薄膜梯度扩散技术结合相研究进展

薄膜梯度扩散(DGT)技术是一种新型原位被动采样技术,已被广泛应用于水体、土壤、沉积物中目标物的采集与测量。结合相是DGT技术的重要组成部分,决定了与目标物的结合能力、结合速度、结合容量以及目标物的形态选择性等。DGT结合相分为固态结合相和液态结合相。本文重点综述了树脂、氧化物、无机盐、活性炭、改性硅胶、分子印迹、共聚物、复合、液态等结合相在DGT技术中的应用,展望了DGT结合相的发展前景。     

抗肿瘤药物替加氟与牛血清白蛋白相互作用的热化学研究

在298.15 K下, 以等温滴定微量热(ITC)实验数据为依据,根据合理假设和Langmuir结合理论, 应用非线性最小方差拟合方法测定了抗肿瘤药物替加氟(Tegafur)与牛血清白蛋白(BSA)结合过程热力学性质的改变. 研究结果表明, 牛血清白蛋白与替加氟相互作用存在两类结合位点: (1) N=52.00±0.12, K=(9.83±0.13)×104 L/mol, ΔH=(30.10±0.17) kJ/mol>0, ΔS=(196.00±0.65) J/(mol·K)>0, ΔG=(-28.50±0.66) kJ/mol<0, 表现为熵驱动过程, 疏水相互作用为过程的主要推动力; (2) N=86.00±0.14, K=(9.35±0.13)×104 L/mol, ΔH=(-19.80±0.17) kJ/mol<0, ΔS=(28.30±0.50) J/(mol·K)>0, ΔG=(-28.40±0.43) kJ/mol<0, 表现为焓-熵协同过程, 氢键和静电相互作用为过程的主要推动力. 圆二色谱(CD)分析结果表明, 抗肿瘤药物替加氟诱导蛋白质(BSA)二级结构单元的相对含量发生了一定程度的变化.     

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase.

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin-azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.     

Absolute Protein Binding Free Energy Simulations for Ligands with Multiple Poses,a Thermodynamic Path That Avoids Exhaustive Enumeration of the Poses

We propose a free energy calculation method for receptor–ligand binding, which have multiple binding poses that avoids exhaustive enumeration of the poses. For systems with multiple binding poses, the standard procedure is to enumerate orientations of the binding poses, restrain the ligand to each orientation, and then, calculate the binding free energies for each binding pose. In this study, we modify a part of the thermodynamic cycle in order to sample a broader conformational space of the ligand in the binding site. This modification leads to more accurate free energy calculation without performing separate free energy simulations for each binding pose. We applied our modification to simple model host–guest systems as a test, which have only two binding poses, by using a single decoupling method (SDM) in implicit solvent. The results showed that the binding free energies obtained from our method without knowing the two binding poses were in good agreement with the benchmark results obtained by explicit enumeration of the binding poses. Our method is applicable to other alchemical binding free energy calculation methods such as the double decoupling method (DDM) in explicit solvent. We performed a calculation for a protein–ligand system with explicit solvent using our modified thermodynamic path. The results of the free energy simulation along our modified path were in good agreement with the results of conventional DDM, which requires a separate binding free energy calculation for each of the binding poses of the example of phenol binding to T4 lysozyme in explicit solvent. © 2019 Wiley Periodicals, Inc.     

The binding of organic anions by polyvinylpyrrolidone: Determination of intrinsic binding constant and number of binding sites by competitive binding

The binding of 4′-dibutylaminoazobenzene-4-sulfonate (butyl orange, BO) and 1-amino-4-methylaminoanthraquinone-2-sulfonate (AQ) by crosslinked polyvinylpyrrolidone and the competition between BO and AQ for binding sites of the polymer were examined. The equilibrium data showed competitive binding. Thus the intrinsic binding constants and the number of binding sites can be evaluated easily and precisely by competitive binding. The thermodynamic parameters that accompained the binding were calculated from the intrinsic binding constant and its temperature-dependence. The thermodynamic data for BO showed that the binding process is athermal and stabilized entirely by the entropy term. The binding of BO to the polymer is entropically favorable as a result of the operation of the hydrophobic effect. In contrast, with AQ a favorable free energy for a dye-polymer complex formation is associated with a large negative enthalpy and a small entropy. Therefore it is likely that the binding of AQ occurs by energetic forces and that the large aromatic ring of the dye contributes to the binding energy. On the average, BO and AQ can bind to the crosslinked polymer to the extent of 1 dye/ca 73 basemol in 0.1M tris-acetate buffer, pH 7.0.     

Multiple binding modes for dicationic Hoechst 33258 to DNA

The binding of dicationic Hoechst 33258 (ligand) to DNA was characterized by means of the fluorescence spectra, fluorescence intensity titration, time-resolved fluorescence decay, light scattering, circular dichroism, and fluorescence thermal denaturation measurements, and two binding modes were distinguished by the experimental results. Type 1 binding has the stoichiometry of one ligand to more than 12 base pairs, and it is defined as quasi-minor groove binding which has the typical prolonged fluorescence lifetime of about 4.4 ns. In type 1 binding, planar conformation of the ligand is favorable. Type 2 binding with phosphate to ligand ratio (P/L) < 2.5 has the stoichiometry of one ligand to two phosphates. It is defined as a highly dense and orderly stacked binding with DNA backbone as the template. Electrostatic interactions between doubly protonated ligands and negatively charged DNA backbone play a predominant role in the type 2 binding mode. The characteristics of this type of binding result in a twisted conformation of the ligand that has a fluorescence lifetime of less than 1 ns. The results also indicate that the binding is in a cooperative manner primarily by stacking of the aromatic rings of the neighboring ligands. Type 1 binding is only observed for double-stranded DNA (dsDNA) with affinity constant of 1.83 x 10(7) M-1. In the type 2 binding mode, the binding affinity constants are 4.9 x 10(6) and 4.3 x 10(6) M-1 for dsDNA and single-stranded DNA (ssDNA), respectively. The type 2 binding is base pair independent while the type 1 binding is base pair related. The experiments described in this paper revealed that the dication bindings are different from the monocation bindings reported by previous study. The dication binding leads to stronger aggregation at low ligand concentration and results in orderly arrangements of the ligands along DNA chains. Furthermore the dication binding is demonstrated to be beneficial for enhancing the DNA's stability.     

Water‐Soluble Molecularly Imprinted Nanoparticles (MINPs) with Tailored,Functionalized, Modifiable Binding Pockets

Construction of receptors with binding sites of specific size, shape, and functional groups is important to both chemistry and biology. Covalent imprinting of a photocleavable template within surface–core doubly cross‐linked micelles yielded carboxylic acid‐containing hydrophobic pockets within the water‐soluble molecularly imprinted nanoparticles. The functionalized binding pockets were characterized by their binding of amine‐ and acid‐functionalized guests under different pH values. The nanoparticles, on average, contained one binding site per particle and displayed highly selective binding among structural analogues. The binding sites could be modified further by covalent chemistry to modulate their binding properties.     

Revisiting the Rate-Limiting Step of the ANS–Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS–protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS–protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective.     

纳米粒子尺寸效应引起的内层电子结合能位移

用XPS测定了氩离子溅射前后的Pt-PVP纳米粒子和TiO2、ZnO及SiO2纳米粒子的内层电子结合能, 并与其对应的体材料进行了比较. 结果表明, Ar+溅射前Pt-PVP的Pt 4f结合能比体材料Pt的稍低, 但Ar+溅射后由于PVP包覆层被除去, 裸露的Pt 纳米粒子的结合能明显高于Pt体材料. 与非纳米氧化物比较, 纳米氧化物TiO2、ZnO 和SiO2的内层电子结合能也向高结合能方向位移, 其位移大小的顺序为TiO2相似文献   

蛋白质分子与配体结合过程构象变化的研究

蛋白质分子与配体的作用模式主要有直接的环区结合及铰链式结合两种方式。针对这两种不同的作用方式,我们提出采用不同的策略进行结合过程的构象研究。对于直接的环区结合模式,通过建立环区主链构象库,来实现蛋白质环区与配体的准柔性对接,并以链霉抗生物素蛋白体系为例对构象库建立的可行性进行了验证计算。对铰链结合方式,采用分步对接的方法进行计算,并具体应用于HIV蛋白酶与其小分子配体的结合过程。计算结果表明,这两种处理方法分别能较好地模拟不同类型的蛋白质与配体结合的的构象变化。     

Free energy calculations on the two drug binding sites in the M2 proton channel

Two alternative binding sites of adamantane-type drugs in the influenza A M2 channel have been suggested, one with the drug binding inside the channel pore and the other with four drug molecule S-binding to the C-terminal surface of the transmembrane domain. Recent computational and experimental studies have suggested that the pore binding site is more energetically favorable but the external surface binding site may also exist. Nonetheless, which drug binding site leads to channel inhibition in vivo and how drug-resistant mutations affect these sites are not completely understood. We applied molecular dynamics simulations and potential of mean force calculations to examine the structures and the free energies associated with these putative drug binding sites in an M2-lipid bilayer system. We found that, at biological pH (~7.4), the pore binding site is more thermodynamically favorable than the surface binding site by ~7 kcal/mol and, hence, would lead to more stable drug binding and channel inhibition. This result is in excellent agreement with several recent studies. More importantly, a novel finding of ours is that binding to the channel pore requires overcoming a much higher energy barrier of ~10 kcal/mol than binding to the C-terminal channel surface, indicating that the latter site is more kinetically favorable. Our study is the first computational work that provides both kinetic and thermodynamic energy information on these drug binding sites. Our results provide a theoretical framework to interpret and reconcile existing and often conflicting results regarding these two binding sites, thus helping to expand our understanding of M2-drug binding, and may help guide the design and screening of novel drugs to combat the virus.     

Calculation of binding energy differences for receptor–ligand systems using the Poisson-Boltzmann method

An approach using the finite difference solution of the Poisson-Boltzmann equation to estimate binding free energy changes for two receptor–ligand systems, arabinose binding protein and sulfate binding protein, is presented. The eight calculated binding free energy changes agree with experiment, showing a correlation coefficient of 0.92 and energy deviations of 1 kcal/mol or less. More importantly, the decomposition of solvation and assembly energies in this approach provides an understanding of binding mechanisms and therefore could suggest directions to alter binding affinities. The method is demonstrated to be useful in analyzing experimental binding structures and predicting binding effects of mutants or modified ligands for macromolecular systems, in which the electrostatic forces dominate the overall interaction and the structural perturbations upon modifications are small. © 1995 by John Wiley & Sons, Inc.     

The Binding Energy Distribution Analysis Method (BEDAM) for the Estimation of Protein-Ligand Binding Affinities

The Binding Energy Distribution Analysis Method (BEDAM) for the computation of receptor-ligand standard binding free energies with implicit solvation is presented. The method is based on a well established statistical mechanics theory of molecular association. It is shown that, in the context of implicit solvation, the theory is homologous to the test particle method of solvation thermodynamics with the solute-solvent potential represented by the effective binding energy of the protein-ligand complex. Accordingly, in BEDAM the binding constant is computed by means of a weighted integral of the probability distribution of the binding energy obtained in the canonical ensemble in which the ligand is positioned in the binding site but the receptor and the ligand interact only with the solvent continuum. It is shown that the binding energy distribution encodes all of the physical effects of binding. The balance between binding enthalpy and entropy is seen in our formalism as a balance between favorable and unfavorable binding modes which are coupled through the normalization of the binding energy distribution function. An efficient computational protocol for the binding energy distribution based on the AGBNP2 implicit solvent model, parallel Hamiltonian replica exchange sampling and histogram reweighting is developed. Applications of the method to a set of known binders and non-binders of the L99A and L99A/M102Q mutants of T4 lysozyme receptor are illustrated. The method is able to discriminate without error binders from non-binders, and the computed standard binding free energies of the binders are found to be in good agreement with experimental measurements. Analysis of the results reveals that the binding affinities of these systems reflect the contributions from multiple conformations spanning a wide range of binding energies.     

Free energy balance predicates dendrimer binding multivalency at molecular printboards

Self-assembled monolayers (SAMs) terminating in beta-cyclodextrin (beta-CD) cavities can be used to bind ink molecules and so provide a molecular printboard for nanopatterning applications. Multivalent, or multisite, binding strengthens the attachment of large inks to the printboard, yielding more robust patterns. We performed fully atomistic molecular dynamics (MD) simulations in bulk explicit solvent to probe the conformational space available to dendrimer and dendrite ink molecules, in both free and bound environments. We show that accurate treatment of both pH effects and binding conformations gives calculated binding modes in line with known binding multivalencies. We identify and quantify the steric frustration causing small, low-generation dendrimer inks to bind to the printboard using just a subset of the available anchor groups. Furthermore, we show that the enhanced binding energy of multisite attachment offsets the steric strain, the feasibility of a given binding mode thus determined by the relative magnitudes of the unfavorable steric strain and favorable multisite binding free energies. We use our experimentally validated model of dendrimer binding to predict the binding mode of novel fluorophoric dendrites and find divalent binding, consistent with confocal microscopy imaging of pattern formation at molecular printboards.     

Competitive Binding of Drugs to the Multiple Binding Sites on Human Serum Albumin: A calorimetric study

A multiple-site competitive model has been developed to evaluate quantitatively the equilibrium competition of drugs that bind to multiple classes of binding sites on human serum albumin (HSA). The equations, which are based on the multiple-class binding site model, assume that competition exists at individual sites, that the binding parameters for drug or drug competitor pertain to individual sites, and also that the binding parameters for drug or competitor at any given site are independent of drug or competitor bound at other sites. For the drug-competitor pairs, ethacrynic acid (EA) -caproic acid (C6), -lauric acid (C12), and -palmitic acid (C16), the reaction heat of EA binding to HSA was measured in the absence and the presence of fatty acids at the molar ratio of 3:1 with HSA at pH 7.4 and 37°C by isothermal titration microcalorimetry. The calorimetric titration data induced by the presence of fatty acids were directly compaired to the computer simulation curves by the corresponding multiple-site competititve models, which were precedently calculated from binding parameters of EA and fatty acids. In the case of EA-C12 or -C16 competitive binding, EA binding at the first and the second classes of binding sites on HSA were instantaneously inhibited by C12 or C16, resulting that the binding constant of the first class of binding sites of EA were decreased and that the second class of binding sites on HSA entirely disappeared. In the competition between EA and C6, the first class of binding sites of EA was diminished by C6, resulting in the decrease of the binding constants and the number of binding sites in the first class of EA, whereas, the second class of binding sites was unaffected. The multiple-site competitive model assuming site-site competition could be directly comparable to the calorimetric data and be suitable to account for the competitive processes for drugs bound to the multiple-class of binding sites on HSA.This revised version was published online in November 2005 with corrections to the Cover Date.     

Polyoxometalate binding to human serum albumin: a thermodynamic and spectroscopic approach

The molecular recognition of polyoxometalates by human serum albumin is studied using two different polyoxometalates (POMs) at pH 7.5. The results are compared with those obtained at pH 3.5 and 9.0. At pH 7.5, both POMs strongly interact with the protein with different binding behaviors. The Keggin shaped POM, [H(2)W(12)O(40)](6-) (H2W12), specifically binds the protein, forming a complex with a 1:1 stoichiometry with Ka = 2.9 x 10(6) M(-1). The binding constant decreased dramatically with the increase of the ionic strength, thus indicating a mostly electrostatic binding process. Isothermal titration calorimetry (ITC) experiments show that the binding is an enthalpically driven exothermic process. For the wheel shaped POM [NaP(5)W(30)O(110)](14-) (P5W30), there are up to five binding sites on the protein. Increasing the ionic strength changes the binding behavior significantly, leading to a simple exothermic process, with several binding sites. Competitive binding experiments indicate that the two POMs share one common binding site. In addition, they show the existence of another important binding site for P5W30. The two POMs exhibit different binding dependences on the pH. The combination of the experimental results with the knowledge of the surface map of the protein in its N-B conformation transition domain leads to the proposal for the probable binding site of POMs. The present work reveals a protein conformation change upon P5W30 binding, a new feature not explicitly documented in previous studies.     

Protein surface conservation in binding sites

An algorithm is described which uses the conservation of the 3D structure of protein surfaces, as opposed to their sequences, to detect protein-protein binding sites. The protein in which protein-protein binding sites are sought is compared with structures of multiple structurally related proteins and the surface that is conserved at least once is considered to be a part of the binding site. The binding site predictions obtained in this way for a set of protein-protein complexes correspond well with the actual protein-protein binding sites. A comparison of this method with an algorithm using the support vector machine approach for predicting protein-protein binding sites shows structural conservation to be an important characteristic that distinguishes binding sites from the remainder of protein surfaces.     

Interaction of atrazine with Laurentian humic acid

Ultrafiltration fractionation and liquid chromatography have been applied to study the binding and hydrolysis of polar herbicide atrazine on a stoichiometrically well characterized Laurentian humic acid. The main advantage of this method over gas chromatography is the simultaneous determination of both free and bound atrazine, hydroxyatrazine and copper(II) ion with satisfactory accuracy and precision. Atrazine binding requires extensive carboxylate site protonation but the binding sites represent only a very small fraction of total carboxylate of humic acid. The results show that binding of atrazine is not competitive with binding of the hydrolysis product hydroxyatrazine, the binding capacity is reduced at higher ionic strength or by cation competition for carboxylate and the atrazine binding constant and free energy of binding can be fitted by a single value at all pH values. The differences between atrazine binding by fulvic acid and humic acid can be ascribed to the structure difference, one being a flexible linear polymer and the other a three-dimensional colloidal gel particle.