Hinge-bending motions in annexins: molecular dynamics and essential dynamics of apo-annexin V and of calcium bound annexin V and I

Annexins are homologous proteins that bind to membranes in a calciumdependent manner, but for which precise physiological roles have yet to bedefined. Most annexins are composed of a planar array of four homologousrepeats, each containing five alpha-helices and associated into twomodules. Annexin V forms a voltage-gated calcium channel in phospholipidbilayers. It has been proposed that the hydrophilic pore in the centre ofthe molecule may represent the ion conduction pathway and that a hingemovement in annexin V causes a variation of the inter- module angle andopens the calcium ion path. Here we present the results of moleculardynamics simulations of apo-annexin V and of calcium-bound annexin V andannexin I. The three simulations show significant differences inconformation and dynamics. The essential dynamics method was used to studythe essential subspace of annexin V and showed that one of the essentialmotions corresponds to the postulated hinge motion. The hinge residues werelocated between repeats but belong to helices rather than to the linksbetween helices. Calcium binding to annexin V led to a limitation of thishinge motion with more open conformations being favoured.     

Computational fluid dynamics simulation of virtual impactor performance: Comparison to experiment

Computational fluid dynamics (CFD) simulations were performed on a slit virtual impactor (SVI). A full-section planar model of the critical zone (nozzle) was constructed based on the measured values of geometrical dimensions of the SVI. Simulation was performed for a flow rate of 20.33 SCFH (standard cubic feet per hour) that corresponded to a Reynolds number value of 448 in the experiment. Impactor efficiency curve was computed and compared to experimental data. Details of internal wall losses were characterized and the wall loss curve was generated. Performance characteristics obtained from simulation results are in good agreement to the observed experimental trend over the complete range of Stokes numbers and the exact value of the cut-point Stokes number. Tracks for lower Stokes number particles faithfully reproduce the trend observed in the experiment, namely the regional demarcation of particle origin in the inlet nozzle, based on their final destination. Further, the effect of crossing trajectories that was visualized in the experiment for higher Stokes number particles of value of ~ 50 was captured. Additional details from the simulation results indicated that the onset of effect of crossing trajectories occurred for particles larger than a Stokes number of 6 and the collection efficiency for the above device was nearly unity over a wide range of Stokes number (Stk ~ 2 to 125) values.     

Hydrophobic charge-induction chromatographic resin with 5-aminobenzimidazol ligand: Effects of ligand density on protein adsorption

Hydrophobic charge-induction chromatography (HCIC) is a highly promising technology for antibody separation. HCIC resins ABI-B-6FF were prepared with 5-aminobenzimidazole (ABI) as the functional ligand. The effects of ligand density on the adsorption of human immunoglobulin G (hIgG) and bovine serum albumin were focused. It was found that the adsorption capacity and dynamic binding capacity (DBC) were improved with the increase of ligand density. The adsorption capacity and DBC of hIgG reached 128.07 mg/g gel and 67.63 mg/g gel. The results indicated that ABI-B-6FF resin has a promising potential for the application of antibody purification.     

Rapid and easy development of versatile tools to study protein/ligand interactions

The system described here allows the expression of protein fragments into a solvent-exposed loop of a carrier protein, the beta-lactamase BlaP. When using Escherichia coli constitutive expression vectors, a positive selection of antibioresistant bacteria expressing functional hybrid beta-lactamases is achieved in the presence of beta-lactams making further screening of correctly folded and secreted hybrid beta-lactamases easier. Protease-specific recognition sites have been engineered on both sides of the beta-lactamase permissive loop in order to cleave off the exogenous protein fragment from the carrier protein by an original two-step procedure. According to our data, this approach constitutes a suitable alternative for production of difficult to express protein domains. This work demonstrates that the use of BlaP as a carrier protein does not alter the biochemical activity and the native disulphide bridge formation of the inserted chitin binding domain of the human macrophage chitotriosidase. We also report that the beta-lactamase activity of the hybrid protein can be used to monitor interactions between the inserted protein fragments and its ligands and to screen neutralizing molecules.     

Dynameomics: mass annotation of protein dynamics and unfolding in water by high-throughput atomistic molecular dynamics simulations

The goal of Dynameomics is to perform atomistic molecular dynamics (MD) simulations of representative proteins from all known folds in explicit water in their native state and along their thermal unfolding pathways. Here we present 188-fold representatives and their native state simulations and analyses. These 188 targets represent 67% of all the structures in the Protein Data Bank. The behavior of several specific targets is highlighted to illustrate general properties in the full dataset and to demonstrate the role of MD in understanding protein function and stability. As an example of what can be learned from mining the Dynameomics database, we identified a protein fold with heightened localized dynamics. In one member of this fold family, the motion affects the exposure of its phosphorylation site and acts as an entropy sink to offset another portion of the protein that is relatively immobile in order to present a consistent interface for protein docking. In another member of this family, a polymorphism in the highly mobile region leads to a host of disease phenotypes. We have constructed a web site to provide access to a novel hybrid relational/multidimensional database (described in the succeeding two papers) to view and interrogate simulations of the top 30 targets: http://www.dynameomics.org. The Dynameomics database, currently the largest collection of protein simulations and protein structures in the world, should also be useful for determining the rules governing protein folding and kinetic stability, which should aid in deciphering genomic information and for protein engineering and design.     

FK506-binding protein mutational analysis: defining the active-site residue contributions to catalysis and the stability of ligand complexes

The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolylisomerase that binds the macrolides FK506 and rapamycin. Wehave examined the role of the binding pocket residues of FKBP12in protein–ligand interactions by making conservativesubstitutions of 12 of these residues by site-directed mutagenesis.For each mutant FKBP12, we measured the affinity for FK506 andrapamycin and the catalytic efficiency in the cis–transpeptidyl-prolyl isomerase reaction. The mutation of Trp59 orPhe99 generates an FKBP12 with a significantly lower affinityfor FK506 than wild-type protein. Tyr26 and Tyr82 mutants areenzymatically active, demonstrating that hydrogen bonding bythese residues is not required for catalysis of the cis–transpeptidyl-prolyl isomerase reaction, although these mutationsalter the substrate specificity of the enzyme. We conclude thathydrophobic interactions in the active site dominate in thestabilization of FKBP12 binding to macrolide ligands and tothe twisted-amide peptidyl-prolyl substrate intermediate.     

酶/酸水解毛叶木姜子中键合态香味成分的比较

采用AmberliteXAD-2树脂吸附洗脱分离毛叶木姜子中的键合态香气物质前体,将分离得到的键合态香气物质前体在AR2000酶、果胶酶和酸3种条件下分别进行水解释放,采用气相色谱-质谱联用法(GC-MS)对水解后的键合态香气物质进行分离分析。结果表明:3种水解条件得到的键合态香气物质共有90种,主要为萜烯类和含氧萜类物质。其中,香叶醇在2种酶水解条件下均含量高,果胶酶水解条件下含量最高达21798.79μg/L;共34种香气物质具有明显香气特征,2种酶水解下最显著的香气特征为花香和甜香;酸水解条件下最显著的香气特征为花香。酶水解与酸水解条件下得到的键合态香气物质存在明显差异,果胶酶水解条件下所检出的香气物质种类多且含量较高。因此,果胶酶处理更有利于毛叶木姜子键合态香气成分的释放。     

Comparison of proton field-cycling relaxometry and molecular dynamics simulations for proton-water surface dynamics in cement-based materials

Diffusion coefficients of water in hydrated cement pastes and mortars obtained from proton field cycling NMR spin lattice relaxation over three orders of magnitude in magnetic field strength are in good agreement with values from molecular dynamics simulations of water on the surface of tobermorite. The level of agreement from these two independent approaches provides mutual support for their validity.     

Integrated approach using protein and ligand information to analyze selectivity- and affinity-determining features of carbonic anhydrase isozymes

The application and comparison of selected protein- and ligand-based approaches to elucidate factors important for affinity and selectivity towards the carbonic anhydrase isozymes I, II, and IV are described. Carbonic anhydrases are abundant in pro- and eukaryotes. These enzymes catalyze the reversible hydration of carbon dioxide to bicarbonate and H(+) ions and are thus involved in many important physiological and pathophysiological processes. Due to the fact that the human carbonic anhydrase family consists of 16 closely related isozymes, the design of selective inhibitors is a special challenge for medicinal chemists. In order to extract selectivity-determining features, we applied purely ligand-based 3D QSAR techniques as well as qualitative comparative molecular field analyses of the targets' binding sites using consensus principal component analysis (CPCA). The dataset for the QSAR studies was deliberately compiled from 1,748 inhibitors and comprises about 140 ligands, mainly of the sulfonamide type. Additionally, we employed the novel AFMoC approach, which intrinsically combines protein and ligand information. The simultaneous use of these different techniques gives deeper insight into selectivity and affinity-determining features and provides quantitative models for prediction.     

Comparison of bound rubber and swelling in silicone rubber/silica mixes and in silicone rubber vulcanizates

Unextracted polymer and liquid absorption values have been determined on unvulcanized silicone rubber/silica mixes and on unfilled silicone rubber vulcanizates, for comparison of the two different types of three-dimensional structure which must exist. In both types, the amount of rubber extracted on immersion in liquid is dependent on the expansion of the respective network but only in the former is the amount dependent on the method of extraction; this is attributed to interaction between filler and rubber continuing as a result of immersion. Even at equilibrium, the amount of unextracted polymer can be greater than that of bound rubber. In some mixes, liquid absorption is substantially constant during extraction of considerable amounts of soluble rubber. This is attributed, in the presence of filler, to a dependence of the absorption on the previously postulated interparticular rubber which, in turn, is dependent on the mean distance between filler particles. Although unextracted polymer is also dependent on that mean distance, it is proposed that this is a secondary effect of network expansion by the absorbed liquid. As some published values of bound rubber have undoubtedly been of unextracted polymer, these results help to explain published differences of opinion on the value of bound rubber for investigation of filler reinforcement phenomena.     

Analysis of isothermal annular jets: Comparison of computational fluid dynamics and experimental data

A computational fluid dynamics technique was developed for the simulation of airflow through an annular jet. The technique used a commercial simulation package with a Reynolds stress model for the simulation of turbulent flows. The model parameters were calibrated using available experimental data for circular and annular jets. It was found that, after this calibration, the computational results agreed well with experimental data (specifically, with the velocity magnitude, velocity decay rate, and the velocity spreading rate). The jet geometry studied was based on industrial melt‐blowing nozzles. The velocities studied varied from the low subsonic incompressible range to nearly sonic conditions. Based on both the computational and experimental results, a correlation was proposed that predicts the centerline velocity profiles in both the near‐ and far‐field regions. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 94: 909–922, 2004     

Protein flexibility and ligand rigidity: a thermodynamic and kinetic study of ITAM-based ligand binding to Syk tandem SH2

The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI-MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the gamma chain of the FcepsilonRI-receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 degrees C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of approximately 9 kcal mol-1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI-MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two-step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal-transduction processes.     

Modeling of protein adsorption to DEAE sepharose FF: Comparison of data with model simulation

The equilibrium and kinetic characteristics of the adsorption of human serum albumin (HSA) and ovalbumin (OVA) to the DEAE Sepharose FF weak anion exchanger were experimentally determined. The rate for protein adsorption was simulated with two different models, the first being based on a single lumped kinetic parameter, while the second includes the individual mass transfer processes occurring prior to the adsorption intervention, i.e., diffusion across the liquid film surrounding individual particles and diffusion within the ion exchanger particle itself. The actual adsorption of OVA to DEAE Sepharose FF in fully mixed stirred vessels and in packed bed columns was consistent with both models. In the case of HSA, however, the adsorption profile in an agitated vessel was consistent only with the pore diffusion model and neither model could correctly predict the latter part of the breakthrough profile observed in packed bed experiments.     

Fluorescent dyes bound to hydrophilic copolymers: Applications in aqueous halide sensing

A range of halide‐sensitive fluorophores were bound to two hydrophilic copolymers. Thin films of the copolymers swelled in the aqueous media, allowing dye fluorescence to be dynamically quenched by the diffusion of halide ions. The resultant sensor films were characterized in terms of their hydrophilicity, sensitivity, and selectivity toward halide. The sensor films were reversibly capable of determining aqueous bromide and iodide at a mildly alkaline pH with typical 90% response times of 30–70 s and a shelf life in excess of 2 years. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 603–615, 2000     

A new combined computational and NMR-spectroscopical strategy for the identification of additional conformational constraints of the bound ligand in an aprotic solvent

This study documents the feasibility of switching to an aprotic medium in sugar receptor research. The solvent change offers additional insights into mechanistic details of receptor--carbohydrate ligand interactions. If a receptor retained binding capacity in an aprotic medium, solvent-exchangeable protons of the ligand would not undergo transfer and could act as additional sensors, thus improving the level of reliability in conformational analysis. To probe this possibility, we first focused on hevein, the smallest lectin found in nature. The NMR-spectroscopic measurements verified complexation, albeit with progressively reduced affinity by more than 1.5 orders of magnitude, in mixtures of up to 50% dimethyl sulfoxide (DMSO). Since hevein lacks the compact beta-strand arrangement of other sugar receptors, such a structural motif may confer enhanced resistance to solvent exchange. Two settings of solid-phase activity assays proved this assumption for three types of alpha- and/or beta-galactoside-binding proteins, that is, a human immunoglobulin G (IgG) subfraction, the mistletoe lectin, and a member of the galectin family of animal lectins. Computer-assisted calculations and NMR experiments also revealed no conspicuous impact of the solvent on the conformational properties of the tested ligands. To define all possible nuclear Overhauser effect (NOE) contacts in a certain conformation and to predict involvement of exchangeable protons, we established a new screening protocol applicable during a given molecular dynamics (MD) trajectory and calculated population densities of distinct contacts. Experimentally, transferred NOE (tr-NOE) experiments with IgG molecules and the disaccharide Gal'alpha1-3Galbeta1-R in DMSO as solvent disclosed that such an additional crosspeak, that is, Gal'OH2--GalOH4, was even detectable for the bound ligand under conditions in which spin diffusion effects are suppressed. Further measurements with the plant lectin and galectins confirmed line broadening of ligand signals and gave access to characteristic crosspeaks in the aprotic solvent and its mixtures with water. Our combined biochemical, computational, and NMR-spectroscopical strategy is expected to contribute notably to the precise elucidation of the geometry of ligands bound to compactly folded sugar receptors and of the role of water molecules in protein--ligand (carbohydrate) recognition, with relevance to areas beyond the glycosciences.     

重组H21G蛋白衍生物的制备及其免疫原性

目的分析重组H21G蛋白的化学修饰方法及化学修饰与免疫原性之间的相关性。方法应用琥珀酸酐和己二酸二酰肼(adipic acid dihydrazide,ADH)分别修饰重组H21G蛋白制备衍生物,赖氨酸单位减少率法测定重组H21G蛋白琥珀酰化物的琥珀酰化率;2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic acid,TNBS)法测定重组H21G蛋白衍生物的-AH衍生率;SDS-PAGE及HPLC法分析重组H21G蛋白各衍生物的纯度及分子量分布。用各衍生物分别经皮下免疫小鼠,免疫浓度为25μg/ml,共免疫3次,于末次免疫后2周,经小鼠眼眶采血,分离血清,采用间接ELISA法检测血清IgG含量。采用非还原型SDS-PAGE分析重组H21G蛋白衍生物在制备初期、制备后4℃储存2周及6个月的稳定性。结果随着重组H21G蛋白与琥珀酸酐质量比(10∶1、10∶3、10∶4和10∶5)的提高,琥珀酰化率也逐渐升高,但质量比为10∶4和10∶5时无明显差异。重组H21G蛋白ADH衍生1和2 h的衍生率无明显差异。质量比为10∶4及10∶5的琥珀酰化物与原蛋白分子量分布差异较大;ADH衍生后,原蛋白的分子量分布也发生改变。重组H21G蛋白与琥珀酸酐质量比高于10∶3时,对蛋白的降解作用明显,当质量比达到10∶5时,重组H21G蛋白几乎不再以单体形式存在;而ADH衍生1和2 h对蛋白聚合作用无明显差异,均产生了二聚体和多聚体。重组H21G蛋白及其衍生物免疫小鼠后表现明显的剂次加强效应,且ADH衍生的产物的免疫原性高于琥珀酰化产物。琥珀酰化产物的稳定性较差,目标蛋白均明显降解,而ADH衍生的产物在4℃储存6个月后未见明显聚合或降解。结论重组H21G蛋白经ADH衍生的产物免疫原性及稳定性均优于琥珀酰化产物。