results in coherent with fluorescence measurements illustrated that Pt(II) complex can bind to HSA at one position that located in the hydrophobic cavity of groove between drug site I and II. Also, experimental data on driving force in binding site was confirmed whereas theoretical results demonstrated Pt(II) complexinteract to HSA by hydrophobic interaction. Far-UV-CD results showed that Pt(II)-complex induced an increasing in the content of α-helical structure of the protein and stabilized it. Also, MTT assay represented growth inhibitory effect of the complex toward the breast cancer cell line.     

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.     

Study on the effect of protein on drug efficacy: cefonicid sodium–pepsin binding mechanism

The mechanism of interaction between cefonicid sodium and pepsin was investigated by various spectroscopic methods and molecular docking. Cefoncid sodium quenched the intrinsic fluorescence of pepsin at pH of 2.0 to form a new complex in a 1:1 binding mode driven by Van der Waals and hydrogen bonds. The mechanism of quenching was static. The results of molecular docking indicated that the cefonicid sodium-binding site was located in the active site of pepsin. The protein binding rates of cefonicid sodium in gastric juice was calculated and the binding model was established. It is concluded that cefonicid sodium is not suitable for oral administration.     

Specific binding of a dihydropyrimidinone derivative with DNA: Spectroscopic,calorimetric and modeling investigations

One of the dihydropyrimidinone derivative 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one (EMMD) was synthesized, and its binding properties with calf-thymus DNA (ctDNA) were investigated using spectroscopic, viscometric, isothermal titration calorimetric (ITC) and molecular modeling techniques. Fluorescence spectra suggested that the fluorescence enhancement of the binding interaction of EMMD to ctDNA was a static process with ground state complex formation. The binding constant determined with spectroscopic titration and ITC was found to be in the same order of 10⁴ M^?1. According to the results of the viscosity analysis, fluorescence competitive binding experiment, fluorescence quenching studies, absorption spectral and ITC investigations, it can be concluded that EMMD is intercalative binding to ctDNA. Furthermore, the results of molecular modeling confirmed those obtained from spectroscopic, viscosimetric and ITC investigations. Additionally, ITC studies also indicated that the binding interaction is predominantly enthalpy driven.     

Synthesis,Spectral Characterization,DNA/ Protein Binding,DNA Cleavage,Cytotoxicity, Antioxidative and Molecular Docking Studies of Cu(II)Complexes Containing Schiff Base-bpy/Phen Ligands

Ternary Cu(II) complexes [Cu(II)(L)(bpy)Cl] 1, [Cu(II)(L)(Phen)Cl] 2 [L = 2,3–dimethyl-1-phenyl-4(2 hydroxy-5-methyl benzylideneamino)-pyrazol-5-one, bpy = 2,2^′ bipyridine, phen =1,10 phenanthroline) were synthesized and characterized by elemental analyses, UV-Visible, FT-IR, ESR, Mass, thermogravimetric and SEM EDAX techniques. The complexes exhibit octahedral geometry. The interaction of the Cu(II) with cailf thymus DNA (CT-DNA) was explored by using absorption and fluorescence spectroscopic methods. The results revealed that the complexes have an affinity constant for DNA in the order of 10⁴ M^?1 and mode of interaction is intercalative mode. The DNA cleavage study showed that the complexes cleaved DNA without any external agent. The interaction of Cu(II) complexes with bovine serum albumin (BSA) was also studied using absorption and fluorescence techniques. The cytotoxic activity of the Cu(II) complexes was probed in HeLa (human breast adenocarcinoma cell line), B16F10 (Murine melanoma cell line) and HEPA1–6 celllines, complex 1 has good cytotoxic activity which is comparable with the doxarubicin drug, with IC₅₀ values ranging from 3 to 12.6 μM. A further molecular docking technique was employed to understand the binding of the complexes towards the molecular target DNA. Investigation of the antioxidative properties showed that the metal complexes have significant radical scavenging activity potency against DPPH radical.     

Thermally Stable Schiff Base and its Metal Complexes: Molecular Docking and Protein Binding Studies

In this paper, interaction of Schiff base and its metal complexes carrying naphthalene ring in the structure with bovine serum albumin (BSA) were investigated using UV-vis absorption, fluorescence spectroscopies and molecular docking methods. The effect on the binding mechanism and properties of these compounds containing metal-free, iron and copper ions were also investigated. The fluorescence spectroscopy results showed that fluorescence intensity of BSA in the presence of different concentration of ligands was decreased through a static quenching mechanism. Binding constants (KSV, Kbin and Ka) and thermodynamic parameters (ΔG, ΔH and ΔS) for the ligand-protein interactions were also determined. ΔG values of ligand-protein interaction were calculated in the range ? 6.3 to ?5.5 kcal/mol. These negative values showed that binding process is spontaneous and, hydrogen bonding and van der Waals force were main interaction of the protein and ligands. ΔH and ΔS value were also calculated in the range of 1.10 to 1.26 kJ/mol and 0.133 to 0.135 kJ/mol. K, respectively. These positive values indicated that the binding process between ligands and BSA are endothermic and electrostatic interaction, respectively.     

用分子对接方法预测HIV-1整合酶与金精三羧酸抑制剂的相互作用

用分子对接方法 (Docking)研究了HIV 1整合酶与其抑制剂金精三羧酸的结合过程 .为弄清金属离子在结合中所起的作用 ,选择含有一个Mg+ 2 或不含Mg+ 2 的两种不同的整合酶受体分别与金精三羧酸对接 .结果表明 ,Mg+ 2 对稳定配体与受体的结合起了重要作用 .金精三羧酸配体与含有一个金属Mg+ 2 的整合酶受体对接 ,最优结合自由能为 - 4 5 .19kJ/mol.当Mg+ 2 失去后 ,整合酶的活性中心构象将发生变化 ,使金精三羧酸抑制剂与整合酶的结合自由能 (- 2 4 .35kJ/mol)明显增加 .预测了未知的HIV 1整合酶与其抑制剂金精三羧酸的复合物结构 ,并可对基于结构的抗HIV 1整合酶的药物设计提供重要信息     

Mangiferin binding to serum albumin is non-saturable and induces conformational changes at high concentrations

The binding interaction between mangiferin (MGF), which a natural xanthone isolated from mangoes, and bovine serum albumin (BSA) was studied with absorbance and fluorescence spectroscopy, cyclic voltammetry and molecular modeling. The data were analyzed to assess the binding mechanism, effect of pH and ionic strength, conformational changes in the protein and electrical charge transfer involved. The MGF–BSA complex exhibited positive cooperativity with a 1:1 stoichiometry (K_d=0.38 mmol L^?1) for the first binding site and a non-saturable binding at high ligand concentrations. Furthermore, the data also suggest an increase in drug bioavailability in the acidic region and relatively low ionic strength values, which are close to physiological levels. The data suggest a specific electrostatic interaction together with hydrophobic effects and H-bonding displayed in MGF binding to the BSA IIA subdomain. Synchronous fluorescence spectra indicate that there are conformational changes in the polypeptide backbone upon ligand binding. Cyclic voltammetry indicates that there is an irreversible charge transfer between MGF and BSA that is modulated by diffusion on the electrode surface, where two electrons are transferred. These results can help the knowledge of the pharmacokinetic activities of natural or chemical xanthone-based drugs.     

Comparison of ligand migration and binding in heme proteins of the globin family

The binding of small diatomic ligands such as carbon monoxide or dioxygen to heme proteins is among the simplest biological processes known. Still, it has taken many decades to understand the mechanistic aspects of this process in full detail. Here, we compare ligand binding in three heme proteins of the globin family, myoglobin, a dimeric hemoglobin, and neuroglobin. The combination of structural, spectroscopic, and kinetic experiments over many years by many laboratories has revealed common properties of globins and a clear mechanistic picture of ligand binding at the molecular level. In addition to the ligand binding site at the heme iron, a primary ligand docking site exists that ensures efficient ligand binding to and release from the heme iron. Additional, secondary docking sites can greatly facilitate ligand escape after its dissociation from the heme. Although there is only indirect evidence at present, a preformed histidine gate appears to exist that allows ligand entry to and exit from the active site. The importance of these features can be assessed by studies involving modified proteins(via site-directed mutagenesis) and comparison with heme proteins not belonging to the globin family.     

微乳液介质-配合物光谱探针高灵敏光度法测定微量蛋白质的研究

研究了在OP微乳液介质中钼(Ⅵ)-邻氯苯基荧光酮(o-CPF)-蛋白质体系的相互作用和吸收光谱情况。在最佳条件下,体系的摩尔吸光系数ε_{533 nm}=6.12×106 mol-1·cm-1,牛血清蛋白质含量在0～14 μg·mL-1范围内服从比尔定律;同时采用摩尔比法和斜率比法测得配合物与蛋白质的结合数n=91。初步探讨了蛋白质与o-CPF-Mo(Ⅵ)配合物相互作用机理。将OP微乳液引入到蛋白质的测定中,显著地提高了体系的灵敏度。实验结果表明： 此法具有很高的灵敏度、选择性和稳定性,可直接用于尿样的定量测定。     

Spectroscopy and Molecular Docking Study on the Interaction Behavior Between Nobiletin and Pepsin

In this study, the binding mode of nobiletin (NOB) with pepsin was investigated by spectroscopic and molecular docking methods. NOB can interact with pepsin to form a NOB-pepsin complex. The binding constant, number of binding sites and thermodynamic parameters were measured, which indicated that NOB could spontaneously bind with pepsin through hydrophobic and electrostatic forces with one binding site. Molecular docking results revealed that NOB bound into the pepsin cavity. Synchronous and three-dimensional fluorescence spectra results provide data concerning conformational and some micro-environmental changes of pepsin. Furthermore, the binding of NOB can inhibit pepsin activity in vitro. The present study provides direct evidence at a molecular level to show that NOB could induce changes in the enzyme pepsin structure and function.     

Effect of temperature and pH on interaction between bovine serum albumin and cetylpyridinium bromide: Fluorescence spectroscopic approach

The fluorescence spectroscopic technique has been efficiently employed to investigate the interaction between bovine serum albumin (BSA) and cetylpyridinium bromide (CPB) under different pH and temperature conditions. The binding constant, number of binding sites, thermodynamic parameters such as ΔG, ΔH, ΔS, and nature of binding forces between BSA and CPB were obtained by measuring the steady state fluorescence quenching of BSA by CPB. The experimental results showed that the fluorescence quenching of BSA by CPB was a result of the formation of CPB-BSA complex. The static quenching was confirmed from the Stern-Volmer quenching constant at different temperatures. The effect of CPB on the conformation of BSA was analyzed using synchronous and three-dimensional fluorescence spectroscopy. pH dependence complex formation between BSA-CPB is due to the interaction between cationic side chain of CPB and the net charge developed on BSA. The distance ‘r’ between BSA and CPB was obtained according to the fluorescence resonance energy transfer.