Single-site Model Research Articles

Probing the mechanism of drug/lipid membrane interactions using Biacore

Assay conditions were established to screen a panel of drugs for binding to liposome surfaces using a surface plasmon resonance (SPR) biosensor. Drugs were found to bind negligibly or reversibly or were retained on the liposome surface. Cationic amphiphilic drugs fell into the last class and correlated with drugs that induce phospholipidosis in vivo. To a first approximation, a single-site model yielded apparent binding affinities that adequately described a drug’s dose-dependent binding to liposome surfaces. Affinities ranged at least 1000-fold within the drug panel. A liposome’s drug-binding capacity and affinity depended on both the lipid headgroup and the drug’s structure. Although a drug’s charge state generally dominated whether or not it remained bound to the liposome, subtle structural differences between members of certain drug families led to them having widely differing binding affinities. A comparison between the dissociation of drugs from liposome surfaces by Biacore and the lipid retention measurements determined by a parallel artificial membrane permeability assay was drawn. The results from this study demonstrate the potential of using SPR-based assays to characterize drug/liposome-binding interactions.

Crystal structure of the quorum-sensing protein TraM and its interaction with the transcriptional regulator TraR.

Transfer of the tumor-inducing plasmid in Agrobacterium tumefaciens is controlled by a quorum-sensing system whose main components are the transcriptional regulator TraR and its autoinducer. This system allows bacteria to synchronize infection of the host plant when a "quorum" of cells has been reached. TraM is an A. tumefaciens protein involved in the regulation of this system because it binds to TraR and prevents it from binding DNA. As a first step to understanding the molecular basis for the regulation of TraR by TraM, we have determined the crystal structure of TraM at 1.65 A resolution. This protein is packed as a dimer, with each monomer consisting mainly of two antiparallel alpha helices. Monomers are tightly associated, with a large hydrophobic area buried upon dimerization. Secondly, we characterized the TraR-TraM complex in vitro. TraM (11.4 kDa, monomer molecular mass) binds tightly TraR (27 kDa, monomer molecular mass) forming a stable oligomeric complex that likely accounts for two TraR and two TraM dimers.

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

The Wilson disease protein (WND) is a transport ATPase involved in copper delivery to the secretory pathway. Mutations in WND and its homolog, the Menkes protein, lead to genetic disorders of copper metabolism. The WND and Menkes proteins are distinguished from other P-type ATPases by the presence of six soluble N-terminal metal-binding domains containing a conserved CXXC metal-binding motif. The exact roles of these domains are not well established, but possible functions include exchanging copper with the metallochaperone Atox1 and mediating copper-responsive cellular relocalization. Although all six domains can bind copper, genetic and biochemical studies indicate that the domains are not functionally equivalent. One way the domains could be tuned to perform different functions is by having different affinities for Cu(I). We have used isothermal titration calorimetry to measure the association constant (K(a)) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to their metallochaperone Atox1. The association constants for both the chaperone and target domains are approximately 10(5) to 10(6) m(-1), suggesting that the handling of copper by Atox1 and copper transfer between Atox1 and WND are under kinetic rather than thermodynamic control. Although some differences in both n and K(a) values are observed for variant proteins containing less than the full complement of six metal-binding domains, the data for domains 1-6 were best fitted with a single site model. Thus, the individual functions of the six WND metal-binding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties.

Modeling benzene with single-site potentials from ab initio calculations: a step toward hybrid models of complex molecules.

Extensive ab initio calculations at the MP2/6-31G* level have been carried out to sample the energy surface for the interactions of the benzene dimers. This database has been used to parameterize two anisotropic single-site models, meant to be used as building blocks in hybrid models of complex, liquid crystal forming molecules. A quadrupolar Gay-Berne (GBQIII) and an S-function (SF) Corner potentials have been obtained in this way. Their ability to reproduce, qualitatively at least, the phase diagram as well as energetic and structural properties of benzene has been tested with Monte Carlo simulations and compared with previous literature potentials, GBQI [S. Gupta et al., Mol. Phys. 65, 961 (1988)] and GBQII [T. R. Walsh, Mol. Phys. 100, 2867 (2002)]. It turned out that GBQI showed no melting transition in the temperature range explored (100-400 K), while GBQII underwent a phase transition from solid to gas, with no liquid phase. Conversely, both models parameterized on our database of ab initio interaction energies (GBQIII and SF) gave rise to a stable liquid phase. Melting has been observed between 100 and 150 K (GBQIII) and in the range 300-350 K (SF), i.e., substantially below and slightly above the experimental value at ambient pressure, 278 K. The description of the crystal structure of benzene at atmospheric pressure is also in better agreement with experimental data if the SF model is used, while positional correlations in the liquid are better described by the GBQIII potential. The S-function potential is also computationally more convenient. These results could be useful in the semirealistic modeling of more complex molecules.

Multi-Site Performance Monitoring in Batch Pharmaceutical Production

Abstract A challenge facing the pharmaceutical and chemical industries is how to understand and identify differences in process behaviour where a product is manufactured at two different sites. Three approaches based on multi-group principal component analysis are investigated and benchmarked against single site models. The multi-group approach is shown to remove differences between sites such as operational scale thereby enabling the analysis to focus on identifying differences in variation between the two sites that are not a consequence of process configurations. From the analysis it is observed that the multi-group approach can assist in the understanding of manufacturing performance.

Cadmium Adsorption by Chlamydomonas reinhardtii and its Interaction with the Cell Wall Proteins

Environmental Context. In natural waters, trace metals levels are largely controlled by microbiology; organisms take up, metabolize, store, and detoxify the metals. However, aquatic organisms may regulate their own uptake via dynamic processes that result in a system that is far from equilibrium. By examining the model title alga with a battery of techniques, a more realistic assessment of metal uptake and metal regulatory processes could be gained. Abstract. Cadmium adsorption by a wild type strain of Chlamydomonas reinhardtii and a cell wall-less mutant was quantified as a function of Cd speciation in a well-defined aqueous medium. For both strains, Cd adsorption to the cell surface was not predicted by a single-site (Langmuirian) model. Indeed, no saturation of the cell wall was observed, even for Cd concentrations in excess of 5 × 10−3 M. A continual production of Cd binding sites appeared to be responsible for the observed increase of Cd adsorption with time. SDS-page separations and measurements of the protein content of algal supernatants demonstrated that organic matter was released by the algae, both in the presence and absence of Cd. Both the nature (e.g. polysaccharides, proteins) and the quantity of exudate production was influenced by the physicochemistry of the external medium. Measurements using the permeation liquid membrane (PLM) and anodic stripping voltammetry (ASV) demonstrated that dissolved cadmium was rapidly complexed by the organic exudates produced by the algae.

Imaging Dopamine Receptors in the Rat Striatum with the MicroPET R4: Kinetic Analysis of [11C]Raclopride Binding Using Graphical Methods

Publisher Summary This chapter describes the binding potential computed for 15 [11C]raclopride studies in the rat striatum using the micro-PET R4. In two of these studies, a 1-mg/kg blocking dose of raclopride was coadministered with the tracer to measure nonspecific binding of the tracer. The remaining studies were carried out with varying doses of [11C]raclopride of varying specific activity. Data are summarized as the binding potential as a function of raclopride mass injected. The curve is the best fit to a single-site ligand receptor model allowing for fitting of the modified equilibrium constants. Image data were corrected for spillover from the intraorbital lachrymal glands but were not corrected for partial volume averaging. The microPET R4, with a FWHM resolution is expected to give a truer measure of striatal [11C]raclopride concentration, and therefore, gives a larger binding potential. Determinations of binding potential using the graphical method were compared with the values obtained using a compartmental model without blood sampling. The results support the use of the noninvasive graphical analysis methodology with [11C]raclopride, and the microPET R4 to measure dopamine receptor availability in the rat striatum.

Fibronectin interactions with osteoblasts: identification of a non-integrin-mediated binding mechanism using a real-time fluorescence binding assay.

Fibronectin (Fn) is an extracellular matrix protein that interacts with specific integrins on the cell surface, initiating signal transduction processes that lead to a reorganization of the cytoskeleton and the assembly of focal adhesions. Cell surface proteoglycans or glycosaminoglycans (GAGs) such as heparan sulfate are also known to participate in the interaction of Fn with the cell surface by binding to two different heparin-binding domains. The influence of Fn and GAGs on the spreading and differentiation of human osteoblasts was also previously described. In the current work, a method developed in our laboratory is established to evaluate the interaction between Fn and human osteoblasts and the influence of GAGs on such interactions. This technique makes use of fluoresceinphosphatidylethanolamine (FPE) such that when inserted into the lipidic bilayer, it acts as a fluorescent indicator of membrane interactions. The results indicate that the binding profile of Fn with the osteoblast cell surface is best represented by a hyperbolic single binding site model with a membrane affinity of 120 nM. Removal of cell surface heparan sulfate by treatment with heparitinase indicates that the cell surface moiety is directly involved in the binding process. Studies directed to assess the influence of heparin on the interaction of Fn with osteoblasts reveal that although it does not hamper Fn binding to the cell surface, it blocks the initial attachment to Fn-coated surfaces, indicating that binding to the integrin receptor alone is not enough to promote cell attachment but that the participation of the cell-surface GAGs is also a necessary condition.

The effect of pH, ion strength and reactant content on the complexation of Cu 2+ by various natural organic ligands from water and soil in Hong Kong

The complexation constants for copper associated with different natural organic ligands, including dissolved organic carbon (DOC) extracted from water, water soluble organic carbon (WSOC), fulvic acid (FA) and humic acid (HA) from soil, were determined and then compared based on discrete single site model. Both ion-selective electrode (ISE) and anodic stripping voltammetry were used to determine the content of free copper ions, while the relative number of complexation sites was estimated using a fluorescence quenching (FQ) at the same time. ISE proved to be the most applicable technique when the concentration of copper was above 10 −7 mol l −1. The log k values for two WSOC sample sites (Song Ziyuan and Xin Niangtan) were 4.64 and 4.66; higher than both the DOC and HA values yet lower than the FA values, which were unusually high due to unavoidable pollution from the cation exchange resin used during the purification process. Binding affinities between the copper ions and the organic ligands obtained from streams in Yong Wei (DOC) and Song Ziyuan (WSOC) were influenced by pH, ion strength, and reactant concentration. Values for log k increased with increases in pH (ion strength of 0.1 N). At pH 4, the log k values decreased with increases in the supporting electrolyte concentration and total copper ion additions.

Model selection for local and regional meteorological normalisation of background concentrations of tropospheric ozone

Meteorological normalisation of time series of air quality data aims to extract anthropogenic signals by removing natural fluctuations in the collected data. We showed that the currently used procedures to select normalisation models can cause over-fitting to observed data and undesirable smoothing of anthropogenic signals. A simulation study revealed that the risk of such effects is particularly large when: (i) the observed data are serially correlated, (ii) the normalisation model is selected by leave-one-out cross-validation, and (iii) complex models, such as artificial neural networks, are fitted to data. When the size of the test sets used in the cross-validation was increased, and only moderately complex linear models were fitted to data, the over-fitting was less pronounced. An empirical study of the predictive ability of different normalisation models for tropospheric ozone in Finland confirmed the importance of using appropriate model selection strategies. Moderately complex regional models involving contemporaneous meteorological data from a network of stations were found to be superior to single-site models as well as more complex regional models involving both contemporaneous and time-lagged meteorological data from a network of stations.

Insights into ssDNA recognition by the OB fold from a structural and thermodynamic study of Sulfolobus SSB protein.

Information processing pathways such as DNA replication are conserved in eukaryotes and archaea and are significantly different from those found in bacteria. Single-stranded DNA-binding (SSB) proteins (or replication protein A, RPA, in eukaryotes) play a central role in many of these pathways. However, whilst euryarchaea have a eukaryotic-type RPA homologue, crenarchaeal SSB proteins appear much more similar to the bacterial proteins, with a single OB fold for DNA binding and a flexible C-terminal tail that is implicated in protein-protein interactions. We have determined the crystal structure of the SSB protein from the crenarchaeote Sulfolobus solfataricus to 1.26 A. The structure shows a striking and unexpected similarity to the DNA-binding domains of human RPA, providing confirmation of the close relationship between archaea and eukaryotes. The high resolution of the structure, together with thermodynamic and mutational studies of DNA binding, allow us to propose a molecular basis for DNA binding and define the features required for eukaryotic and archaeal OB folds.

RNA-based phylogenetic methods: application to mammalian mitochondrial RNA sequences

The PHASE software package allows phylogenetic tree construction with a number of evolutionary models designed specifically for use with RNA sequences that have conserved secondary structure. Evolution in the paired regions of RNAs occurs via compensatory substitutions, hence changes on either side of a pair are correlated. Accounting for this correlation is important for phylogenetic inference because it affects the likelihood calculation. In the present study we use the complete set of tRNA and rRNA sequences from 69 complete mammalian mitochondrial genomes. The likelihood calculation uses two evolutionary models simultaneously for different parts of the sequence: a paired-site model for the paired sites and a single-site model for the unpaired sites. We use Bayesian phylogenetic methods and a Markov chain Monte Carlo algorithm is used to obtain the most probable trees and posterior probabilities of clades. The results are well resolved for almost all the important branches on the mammalian tree. They support the arrangement of mammalian orders within the four supra-ordinal clades that have been identified by studies of much larger data sets mainly comprising nuclear genes. Groups such as the hedgehogs and the murid rodents, which have been problematic in previous studies with mitochondrial proteins, appear in their expected position with the other members of their order. Our choice of genes and evolutionary model appears to be more reliable and less subject to biases caused by variation in base composition than previous studies with mitochondrial genomes.

Combining density-functional calculations with kinetic models: NO/Rh(111)

We present a dynamic Monte-Carlo model involving lateral interactions and different adsorption sites (top, fcc and hcp). Using this model in combination with kinetic parameters from UHV experiments and lateral interactions derived from DFT calculations we have reproduced the ordering behavior of NO on Rh(111) during adsorption and the temperature programmed desorption (TPD) of NO from Rh(111) under UHV conditions. The formation of c(4×2)-2NO domains at 0.50 ML coverage is shown to depend strongly on the next-next-nearest-neighbor repulsion between the NO adsorbates in our model. The formation of the (2×2)-3NO structure at higher coverage follows from the avoidance of the strong next-nearest-neighbor repulsion in favor of the occupation of the top sites. A single-site model was able to reproduce the experimental TPD, but the lateral interactions were at odds with the values of the DFT calculations. A three-site model resolved this problem. It was found that all NO dissociates during TPD for initial coverages of NO below 0.20 ML. The nitrogen atoms recombine at higher temperatures. For NO coverages larger than 0.20 ML, 0.20 ML NO dissociates while the rest desorbs. This is due to a lack of accessible sites on the surface, i.e., sites where a molecule can bind without experiencing large repulsions with neighboring adsorbates. For NO coverages above 0.20 ML, the dissociation of NO causes a segregation into separate NO and N+O islands. The dissociation causes the surface to be filled with adsorbates, and the adsorbates are therefore pushed closer together. NO on one hand can easily be compressed into islands of 0.50 ML coverage, because there is no large next-next-nearest-neighbor repulsion. N+O on the other hand form islands with a lower coverage (0.30–0.35 ML) due to the considerable next-next-nearest-neighbor repulsion. Top bound NO (above 0.50 ML initial coverage) does not dissociate during TPD. It desorbs in a separate peak at 380 K.

The PrP-like Protein Doppel Binds Copper

Doppel (Dpl) is a glycosylphosphatidylinositol-anchored protein expressed in the testis. It exhibits 26% sequence identity with the prion protein (PrP) but lacks the octarepeat region implicated as the major copper-binding domain. Contrary to expectations, Cu(II) induced a 26% reduction in the intrinsic fluorescence of Dpl(27-154) and a calculated K(d) for a single-site model of 0.16 +/- 0.08 microm. Other metals had minimal effects on fluorescence quenching. Matrix-assisted laser desorption ionization mass spectrometry of a Dpl peptide revealed binding of copper (but not other metals) to the helical alphaB/B'-loop-alphaC subregion of Dpl. Fluorescence quenching and equilibrium dialysis analyses of this Dpl(101-145) peptide were compatible with a binding site of K(d) = 0.4 microm. Diethylpyrocarbonate footprinting (Qin, K., Yang, Y., Mastrangelo, P., and Westaway, D. (2002) J. Biol. Chem. 277, 1981-1990) of Dpl(27-154) defined one residue/molecule was protected by copper from diethylpyrocarbonate adduct formation, and reiteration of this analysis with Dpl(101-145) suggested that His(131) may contribute to Cu(II) binding. Taken together, our data indicate that the alpha-helical region of mouse Dpl possesses a selective copper-binding site with a submicromolar K(d) and perhaps one or more lower affinity sites. Although metallated forms of Dpl might exist in vivo, analyses of Tg(Dpl)10329 mice were inconsistent with reports that Dpl expression is associated with increased carbonylation and nitrosylation of brain proteins. Thus, rather than comprising an important source of free radical damage, copper binding may serve to modulate the activity, stability, or localization of the Dpl protein.

Temporal correlation in recreation demand models with limited data

Typical single-site recreation demand models assume the decision to take a recreation trip on a particular choice occasion is independent of all past trip decisions. This paper develops a model of recreation behavior that allows for temporal correlation between choice occasions, but can be estimated on standard recreation demand data (i.e. seasonal summaries of behavior). Nested within the temporally correlated model is the Poisson count data model often employed in single-site recreation demand studies.

Modelling extreme rainfall data within a catchment region

AbstractThe present work examines the extreme behaviour of monthly rainfall in the Reno River catchment area (Italy). The data are recorded by a network of monitoring sites for which a single model, valid for the whole region, can be built. As a first step, a generalized extreme value (GEV) model is fitted to monthly rainfall maxima for each site. As a further step, we have introduced a second level into the model whereby the GEV site‐specific parameters depend on covariates by means of hyperparameters common to the area. The maximum likelihood method is used for the purposes of estimation. We employed an extra‐sample set of monitoring sites in order to compare the results obtained using the catchment model with the single‐site models estimated by classical one‐level extreme value analysis. Copyright © 2003 John Wiley & Sons, Ltd.

Surface Site Densities of Uranium Oxides: UO2, U3O8

ABSTRACTThe estimation of the surface site density for UO2 (main component of the SF matrix) is an important aspect to take into account in the development of radiolytical models for the dissolution of the spent nuclear fuel (SF). Also, other oxides can be formed on the SF surface due to the effect of radiolytically-formed oxidizing species. Due to the lack or reliable data in literature we have studied the surface site densities of two uranium oxides: UO2 and U3O8.For this determination, the knowledge of both the reactive surface area and the surface charge of the solid are necessary. In this work the reactive surface area of the two solids was determined by the BET method while the surface charge was determined by potentiometric acid-base titrations of suspensions of the solids at different ionic strengths (0.001, 0.01, and 0.1 mol·dm−3) under N2 atmosphere.The amount of adsorbed protons was calculated by subtracting blank titration from thesolid suspension titration. The single-site nonelectrostatic model (NEM) was used to describe titration data. Uranium speciation in solution was included in the model as well.The surface area values obtained were 0.15 ± 0.01 m2·g−1 for UO2 and 0.77 ± 0.02 m2·g−1 for U3O8, while the surface acidic site densities were determined to be 165 ± 10 sites·nm−2 and 48 ± 3 sites·nm−2 for UO2 and U3O8, respectively.

Thermodynamical Study on the Heavy-Fermion Superconductor PrOs4Sb12: Evidence for Field-Induced Phase Transition

We report measurements of low-temperature specific heat on the 4f^2-based heavy-fermion superconductor PrOs4Sb12. In magnetic fields above 4.5 T in the normal state, distinct anomalies are found which demonstrate the existence of a field-induced ordered phase (FIOP). The Pr nuclear specific heat indicates an enhancement of the 4f magnetic moment in the FIOP. Utilizing a Maxwell relation, we conclude that anomalous entropy, which is expected for a single-site quadrupole Kondo model, is not concealed below 0.16 K in zero field. We also discuss two possible interpretations of the Schottky-like anomaly at ~3 K, i.e., a crystalline-field excitation or a hybridization gap formation.

A Single-Site Model for Divalent Transition and Heavy Metal Adsorption over a Range of Metal Concentrations

Metal adsorption data over a range of surface coverages typically are characterized by curvilinear metal adsorption isotherms. These isotherms generally have a slope of 1 at low surface coverage and a shallower slope at higher surface coverages. The curvature of metal adsorption isotherms with increasing surface coverage is frequently interpreted in terms of sequential adsorption onto different types of surface sites, multinuclear surface complexation, or nonideality of metal adsorption. We demonstrate that the curvature of metal adsorption isotherms can also be attributed to changes in surface charge and potential that depend on the predominant type of metal surface complex. A single-site extended triple-layer model is used to reinterpret previously studied metal adsorption isotherms and pH edges for a wide variety of metals (Cd 2+, Co 2+, Cu 2+, Pb 2+, and Zn 2+) and solids (goethite, hydrous ferric oxide, corundum, and magnetite) in different electrolyte solutions (NaNO 3 and NaClO 4). Only metal adsorption on ferrihydrite at very low surface coverages is not consistent with the single-site triple-layer model. This discrepancy might be explained if ferrihydrite is in fact not a single phase but a mixture of two or more phases. Metal surface coverages ranging from 10 −4 to 10.2 mmol/m 2 on the other minerals can be accounted for with a single-site extended triple-layer model if appropriate metal adsorption reactions are chosen. In addition, several examples suggest that, within the context of the model, surface complexation schemes can be established that describe metal adsorption over both a wide range of surface coverage and a wide range of ionic strength.

A space‐time Neyman‐Scott model of rainfall: Empirical analysis of extremes

A spatial‐temporal model of rainfall, based on a Neyman‐Scott stochastic point process, is fitted to hourly data taken from nine sites in the Arno Basin, Italy. The stochastic model is an extension of the temporal Neyman‐Scott rectangular pulses model into two‐dimensional space and introduces a further parameter into the model. In the model, storms arrive in a Poisson process, where each storm consists of discs representing rain cells, with centers distributed over an area according to a spatial Poisson process. The cells have a random radius, lifetime, and intensity, with the intensity remaining constant over the area of the disc and cell lifetime. A fitting procedure is proposed which couples the results obtained in two preceding papers: the second‐order properties of the spatial‐temporal model and the third moment function of the single site model [Cowpertwait, 1995, 1998]. The model is validated by comparing extreme historical hourly data and equivalent data simulated using the fitted spatial‐temporal model. These comparisons are made using a regional frequency analysis, based on L moments, and log‐log plots of the upper distribution tail. The results indicate that the model is able to preserve regional extremes and support the use of the model in hydrological applications.