Loose Binding Research Articles

Modified cellobiohydrolase–cellulose interactions following treatment with lytic polysaccharide monooxygenase CelS2 (ScLPMO10C) observed by QCM-D

The beneficial mechanisms of the lytic polysaccharide monooxygenase CelS2 (ScLPMO10C) from Streptomyces coelicolor on interactions between cellulose and the processive cellulase cellobiohydrolase I (Cel7A) were investigated by quartz crystal microbalance with dissipation. Initial binding of CelS2 to cellulose coated SiO2 sensors at 40 °C was rapid, but minimal, and was followed by modest positive changes in oscillation frequencies of the quartz crystal sensors that were attributed to mass loss from the cellulose surface. The presence of oxidized cellulose on the CelS2 treated sensors was verified by X-ray photoelectron spectroscopy. Subsequent binding of purified Cel7A from Trichoderma longibrachiatum at 23 °C was significantly less in overall extent to CelS2 treated sensors than to untreated controls despite identical initial binding rates and dissipation-change: frequency-change ratios (an indication of the rigidity of the newly forming layer). Moreover, initial Cel7A binding to the treated sensors was followed by positive overall frequency changes indicating potential increased hydrolytic removal of cellulose mass by cellobiohydrolase action at 23 °C. Furthermore, secondary binding of Cel7A to the control sensors was coupled with considerable changes in dissipation (indicating greater surface viscoelasticity) not observed during the initial binding phase; the extent of this secondary binding was observed to be negligible during Cel7A interaction with the CelS2 treated sensors. This drastic difference in more viscoelastic secondary binding suggests a reduction in loose non-productive cellobiohydrolase binding following CelS2 treatment.

Improvement of Engine Coolant Ultrasonic Cavitation Apparatus

The structure of cast iron specimen and the temperature control unit of the engine coolant ultrasonic cavitation apparatus were improved in this paper. The broken problem of cast iron specimen was solved by split-structure design, which also solved the loose binding problem between specimen and sonotrode. The temperature control unit included a beaker with water circulating jacket, water bath, circulating pump and connection hose. The temperature control unit overcame the difficulty of the temperature control of test coolant solution. More important, the temperature control inside the soundproof box was realized, which avoided the effect of ultrasonic vibration noise on the health of operator. The unit has the characteristics of simple, practical and easy to use.

Efficient run-time verification of web service composition

Service-oriented architecture SOA is an architectural solution that underlined loose coupling and dynamic binding between web services. SOA layers consist of client interface layer, web service composition WSC layer, web services layer, and web services-components layer. Web service composition realises SOA's promise of ease of development, high flexibility, reusability, and interoperability. However, current web service composition practice has a major drawback where the behaviour and the quality of web services consumed could deviate from what is anticipated, particularly during run-time. In this proposed work, we would like to develop a run-time verification system for web service composition to detect violation on system behaviour during run time. We highly emphasise on the efficiency attribute in order to develop a powerful verification system with fast response time.

Optic nerve sheath diameter measurement using bedside ultrasound: Is it accurate?

Optic nerve sheath diameter measurement using bedside ultrasound: Is it accurate?

Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where “nonspecific” interactions contribute to biological function.

Ultrafast water dynamics at the interface of the polymerase-DNA binding complex.

DNA polymerases slide on DNA during replication, and the interface must be mobile for various conformational changes. The role of lubricant interfacial water is not understood. In this report, we systematically characterize the water dynamics at the interface and in the active site of a tight binding polymerase (pol β) in its binary complex and ternary state using tryptophan as a local optical probe. Using femtosecond spectroscopy, we observed that upon DNA recognition the surface hydration water is significantly confined and becomes bound water at the interface, but the dynamics are still ultrafast and occur on the picosecond time scale. These interfacial water molecules are not trapped but are mobile in the heterogeneous binding nanospace. Combining our findings with our previous observation of ultrafast water motions at the interface of a loose binding polymerase (Dpo4), we conclude that the binding interface is dynamic and the water molecules in various binding clefts, channels, and caves are mobile and even fluid with different levels of mobility for loose or tight binding polymerases. Such a dynamic interface should be general to all DNA polymerase complexes to ensure the biological function of DNA synthesis.

New method for solution of coupled radial Schrödinger equations: application to the Borromean two-neutron halo nucleus 22C

A generalized Prufer transformation within the framework of the modified variable phase method has been used for numerical solution of coupled radial Schrodinger equations at negative energies. The method has been applied to calculations of the Borromean two-neutron halo nucleus C-22, for which an unusually large value of the matter radius has recently been extracted from measured reaction cross sections. The giant size can only be explained by an extremely loose binding that is, however, not yet known experimentally. Within the three-body cluster model we have explored the sensitivity of the C-22 matter and charge radii and soft dipole mode excitations to the two-neutron separation energy.

Dissociation Free-Energy Profiles of Specific and Non-Specific DNA-Lac Repressor Complexes: Adaptive Biasing Force Molecular Dynamics Study

DNA-binding proteins recognize DNA sequences with at least two different binding modes, specific and non-specific. Experimental structures of such complexes provide us a static view of the bindings. However, it is difficult to reveal further mechanisms of their target-site search and recognition only from static information because the transition process between the bound and unbound states is not clarified by static information. What is the difference between specific and non-specific bindings? Here we performed adaptive biasing force molecular dynamics simulations with the specific and non-specific structures of DNA-Lac repressor complexes to investigate the dissociation process. The resultant free-energy profiles showed that the specific complex has a sharp, deep well consistent with tight binding, whereas the non-specific complex has a broad, shallow well consistent with loose binding. The difference in the well depth, ∼5 kcal/mol, was in fair agreement with the experimentally obtained value and was found to mainly come from the protein conformational difference, particularly in the C-terminal tail. Also, the free-energy profiles were found to be correlated with changes in the number of protein-DNA contacts and that of surface water molecules. The derived protein spatial distributions around the DNA indicate that any large dissociation occurs rarely, regardless of the specific and non-specific sites. Comparison of the free-energy barrier for sliding [∼ 8.7 kcal/mol] and that for dissociation (at least ∼16 kcal/mol) calculated in this study suggests that sliding is much preferred to dissociation.

Loose Binding of the DF Axis with the A3B3 Complex Stimulates the Initial Activity of Enterococcus hirae V1-ATPase

Vacuolar ATPases (V-ATPases) function as proton pumps in various cellular membrane systems. The hydrophilic V1 portion of the V-ATPase is a rotary motor, in which a central-axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex by using ATP hydrolysis energy. We have previously reported crystal structures of Enterococcus hirae V-ATPase A3B3 and A3B3DF (V1) complexes; the result suggested that the DF axis induces structural changes in the A3B3 complex through extensive protein-protein interactions. In this study, we mutated 10 residues at the interface between A3B3 and DF complexes and examined the ATPase activities of the mutated V1 complexes as well as the binding affinities between the mutated A3B3 and DF complexes. Surprisingly, several V1 mutants showed higher initial ATPase activities than wild-type V1-ATPase, whereas these mutated A3B3 and DF complexes showed decreased binding affinities for each other. However, the high ATP hydrolysis activities of the mutants decreased faster over time than the activity of the wild-type V1 complex, suggesting that the mutants were unstable in the reaction because the mutant A3B3 and DF complexes bound each other more weakly. These findings suggest that strong interaction between the DF complex and A3B3 complex lowers ATPase activity, but also that the tight binding is responsible for the stable ATPase activity of the complex.

Dissociation Free-Energy Profiles of Specific and Nonspecific DNA–Protein Complexes

DNA-binding proteins recognize DNA sequences with at least two different binding modes: specific and nonspecific. Experimental structures of such complexes provide us a static view of the bindings. However, it is difficult to reveal further mechanisms of their target-site search and recognition only from static information because the transition process between the bound and unbound states is not clarified by static information. What is the difference between specific and nonspecific bindings? Here we performed adaptive biasing force molecular dynamics simulations with the specific and nonspecific structures of DNA-Lac repressor complexes to investigate the dissociation process. The resultant free-energy profiles showed that the specific complex has a sharp, deep well consistent with tight binding, whereas the nonspecific complex has a broad, shallow well consistent with loose binding. The difference in the well depth, ~5 kcal/mol, was in fair agreement with the experimentally obtained value and was found to mainly come from the protein conformational difference, particularly in the C-terminal tail. Also, the free-energy profiles were found to be correlated with changes in the number of protein-DNA contacts and that of surface water molecules. The derived protein spatial distributions around the DNA indicate that any large dissociation occurs rarely, regardless of the specific and nonspecific sites. Comparison of the free-energy barrier for sliding [~8.7 kcal/mol; Furini J. Phys. Chem. B 2010, 114, 2238] and that for dissociation (at least ~16 kcal/mol) calculated in this study suggests that sliding is much preferred to dissociation.

Energetic basis of catalytic activity of layered nanophase calcium manganese oxides for water oxidation

Previous measurements show that calcium manganese oxide nanoparticles are better water oxidation catalysts than binary manganese oxides (Mn3O4, Mn2O3, and MnO2). The probable reasons for such enhancement involve a combination of factors: The calcium manganese oxide materials have a layered structure with considerable thermodynamic stability and a high surface area, their low surface energy suggests relatively loose binding of H2O on the internal and external surfaces, and they possess mixed-valent manganese with internal oxidation enthalpy independent of the Mn(3+)/Mn(4+) ratio and much smaller in magnitude than the Mn2O3-MnO2 couple. These factors enhance catalytic ability by providing easy access for solutes and water to active sites and facile electron transfer between manganese in different oxidation states.

Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation

Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

Binding energy constraint on matter radius and soft dipole excitations of22C

An unusually large value of the C-22 matter radius has recently been extracted from measured reaction cross sections. The giant size can be explained by a very loose binding that is, however, not known experimentally yet. Within the three-body cluster model we have explored the sensitivity of the s-motion-dominated C-22 geometry to the two-neutron separation energy. A low energy of a few tens of keV is required to reach the alleged experimental lower value of the matter radius, while the experimental mean radius requires an extremely tiny binding. The dependence of the C-22 charge radius on the two-neutron separation energy is also presented. The soft dipole mode in C-22 is shown to be strongly affected by the loose binding and should be studied in the process of Coulomb fragmentation.

Human aldosterone synthase: Recombinant expression in E. coli and purification enables a detailed biochemical analysis of the protein on the molecular level

Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the blood pressure and has been reported to play a key role in the formation of arterial hypertension, heart failure and myocardial fibrosis. Aldosterone synthase (CYP11B2) catalyzes the biosynthesis of aldosterone by successive 11β- and 18-hydroxylation followed by an 18-oxidation of 11-deoxycorticosterone and thus comprises an important drug target. For more than 20 years, all attempts to purify recombinant human CYP11B2 in significant amounts for detailed analysis failed due to its hydrophobic nature as a membrane protein. Here, we present the successful expression of the protein in E. coli yielding approx. 90nmol/l culture, its purification and detailed enzymatic characterization. Biochemical analyses have been performed using in vitro conversion assays which revelead a Vmax of 238±8nmolproducts/nmolhCYP11B2/min and a Km of 103±8μM 11-deoxycorticosterone. Furthermore, binding analyses indicated a very loose binding of the first intermediate of the reaction, corticosterone with a Kd value of 115±6μM whereas for 11-deoxycorticosterone a Kd of 1.34±0.13μM was estimated. Upon substrate conversion of 11-deoxycorticosterone, new intermediates have been identified as 19- and 18-hydroxylated products not described before for the human enzyme. To understand the differences in substrate conversion, we constructed a new homology model based on the 3D structure of CYP11A1, performed docking studies and calculated the activation energy for hydrogen abstraction of the different ligands. The data demonstrated that the 11β-hydroxylation requires much less abstraction energy than hydroxylation at C18 and C19. However, the C18 and C19 hydroxylated products might be of clinical importance. Finally, purified CYP11B2 represents a suitable tool for the investigation of potential inhibitors of this protein for the development of novel drugs against hypertension and heart failure as was shown using ketoconazole.

Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism

FsrC is the membrane-bound histidine kinase component of the Fsr two-component signal transduction system involved in quorum sensing in the hospital-acquired infection agent Enterococcus faecalis. Synchrotron radiation circular dichroism spectroscopy was used here to study the intact purified protein solubilised in detergent micelles. Conditions required for FsrC stability in detergent were firstly determined and tested by prolonged exposure of stabilised protein to far-ultraviolet radiation. Using stabilised purified protein, far-ultraviolet synchrotron radiation circular dichroism revealed that FsrC is 61% α-helical and that it is relatively thermostable, retaining at least 57% secondary structural integrity at 90°C in the presence or absence of gelatinase biosynthesis-activating pheromone (GBAP). Whilst binding of the quorum pheromone ligand GBAP did not significantly affect FsrC secondary structure, near-ultraviolet spectra revealed that the tertiary structure in the regions of the Tyr and Trp residues was significantly affected. Titration experiments revealed a calculated kd value of 2μM indicative of relatively loose binding of gelatinase biosynthesis-activating pheromone to FsrC. Although use of synchrotron radiation circular dichroism has been applied to membrane proteins previously, to our knowledge this is the first report of its use to determine a kd value for an intact membrane protein. Based on our findings, we suggest that synchrotron radiation circular dichroism will be a valuable technique for characterising ligand binding by other membrane sensor kinases and indeed other membrane proteins in general. It further provides a valuable screening tool for membrane protein stability under a range of detergent conditions prior to downstream structural methods such as crystallisation and NMR experiments particularly when lower detergent concentrations are used.

Propane Ammoxidation Over the Mo–V–Te–Nb–O M1 Phase: Reactivity of Surface Cations in Hydrogen Abstraction Steps

Density functional theory calculations (GGA-PBE) have been performed to investigate the adsorption of C3 (propane, isopropyl, propene, and allyl) and H species on the proposed active center present in the surface ab planes of the bulk Mo–V–Te–Nb–O M1 phase in order to better understand the roles of the different surface cations in propane ammoxidation. Modified cluster models were employed to isolate the closely spaced V=O and Te=O from each other and to vary the oxidation state of the V cation. While propane and propene adsorb with nearly zero adsorption energy, the isopropyl and allyl radicals bind strongly to V=O and Te=O with adsorption energies, ΔE, being ≤−1.75 eV, but appreciably more weakly on other sites, such as Mo=O, bridging oxygen (Mo–O–V and Mo–O–Mo), and empty metal apical sites (ΔE > −1 eV). Atomic H binds more strongly to Te=O (ΔE ≤ −3 eV) than to all the other sites, including V=O (ΔE = −2.59 eV). The reduction of surface oxo groups by dissociated H and their removal as water are thermodynamically favorable except when both H atoms are bonded to the same Te=O. Consistent with the strong binding of H, Te=O is markedly more active at abstracting the methylene H from propane (Ea ≤ 1.01 eV) than V=O (Ea = 1.70 eV on V5+=O and 2.13 eV on V4+=O). The higher-than-observed activity and the loose binding of Te=O moieties to the mixed metal oxide lattice of M1 raise the question of whether active Te=O groups are in fact present in the surface ab planes of the M1 phase under propane ammoxidation conditions.

Photodynamic antimicrobial activity of hypocrellin A

Antimicrobial photodynamic therapy is a recently developed therapeutic option that combines a non-toxic photosensitizer with harmless visible light to damage the microbial cell. Hypocrellin A (HA), a natural occurring lipid-soluble perylenequinone pigment, has gained considerable interest since its anticancer and antiviral activities have been reported. Here, we examined the antimicrobial activity of HA against Gram-positive ( Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria ( Escherichia coli, Salmonella typhimurium). The results indicate that HA has a photodynamic antimicrobial activity against both Gram-positive and Gram-negative bacteria when CaCl 2 or MgCl 2 was employed. A loose binding has been established between HA and the organisms. Molecular oxygen is significantly involved in the photodynamic action of HA. Furthermore, HA maintains a photodynamic activity in terms of both types I and II reactions. Our results confirm the potential of HA to be used as a photosensitizer in antimicrobial photodynamic therapy.

Emotional Experience and Estimates of D2Receptor Occupancy in Psychotic Patients Treated With Haloperidol, Risperidone, or Olanzapine

Blockade of dopamine D(2) receptors is thought to mediate the therapeutic effects of antipsychotic medication but may also induce social indifference. As antipsychotic drugs differ in D(2) receptor binding, "tight" and "loose" binding drugs may be hypothesized to differentially affect emotional experience. The present study investigates the differential effects of relatively tight versus looser binding drugs on the experience of emotions in the realm of daily life. We assessed positive and negative affect in the daily life of 109 patients with a DSM-IV diagnosis of psychotic disorder who were currently taking antipsychotic medication by using the experience sampling method (a structured diary technique). Antipsychotic medication was classified as loose (olanzapine; n = 35) or tight (haloperidol, risperidone; n = 74) binding, based on the drug's dissociation constants at the D(2) receptor. The study was conducted from 2007 to 2008. Multilevel analyses showed a significant interaction between binding group (loose vs tight) and D(2) receptor occupancy estimates with regard to the experience of positive (P = .008) and negative (P = .019) affect. For tight-binding-agent users, a significant association was found between D(2) receptor binding estimates and both positive affect (P = .040) and negative affect (P = .0001) in the flow of daily life, with increasing levels of estimated D(2) receptor occupancy being associated with decreased feelings of positive affect and increased feelings of negative affect. For loose-binding-agent users, no such association was apparent. These associations were only partly mediated by clinical symptoms. These findings add ecological validity to previous laboratory findings showing an association between D(2) receptor occupancy and emotional experience.

Dissociation and Multiply Charged Silicon Ejection in High Abundance from Hexamethyldisilane

Quadruply charged, neon-like silicon and helium-like carbon were generated by the exposure of hexamethyldisilane to intense femtosecond laser pulses. Dissociation of the silicon-silicon bond, the formation of highly charged silicons, as well as the saturation intensity of their formation were studied by mass spectroscopy. The production of these ions in high abundance, but also with lower laser intensity than theoretically expected for the element, was accomplished by using organosilicon compounds. Multiply charged silicon was generated at low laser intensity because stripping electrons from organosilicon compounds is much easier than from pure silicon due to the loose binding of electrons belonging to molecular orbitals. Femtosecond laser ionization is a valuable methodology for producing highly charged ions in high abundance and is useful in many fields of interest.

Clean STD‐NMR spectrum for improved detection of ligand‐protein interactions at low concentration of protein

Saturation transfer difference (STD)-NMR has been widely used to screen ligand compound libraries for their binding activities to proteins and to determine the binding epitopes of the ligands. We report herein, a Clean STD-NMR method developed to overcome false positives (artifacts) observed in the STD-NMR spectrum due to the power spillover of RF irradiation. The method achieved higher degree of resonance saturation through digital editing of two STD-NMR spectra to generate a concatenated difference spectrum and three times of sensitivity enhancement for a loose binding complex involving DNA oligonucleotide and an RNA-binding protein, CUGBP-1ab (25.2 kDa). The interesting binding characteristics of the complex dCTGTCT-CUGBP1ab were obtained. The method was applied to a mixture of small ligand and bovine serum albumin protein (BSA, 66.3 kDa), and detected the intermolecular contacts at a BSA concentration as low as 0.1 µM, a working concentration useful for the detection of proteins of low solubility at biologically relevant conditions.