Sites In Macromolecules Research Articles

InterMetalDB: A Database and Browser of Intermolecular Metal Binding Sites in Macromolecules with Structural Information.

InterMetalDB is a free-of-charge database and browser of intermolecular metal binding sites that are present on the interfaces of macromolecules forming larger assemblies based on structural information deposited in Protein Data Bank (PDB). It can be found and freely used at https://intermetaldb.biotech.uni.wroc.pl/. InterMetalDB collects the interfacial binding sites with involvement of metal ions and clusters them on the basis of 50% sequence similarity and the nearest metal environment (5 Å radius). The data are available through the web interface where they can be queried, viewed, and downloaded. Complexity of the query depends on the user, because the questions in the query are connected with each other by a logical AND. InterMetalDB offers several useful options for filtering records including searching for structures by particular parameters such as structure resolution, structure description, and date of deposition. Records can be filtered by coordinated metal ion, number of bound amino acid residues, coordination sphere, and other features. InterMetalDB is regularly updated and will continue to be regularly updated with new content in the future. InterMetalDB is a useful tool for all researchers interested in metalloproteins, protein engineering, and metal-driven oligomerization.

Data mining of iron(II) and iron(III) bond-valence parameters, and their relevance for macromolecular crystallography.

The bond-valence model is a reliable way to validate assumed oxidation states based on structural data. It has successfully been employed for analyzing metal-binding sites in macromolecule structures. However, inconsistent results for heme-based structures suggest that some widely used bond-valence R0 parameters may need to be adjusted in certain cases. Given the large number of experimental crystal structures gathered since these initial parameters were determined and the similarity of binding sites in organic compounds and macromolecules, the Cambridge Structural Database (CSD) is a valuable resource for refining metal-organic bond-valence parameters. R0 bond-valence parameters for iron(II), iron(III) and other metals have been optimized based on an automated processing of all CSD crystal structures. Almost all R0 bond-valence parameters were reproduced, except for iron-nitrogen bonds, for which distinct R0 parameters were defined for two observed subpopulations, corresponding to low-spin and high-spin states, of iron in both oxidation states. The significance of this data-driven method for parameter discovery, and how the spin state affects the interpretation of heme-containing proteins and iron-binding sites in macromolecular structures, are discussed.

Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA, protein and lipid

Fluorescence techniques have drawn increasing attention because they provide crucial information about molecular interactions in protein-ligand systems beyond that obtained by other methods. The advantage of fluorescence spectroscopy stems from the fact that the majority of molecules in biological systems do not exhibit fluorescence, making fluorescent probes useful with high sensitivity. Also, the fluorescence emission is highly sensitive to the local environment, providing a valuable tool to investigate the nature of binding sites in macromolecules. In this review, we discuss some of the important applications of a class of molecules that have been used as fluorescent probes in a variety of studies. Hydroxyphenyl benzazoles (HBXs) show distinct spectroscopic features that make them suitable probes for the study of certain biological mechanisms in DNA, protein and lipid. In particular, the complex photophysics of 2-(2'-hydroxyphenyl)benzoxazole (HBO) and the distinguished fluorescence signatures of its different tautomeric forms make this molecule a useful probe in several applications. Among these are probing the DNA local environment, study of the flexibility and specificity of protein-binding sites, and detecting the heterogeneity and ionization ability of the head groups of different lipidic phases. The spectroscopy of HBX molecules and some of their chemically modified structures is also reviewed.

Solvent Effect on Anthranilic Acid Spectroscopy

The spectroscopy of anthranilic acid (AA) was examined in neat and binary solvents of varying polarity and hydrogen bonding strength in order to understand the role of water in solvating the polar sites of the molecule. With the exception of water, the Stokes shift of AA in different solvents was found to be linearly correlated with the normalized molar transition energy of solvent polarity (ETN), indicating the major role of the hydrogen bonding effect in solution. Analysis of the absorption and fluorescence spectra reveals that AA exists as an anion in neutral water. The pKa (4.50) and pKa* (4.44) values were estimated from the spectral shift in the absorption and fluorescence spectra measured in different pH solutions. The shortest fluorescence lifetime was measured in cyclohexane and is attributed to intramolecular hydrogen dislocation/transfer in the excited state. The lifetime values in polar solvents point to the dominant effect of the hydrogen-bond donating strength (α value) of the solvent. The number of water molecules solvating the polar region of the neutral form of AA was estimated to be three from the absorbance change in dioxane/buffer (pH 3.5) binary mixtures. The structures of AA:water complexes were calculated from density functional theory using the B3LYP method with a 6-311++G(2d,p) basis set. A stepwise addition of water molecules (1–3) to the polar region of AA leads to a preferential solvation of the COOH group of the molecule in a closed-cyclic geometry. It is worth noting that the spectral shift as a function of pH suggests the suitability of AA as a probe to estimate the local acidity of binding sites in macromolecules in the pH range 3.0–7.0.

Comment on “Probing the Thermodynamics of Competitive Ion Binding Using Minimum Energy Structures”

Comment on “Probing the Thermodynamics of Competitive Ion Binding Using Minimum Energy Structures”

Diffusion-influenced ligand binding to buried sites in macromolecules and transmembrane channels

We consider diffusion-influenced binding to a buried binding site that is connected to the surface by a narrow tunnel. Under the single assumption of an equilibrium distribution of ligands over the tunnel cross section, we reduce the calculation of the time-dependent rate coefficient to the solution of a one-dimensional diffusion equation with appropriate boundary conditions. We obtain a simple analytical expression for the steady-state rate that depends on the potential of mean force in the tunnel and the diffusion-controlled rate of binding to the tunnel entrance. Potential applications of our theory include substrate binding to a buried active site of an enzyme and permeant ion binding to an internal site in a transmembrane channel.

Novel polyaromatic ionomers with large hydrophilic domain and long hydrophobic chain targeting at highly proton conductive and stable membranes

A novel dihydroxyl monomer bearing 18 electron rich phenyl rings were synthesized and polymerized with other monomers bearing electron deficient phenyl rings to give dense and selective sites in macromolecules for post-sulfonation, which was successfully conducted in ClSO3H/CH2Cl2 solution at room temperature in a subsequential step. The chemical structures were confirmed by 1H NMR and FT-IR spectra. The ionic exchange capacity (IEC) was controlled to be from 0.65 to 1.21 mequiv g−1 to afford considerable proton conductivity. Distinct phase separation was observed in the resulting membranes from SAXS profiles. The SPAEK-5 with an IEC of 1.21 mequiv g−1 gave better proton conductivity than Nafion 117 at all tested temperatures under 100% relative humidity. The membranes exhibited an exceeding stability when immersing in Fenton's reagent (3 wt.% H2O2 + 2 ppm FeSO4) at 80 °C. These properties make them promising candidates for electrochemical applications.

EUDOC: a computer program for identification of drug interaction sites in macromolecules and drug leads from chemical databases.

The completion of the Human Genome Project, the growing effort on proteomics, and the Structural Genomics Initiative have recently intensified the attention being paid to reliable computer docking programs able to identify molecules that can affect the function of a macromolecule through molecular complexation. We report herein an automated computer docking program, EUDOC, for prediction of ligand-receptor complexes from 3D receptor structures, including metalloproteins, and for identification of a subset enriched in drug leads from chemical databases. This program was evaluated from the standpoints of force field and sampling issues using 154 experimentally determined ligand-receptor complexes and four "real-life" applications of the EUDOC program. The results provide evidence for the reliability and accuracy of the EUDOC program. In addition, key principles underlying molecular recognition, and the effects of structural water molecules in the active site and different atomic charge models on docking results are discussed. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1750-1771, 2001

Multiple‐site ligand binding to flexible macromolecules: Separation of global and local conformational change and an iterative mobile clustering approach

This article concerns the calculation of equilibria of ligand binding to multiple sites in macromolecules in the presence of conformational flexibility and conformation-dependent interaction among the sites. A formulation of this problem is presented in which global conformational changes are distinguished from conformational changes that are confined to “locally flexible regions.” The formalism is quite general in that ligands of different types, multivalent binding sites, tautomeric binding sites, and sites that bind more than one type of ligand can be accommodated. Strictly speaking, the separation of the conformational problem into global and local parts does not impose any loss of generality, although in practice it is necessary to restrict the number of global and local conformers. Because of the combinatorics of binding and conformational states, the computational complexity of a problem having only local conformational flexibility grows exponentially with the number of sites and the number of locally flexible regions. An iterative mobile clustering method for cutting off this exponential growth and obtaining approximate solutions with low computational cost is presented and tested. In this method, a binding site is selected, and a “cluster” of strongly interacting sites is set up around it; within the cluster, the binding and conformational states are fully enumerated, whereas the influences of sites outside the cluster on the sites inside are treated by a mean field approximation. The procedure then moves to the next site around which another (possibly overlapping) cluster is formed and the calculation is repeated. The procedure iterates through the list of sites in this way, using the results of previous iterations for the mean-field terms of current iterations until a convergence criterion is met. The method is tested on a large set of randomly generated problems of varying size, whose geometries are chosen to have protein-like statistical properties. It is found that the method is accurate and rapid with the computational cost scaling linearly to quadratically with the number of sites, except for a minority of cases in which large clusters occur by chance. The new method is more accurate than a Monte Carlo method, and may be faster or slower depending on the clustering criteria and details of the macromolecule. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 1091–1111, 1999

Multiple-site ligand binding to flexible macromolecules: Separation of global and local conformational change and an iterative mobile clustering approach

This article concerns the calculation of equilibria of ligand binding to multiple sites in macromolecules in the presence of conformational flexibility and conformation-dependent interaction among the sites. A formulation of this problem is presented in which global conformational changes are distinguished from conformational changes that are confined to “locally flexible regions.” The formalism is quite general in that ligands of different types, multivalent binding sites, tautomeric binding sites, and sites that bind more than one type of ligand can be accommodated. Strictly speaking, the separation of the conformational problem into global and local parts does not impose any loss of generality, although in practice it is necessary to restrict the number of global and local conformers. Because of the combinatorics of binding and conformational states, the computational complexity of a problem having only local conformational flexibility grows exponentially with the number of sites and the number of locally flexible regions. An iterative mobile clustering method for cutting off this exponential growth and obtaining approximate solutions with low computational cost is presented and tested. In this method, a binding site is selected, and a “cluster” of strongly interacting sites is set up around it; within the cluster, the binding and conformational states are fully enumerated, whereas the influences of sites outside the cluster on the sites inside are treated by a mean field approximation. The procedure then moves to the next site around which another (possibly overlapping) cluster is formed and the calculation is repeated. The procedure iterates through the list of sites in this way, using the results of previous iterations for the mean-field terms of current iterations until a convergence criterion is met. The method is tested on a large set of randomly generated problems of varying size, whose geometries are chosen to have protein-like statistical properties. It is found that the method is accurate and rapid with the computational cost scaling linearly to quadratically with the number of sites, except for a minority of cases in which large clusters occur by chance. The new method is more accurate than a Monte Carlo method, and may be faster or slower depending on the clustering criteria and details of the macromolecule. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 1091–1111, 1999

Nuclear probes in life sciences

The nuclear quadrupole interaction at metal sites in macromolecules monitored by time differential perturbed angular correlation provides information on the local environment of metal ions residing at these sites. This information is obtained in the ultra-trace regime and is, therefore, extremely valuable for research on reaction pathways of enzymatic heavy metal detoxification, for the development of radiopharmaceuticals, for investigations into protein architectures on surfaces, and for environmental research.

Some new developments and challenges in non-covalent molecular imprinting technology.

The technique of molecular imprinting allows the formation of specific recognition and catalytic sites in macromolecules via the use of templates. Molecularly imprinted polymers have been applied in an increasing number of applications where molecular binding events are of interest. These include the use of molecularly imprinted polymers as tailor-made separation materials, antibody and receptor binding site mimics in recognition and assay systems, enzyme mimics for catalytic applications and as recognition elements in biosensors. The stability and low cost of molecularly imprinted polymers make them advantageous for use in analysis as well as in industrial-scale production and application.

The Emerging Technique of Molecular Imprinting and Its Future Impact on Biotechnology

The technique of molecular imprinting allows the formation of specific recognition and catalytic sites in macromolecules by the use of templates. Molecularly imprinted polymers have been applied in an increasing number of applications where molecular binding events are of interest. These include (i) the use of molecularly imprinted polymers as tailor-made separation materials, (ii) antibody and receptor binding site mimics in recognition and assay systems, (iii) enzyme mimics for catalytic applications, and (iv) recognition elements in bio-sensors. The stability and low cost of molecularly imprinted polymers make them advantageous for use in analysis as well as in industrial scale production and application.

Monitoring exposure to acrylonitrile using adducts with N-terminal valine in hemoglobin.

Human exposure to acrylonitrile (ACN), a carcinogen in rats, may occur in industrial settings, through waste water and tobacco smoke. ACN is an electrophilic compound and binds covalently to nucleophilic sites in macromolecules. Measurements of adducts with hemoglobin could be utilized for improved exposure assessments. In this study, a method for quantification of N-(2-cyanoethyl)valine (CEVal), the product of reaction of ACN with N-terminal valine in hemoglobin has been developed. The method is based on the N-alkyl Edman procedure, which involves derivatization of the globin with pentafluorophenyl isothiocyanate and gas chromatographic-mass spectrometric analysis of the resulting thiohydantoin. An internal standard was prepared by reacting valylglycylglycine with [2H3]ACN, spiked with [14C]ACN to a known sp. act. Levels of CEVal were measured in globin from rats exposed to 3-300 p.p.m. ACN in drinking water for 105 days and from humans (four smokers and four non-smokers). CEVal was detected at all exposure levels in the drinking water study. The relationship between adduct level and water concentration was linear at concentrations of 10 p.p.m. (corresponding to an average daily uptake of c. 0.74 mg ACN/kg body wt during the 65 days prior to sacrifice) and below, with a slope of 37.7 pmol CEVal/g globin/p.p.m. At higher concentrations, adduct levels increased sublinearly, indicating saturation of a metabolic process for elimination of ACN. Comparison of adduct formation with the estimated dose (mg/kg/day) of ACN indicated that at low dose (0-10 p.p.m.) CEVal = 0.508 x ACN dose + 0.048 and at high dose (35-300 p.p.m.) CEVal = 1.142 x ACN dose - 1.098. Globin from the smokers (10-20 cigarettes/day) contained about 90 pmol CEVal/g, whereas the adduct levels in globin from non-smokers were below the detection limit. The analytical sensitivity should be sufficient to allow monitoring of occupationally exposed workers at levels well below the current Occupational Safety and Health Administration standard of 2 p.p.m.

Orientation factor in steady-state and time-resolved resonance energy transfer measurements.

Resonance energy transfer measurements provide a way to estimate distances between chromophores attached to different sites of macromolecules. There are two unknowns involved in resonance energy transfer measurements, the distance between two chromophores and their relative orientation. When static orientational disorder exists, the orientation factor, kappa 2, can vary from 0 to 4, leading to considerable uncertainty in estimation of distances. Fluorescence polarization anisotropy measurements can reduce the degree of uncertainty [Dale & Eisinger (1974) Biopolymers 13, 1573]. There may still be substantial error bounds for the average distance measurements. Time-resolved fluorescence measurements provide an "apparent" average distance and distance distribution containing contributions by both distance and orientation. The contribution of orientation to observed "apparent" average distance and distance distribution widths has been estimated for both simulated and real data. With a single unique distance as input in the simulation and with random but static orientation of donor and acceptor, the recovered average distance is very close to that of the input when the input distance is close to or larger than the Förster distance. The recovered width of apparent distance distribution can be substantial and it changes as a function of Förster distance to average distance ratio and as a function of Förster distance. Similar conclusions apply to the case where there is a real distance distribution. Motional averaging of the orientation was simulated by the Monte Carlo method to estimate the contribution of orientation when chromophores have certain degrees of mobility.(ABSTRACT TRUNCATED AT 250 WORDS)

Polarized X-ray emission spectroscopy

Strongly polarized X-ray fluorescence from gas-phase molecules has been observed by selectively exciting near core-level ionization thresholds using monochromatized synchrotron radiation. Both the degree and the direction of the polarization are very sensitive to the incident excitation energy, and the symmetry of the occupied and unoccupied molecular orbitals involved in the excitation/fluorescence process. Illustration of the phenomenon will be made using Cl K-edge excitation on the molecule methyl chloride, CH 3Cl. The possibility of extracting orientational, geometrical, and orbital-symmetry information directly from experiment will be discussed, and the importance of the rapid time scale of the X-ray fluorescence decay (≈ 10 fs), which effectively precludes disorientation effects, will be stressed. Expectations are that polarized X-ray emission spectroscopy will enjoy wide applicability to, for example, solids, surfaces and interfaces, oriented molecules such as surface adsorbates, and active sites in macromolecules.

Fluorescence spectroscopy in biological and medical research

Fluorescence spectroscopy is one of the most sensitive and versatile tools in medical, biological and biochemical research. Here we discuss the use of (1) polarized fluorescence spectroscopy to study the conformational dynamics of proteins, (2) fluorescence recovery after photobleaching to study lateral mobility of proteins and lipids in biological cell membranes and (3) excitation energy transfer to measure distances between interesting sites in macromolecules and biological membranes.

ChemInform Abstract: 155Eu3+ as a Probe of Metal Ion and Cationic Drug Binding Sites on Native and Heat‐denatured DNA

Abstract A flow-dialysis apparatus suitable for the study of high affinity metal ion binding sites in macromolecules has been utilized to study 155Eu3+ exchange processes, as a function of pH, in both ‘native’ and ‘heat-denatured’ DNA. ‘Free exchange’ of 155Eu3+ was found to occur at a significantly faster rate at pH = 7.0 than at pH = 6.0 for both forms of DNA; while non-radioactive Eu3+-induced ‘displacement’ of bound 155Eu3+ occurred at a significantly faster rate at pH = 6.0 than at pH = 7.0 for both species of DNA. These results are consistent with a greater ‘entropic’ driving force for metal ion:DNA complexation at the lower pH value. The effect of ethidium bromide on 155Eu3+ exchange was also examined as a function of pH. The intercalating agent was found to accelerate 155Eu3+ displacement at pH = 6.0 and decelerate displacement at pH = 7.0. All three sets of experiments (i.e., free- exchange of bound 155Eu3+, Eu3+-induced displacement of bound 155Eu3+ and ethidium ion-induced displacement of bound 155Eu3+) indicate that the 155Eu3+ ion can serve as a useful probe of metal ion and drug binding sites in nucleic acid polymers, and constitutes a particularly sensitive probe at pH = 6.0.

155Eu 3+ as a probe of metal ion and cationic drug binding sites on native and heat-denatured DNA

A flow-dialysis apparatus suitable for the study of high affinity metal ion binding sites in macromolecules has been utilized to study 155Eu 3+ exchange processes, as a function of pH, in both ‘native’ and ‘heat-denatured’ DNA. ‘Free exchange’ of 155Eu 3+ was found to occur at a significantly faster rate at pH = 7.0 than at pH = 6.0 for both forms of DNA; while non-radioactive Eu 3+-induced ‘displacement’ of bound 155Eu 3+ occurred at a significantly faster rate at pH = 6.0 than at pH = 7.0 for both species of DNA. These results are consistent with a greater ‘entropic’ driving force for metal ion:DNA complexation at the lower pH value. The effect of ethidium bromide on 155Eu 3+ exchange was also examined as a function of pH. The intercalating agent was found to accelerate 155Eu 3+ displacement at pH = 6.0 and decelerate displacement at pH = 7.0. All three sets of experiments ( i.e., free- exchange of bound 155Eu 3+, Eu 3+-induced displacement of bound 155Eu 3+ and ethidium ion-induced displacement of bound 155Eu 3+) indicate that the 155Eu 3+ ion can serve as a useful probe of metal ion and drug binding sites in nucleic acid polymers, and constitutes a particularly sensitive probe at pH = 6.0.

Remarks on the determination of the fraction of excimer sites in macromolecules

A mathematical model taking into consideration excitation energy migration has been proposed in order to determine the fraction of excimer sites (FES) in synthetic macromolecules. Data for polystyrene and iso- and atactic poly(ar-methyl styrene0 of varying molecular mass have been taken from the work of Ishii, Handa and Matsunaga and re-worked in terms of the proposed model. The FES obtained are compared with those determined from other models.