Hill Constant Research Articles

A highly selective multifunctional naphthalene-labeled polymer sensor for Cu2+ and Fe3+ ions in aqueous medium

In this work, the detection of Cu2+ and Fe3+ with naphthalene labeled poly(acrylic acid) (PAA150Np3) was investigated. Other metal ions such as Ag+, Li+, K+, Na+, Ba2+, Mg2+, Ca2+, Zn2+ and Ni2+ were also considered to analyze the sensor performance. The fluorescence quenching of the naphthalene (Np) graft was assessed from the emission spectra and the fluorescence decays of PAA150Np3 in the presence of Cu2+ or Fe3+. The Stern-Volmer and Hill constants, as well as the detection limits of the studied ions were obtained considering the emissions of both the monomer and the excimer of Np. The polymer conformation was investigated by changing the metal ion concentration or the pH. The polymeric sensor showed high selectivity and sensitivity for Cu2+ or Fe3+. A static mechanism of fluorescence quenching was unveiled for the Np graft. A more stable complex formed between PAA150Np3 and Fe3+, as compared to the counterpart system with Cu2+. The sensor’s performance, including detection limit and selectivity of Cu2+ or Fe3+ ions, is higher than of most currently reported sensors. The study shows that the multifunctional fluorescent polymer sensor has the potential to be used in real-life samples, in bio-systems and environmental applications.

Negative Homotropic Cooperativity in Guest Binding of a Trisporphyrin Double Cleft.

Thenegative homotropic allostery of a triple-layered trisporphyrin cleft with two guest binding sites was confirmed. The X-ray crystal structures of the 1:2 host-guest complexes showed that the trisporphyrin accommodated two guest molecules within the cleft via π-π stacking and donor-acceptor interactions. In solution,1H NMR and Job plots showed 1:2 host-guest complexes. Isothermal titration calorimetry (ITC) and UV/vis absorption spectroscopy were employed to evaluate the binding constants and cooperativities. The guest binding of the trisporphyrin showed negative cooperativity and noncooperativity depending on the structures of the guest molecules. The correlations between the interaction parameters (α) and Hill constants were determined. ITC experiments showed that the host-guest complexation of trisporphyrin with electron-deficient guests incurred an enthalpy penalty in the successive guest binding process. DFT calculations revealed that the first guest binding reduced the electron density of the central porphyrin plane, which led to an energetic penalty that weakened the successive binding process.

A New Method to Self‐Associate Proteins Using a Cooperative DNA‐binding Protein

Many cellular processes depend on proteins that must self‐associate into larger complexes in order to function. This includes membrane receptors that dimerize (form pairs) to transmit transmembrane signals and scaffolding proteins that assemble into structural filaments. This project is developing a new method to experimentally control protein self‐association in order to better study such processes. The method is based on the DNA‐binding protein HAP1 (heme activation protein 1). As an isolated fragment, the DNA binding region of HAP1 is a monomer in solution but binds cooperatively as a dimer to its DNA target site. Using recombinant DNA methods, an 11 kDa fragment of the HAP1 DNA‐binding region was fused to the 43 kDa maltose binding protein (MBP) to test if adding the HAP1 target site DNA could induce dimerization of the fused MBP. The HAP1 DNA‐binding fragment was modified to remove a protease‐sensitive site observed during recombinant expression. In addition, two different lengths of flexible linker were introduced between the MBP and HAP1 proteins to reduce potential steric interference among MBP, HAP1, and bound DNA. Binding of MBP‐HAP1 fusions to DNA was measured by fluorescence anisotropy using 24 base‐pair fluorescein‐labeled DNA duplexes. A titration of the MBP‐HAP1 fusion with 15 residue linker to a labeled target site showed strong binding with half‐maximal binding at 0.3 μM MBP‐HAP1. In addition, the titration was fit with a Hill constant of 2.0 ± 0.3 indicative of cooperative dimer formation on the DNA. Binding to a labeled non‐target DNA was much weaker with half‐maximal binding greater than 2 μM. Gel filtration chromatography of the protease‐sensitive MBP‐HAP1 fusion showed a DNA‐induced shift from monomer to a larger protein‐DNA complex containing at least two proteins per DNA. These tests are being repeated with the protease‐insensitive MBP‐HAP1 fusion. The ability of the HAP1 DNA‐binding region to induce the self‐association of MBP using an inexpensive DNA ligand should be generalizable to other proteins, giving this method wide utility.

Cooperative Demolition: Water's Disruption of Structures in Deep Eutectic Solvents.

We report a study of cooperativity in the transition from a nonaqueous deep eutectic solvent (DES) to an aqueous solution. In some systems, a nonequilibrium region prevails at low water contents, while cooperativity is always observed at high water contents. Catechol-based mixtures exhibit a Hill constant (nH) of ∼ 3 and an overall ΔG° of ca. -3-5 kJ/mol for the transition from DES to aqueous solution. In contrast, o-phenylenediamine-based mixtures exhibit a shift from nH = 0 at low water contents to nH ∼ 12 at high water contents with an overall ΔG° of ca. -13-15 kJ/mol. To the best of our knowledge, this is the first evidence for a shift from nonequilibrium to cooperative binding in a transition from nonaqueous to aqueous solutions.

Anisotropic steady-state creep behavior of Single-crystal β-Sn: A continuum constitutive model based on crystal viscoplasticity

Body-center tetragonal (BCT) β-Sn crystals exhibit highly anisotropic properties such as stiffness and thermal expansion, which significantly affect their thermal-mechanical behavior. The homologous temperature of β-Sn is relatively high under common applications due to its low melting point, which renders Sn and Sn-based alloys viscoplastic even at room temperature. The orientation-dependent creep behavior of β-Sn specimens have been previously measured by different research groups. A dislocation mechanics based crystal viscoplasticity model is applied in this study to describe this anisotropic steady-state creep behavior of β-Sn single crystals. The model constants are calibrated with single-crystal creep test results available in the literature. The resulting creep behavior of β-Sn single-crystal is also represented with a homogenized continuum-scale finite element approach based on the use of a combined Hill-Norton approach where the creep anisotropy is represented with Hill's anisotropic potential and the creep flow rule is represented with Norton's power-law model. Estimation of the six Hill's constants for β-Sn requires multiple creep tests under specific stress states, for single crystals along crystal principal directions. In this study, these physical creep tests are replaced with ‘virtual tests’ conducted with the developed dislocation-based crystal-viscoplasticity model. To assess the ability of the Hill-Norton finite element approach to represent dislocation creep, the finite element simulation results are compared with results of: (i) physical tests on single crystal specimens reported in the literature; and (ii) crystal-viscoplasticity modeling along many crystal orientations (beyond the fundamental calibration cases conducted along crystal principal directions). In future studies, this approach will be used for anisotropic finite element modeling of creep in polycrystalline specimens.

Spectroscopic and docking molecular study of new anticancer Pt complex binding with human serum albumin

After synthesizing and identifying the nature of the new complex based on platinum metal, [Pt(NH3)2(butylgly)]NO3, the interaction of this complex with human serum albumin (HSA) was performed by spectroscopy and molecular docking methods at two temperatures of 27 and 37 °C and under physiological conditions of the body. The toxicity test of this complex was performed on the MCF-7 cell line (IC50 = 300 µM). Enthalpy, entropy, Gibbs free energy, binding constant, number of complex binding sites on the HSA, Scatchard diagrams, Hill coefficient, and Hill constant were calculated and then plotted via UV/Vis. According to the Gibbs free energy obtained at two temperatures of 27 and 37 °C (−20.6, −21.2 kJ mol−1), the interaction was done spontaneously. Moreover, the melting temperature of human serum albumin with this complex; and the kinetics of this interaction (the second-order) were calculated. Using fluorescence at three temperatures of 25, 27, and 37 °C, the binding constant (2.9 × 104, 1.0 × 104, and 5.7 × 103 M−1), the quenching constant, average aggregation number of HSA, and the number of binding sites of the complex on the protein were obtained. As well, the static quenching mechanism was also observed. Molecular docking results showed that the site of binding of this complex to the HSA, is the site II subdomain IIIA, and the hydrogen and hydrophobic bonds are superior.

Uniform description of the helium isoelectronic series down to the critical nuclear charge with explicitly correlated basis sets derived from regularized Krylov sequences.

An efficient computational scheme for the calculation of highly accurate ground-state electronic properties of the helium isoelectronic series, permitting uniform description of its members down to the critical nuclear charge Zc, is described. It is based upon explicitly correlated basis functions derived from the regularized Krylov sequences (which constitute the core of the free iterative CI/free complement method of Nakatsuji) involving a term that introduces split length scales. For the nuclear charge Z approaching Zc, the inclusion of this term greatly reduces the error in the variational estimate for the ground-state energy, restores the correct large-r asymptotics of the one-electron density ρ(Z; r), and dramatically alters the manifold of the pertinent natural amplitudes and natural orbitals. The advantages of this scheme are illustrated with test calculations for Z = 1 and Z = Zc carried out with a moderate-size 12th-generation basis set of 2354 functions. For Z = Zc, the augmentation is found to produce a ca. 5000-fold improvement in the accuracy of the approximate ground-state energy, yielding values of various electronic properties with between seven and eleven significant digits. Some of these values, such as those of the norms of the partial-wave contributions to the wavefunction and the Hill constant, have not been reported in the literature thus far. The same is true for the natural amplitudes at Z = Zc, whereas the published data for those at Z = 1 are revealed by the present calculations to be grossly inaccurate. Approximants that yield correctly normalized ρ(1; r) and ρ(Zc; r) conforming to their asymptotics at both r → 0 and r → ∞ are constructed.

Investigation of a Juxtamembrane Lysine‐rich Region as a Determinant of Intracellular pH Sensitivity in the H+‐conducting Transmembrane Protein Slc4a11

SLC4A11 is a unique H+‐transporting member of the SLC4 family of bicarbonate transporters that is expressed in a variety of cells, including corneal endothelia. The H+ conductance of SLC4A11 increases in response to rises in both intracellular pH (pHi) and extracellular pH (pHe). By aligning the amino‐acid sequences of mammalian Slc4a11 and their bicarbonate‐transporting paralogs, we have identified a unique and highly‐conserved insertion ‘KxKxVGK’ in Slc4a11 at the juxtamembrane boundary between its cytosolic amino‐terminal‐domain and its first transmembrane‐span. The third lysine in this motif (‘K343’ in mouse Slc4a11) brings to mind the lysine at a comparable location in the renal K+‐channel ROMK that underlies the pHi sensitivity of ROMK currents (Fakler et al. EMBO J, 1996). Thus, we hypothesized that K343 might contribute to the pHi sensitivity of Slc4a11.To probe the role of K343, we generated a mutant ‘K343Q’, Q being the non‐titratable residue glutamine that is found at this location in other SLC4s. We expressed wild‐type (WT) or K343Q Slc4a11 from cRNA injected into Xenopus oocytes. We then compared the pH‐sensitivities of the expressed H+ conductances (Gm) at pHe=7.5 by using two‐electrode voltage‐clamp circuitry while raising oocyte pHi (monitored by a H+‐selective microelectrode) using a micro‐injector loaded with 1.2M NaHCO3 solution. As controls we used oocytes that had been injected with water rather than cRNA (‘H2O‐injected’) and oocytes that had been injected with cRNA encoding the mouse equivalent of the human corneal‐dystrophy‐causing SLC4A11 mutant ‘R125H’.ANOVA shows that the Gm of R125H‐expressing cells is indistinguishable from that of H2O‐injected cells over the entire tested pHI range (7.2–8.1), whereas the Gm of WT‐expressing cells is significantly greater than that of H2O‐injected cells over the entire range (n=4–12 data points). On the other hand, the Gm of K343Q‐expressing cells is indistinguishable from H2O‐injected cells over the pHi range 7.2–7.5, greater than H2O‐injected cells but not as great as WT‐expressing cells over the pHi range 7.5–7.9, and ultimately indistinguishable from WT‐expressing cells over the pHi range 7.9–8.1 (n = 4–35 data points). Thus the pHi‐responsiveness of K343Q is altered compared to WT.The H+ conductance of Slc4a11 is much enhanced at pHe=8.5 such that the pHi vs. Gm relationship becomes sigmoidal and can be fit to the Hill equation to determine an apparent pK and Hill constant. We assessed K343Q at pHe=8.5 and calculated a pK of 7.15 ± 0.02 and a Hill constant of 5 ± 1 (n = 4). Both of these values are significantly different from the WT values that we had previously determined (pK= 7.25 ± 0.03, Hill = 8 ± 1, n = 10). The lowered Hill and pK for K343Q are consistent with the loss of a titratable residue that contributes to the pHi sensitivity of Slc4a11. Further work is underway to identify other determinants of pHi and pHe sensitivity.Support or Funding InformationThis work was supported by NIH R01‐EY028580 to MDPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating the Intracellular and Extracellular pH Sensitivities of the H+‐conducting Transmembrane Protein Slc4a11

SLC4A11 is the only member of the SLC4 family that transports H+ rather than bicarbonate (HCO3−) or carbonate (CO3=). SLC4A11 is expressed in a variety of cells including the corneal endothelial cells (CECs) in which it is presumed to mediate H+ influx. CECs help to maintain the optimal hydration status and optical clarity of the cornea by pumping fluid from the corneal stroma. This pump function requires the joint action of NBCe1 (Na+/CO3= influx), MCT1 (H+/lactate− influx), and SLC4A11. Notably, SLC4A11 mutations are sufficient to cause pump failure and the subsequent corneal swelling and opacification that are characteristic of corneal endothelial dystrophy. The role of SLC4A11 in corneal health and disease remains unclear, although we have hypothesized that SLC4A11 action could promote CEC health by correcting pH disturbances caused by imbalanced NBCe1 (base) and MCT1 (acid) import. Consistent with this hypothesis, our laboratory has previously demonstrated that the magnitude of the H+ conductance mediated by SLC4A11 is increased by rises in intracellular pH (pHi) as well as rises in extracellular pH (pHe).To better understand the features of SLC4A11 activation, we studied the pH‐sensitivity of mouse Slc4a11 expressed from cRNA in Xenopus oocytes. Using two electrode voltage clamp circuitry in conjunction with a H+‐selective microelectrode and a micro‐injector loaded with 1.2M NaHCO3 solution, we caused incremental increases in oocyte pHi and followed the accompanying increase in membrane conductance (Gm) while pHe was maintained at 7.5 or 8.5. We find, when pHe = 7.5, that Gm rises steadily over the pHi range 7.1–8.1, reaching a maximum observed conductance (Gm(max)) of 82 ± 5 μS (n = 6, SE) with an apparent intracellular pK ~7.6. On the other hand, when pHe= 8.5, Gm exhibits a steeper dependence on pHi and rises in a sigmoidal fashion over the pHi range 7.0–7.4 achieving a Gm(max) of 78 ± 8 μS (n = 14, SE) at pHi ≥ 7.4. [In comparable experiments performed on oocytes that were not expressing Slc4a11, Gm for was not greater than 4 μS (n=6) at pHi > 7.4.] The data gathered from Slc4a11‐expressing oocytes at pHe=8.5 can be fit to the Hill equation to reveal an average intracellular pK of 7.15 ± 0.02 (n = 10, SE) and an average Hill constant of 8 ± 1 (n = 10, SE).These data reveal a complex connection between pHe and pHi− sensitivity which highlights the challenge of working with such a dynamic substrate. Specifically, our data indicate that the kinetics of pHi sensing by Slc4a11 are modulated by pHe and are governed by numerous titratable residues. Knowledge of the kinetics governing the pH‐sensitivity of SLC4A11 H+ conductance will be valuable for assessing the physiological relevance of pH sensing by SLC4A11, for implementing SLC4A11 in mathematical models of CEC action, and for assessing the consequences of SLC4A11 mutations.Support or Funding InformationThis work was supported by NIH R01‐EY028580 to MDPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anisotropic Gurson‐Tvergaard‐Needleman plasticity and damage model for finite element analysis of elastic‐plastic problems

SummaryImplementation and analysis of the anisotropic version of the Gurson‐Tvergaard‐Needleman (GTN) isotropic damage criterion are performed on the basis of Hill's quadratic anisotropic yield theory with the definition of an effective anisotropic coefficient to represent the elastic‐plastic behavior of ductile metals. This study aims to analyze the extension of the GTN model suitable for anisotropic porous metals and to investigate the GTN model extension. An anisotropic damage model is implemented using the user material subroutine in ABAQUS/standard finite element code. The implementation is verified and applied to simulate a uniaxial tensile test on a commercially produced aluminum sheet material for three‐dimensional and plane stress test cases. Spherical and ellipsoidal micro voids are considered in the matrix material, and their effects on the uniaxial stress‐strain response of the material are analyzed. Hill's quadratic anisotropic yield theory predicts substantially large damage evolution and a low stress‐strain curve compared with those predicted by the isotropic model. An approximate model for anisotropic materials is proposed to avoid increased damage evolution. In this approximate model, Hill's anisotropic constants are replaced with an effective anisotropy coefficient. All model‐generated stress‐strain predictions are compared with the experimental stress‐strain curve of AA6016‐T4 alloy.

Theory of curvature-dependent kinetics of diffusion-limited reactions and its application to ligand binding to a sphere with multiple receptors.

We present a simple theory that explains how surface curvature affects the reaction kinetics of diffusion-limited reactions on spherically curved surfaces. In this theory, we derive a quadratic equation under the conditions that the rate constant satisfies the Hill and Smoluchowski rate constants at the lowest and highest curvatures, respectively, and that at a certain intermediate curvature, there should be a maximum value of the rate constant, which was recently found in our previous work. We find that the result obtained from our theory is in good agreement with the corresponding one obtained from numerical calculation. In addition, we show that our theory can be directly applied to the Šolc-Stockmayer model of axially symmetric reactants, which can be considered as a spherical reactant with a single reaction site. Furthermore, we discuss using our theory to improve the formula for the rate constant in the Berg-Purcell ligand-binding model of a cell membrane covered by multiple receptors. Our simple theory yields insight into the effect of curvature on diffusion-influenced reactions and provides a useful formula for easily and quantitatively evaluating the curvature effect.

Green synthesis of biogenic silver nanoparticles using Solanum tuberosum extract and their interaction with human serum albumin: Evidence of “corona” formation through a multi-spectroscopic and molecular docking analysis

Biogenic silver nanoparticles (AgNPs) have been synthesized by using Solanum tuberosum (potato) extract (PE) as a reducing as well as stabilizing agent which is reasonably cheaper, non-toxic and easily available material. The green synthesis of silver nanoparticles has been carried out by very simple method and the nanoparticles were characterized by surface plasmon band as well as TEM measurements. The PE-AgNPs were highly dispersed in the solution and found to be spherical with around 10nm in size. Interaction of these nanoparticles was studied with plasma protein HSA by means of various spectroscopies, such as, UV–visible, fluorescence, DLS, CD and FTIR spectroscopies. The HSA was found to form the protein “corona” around the starch-capped PE-AgNPs. Absorption spectroscopy revealed that the interaction between HSA and PE-AgNPs resulted in the ground state complex formation. Due to the strong absorption of PE-AgNPs, the inner filter effect was corrected for the fluorescence data. PE-AgNPs were found to quench the fluorescence of HSA with a small blue shift attributed to the increase in the hydrophobicity near tryptophan residue due to the presence of amylopectin and amylose units in the starch. The value of n, Hill's constant, was found to be >1 which determines the existence of a cooperative binding between nanoparticle and albumin. Several parameters such as Stern-Volmer and binding constants in addition to the thermodynamic parameters have been analyzed and discussed which established that the complex formation has taken place via static quenching mechanism and the corona formation between albumin and PE-AgNPs was entropy driven process. Binding of biogenic PE-AgNPs to the HSA slightly affected the secondary structure of latter with a small decrease in α-helical contents resulting in the partial unfolding of the protein, though the structural motif remained the same. Molecular docking simulations revealed various possible binding modes between PE-AgNPs and albumin.

Affinity study on bovine serum albumin’s peptides to amphiphilic gold nanoparticles: A test of epitopes and non-epitopes

It is an inevitable event that nanoparticles (NPs) will encounter proteins/peptides in nano-medicine, so it has been significant to know their interaction mechanism before in vivo applications. Previously, a 105-amino-acid sequence had been reported as the binding site between bovine serum albumin (BSA) and amphiphilic polymer coated gold nanoparticles (AP-AuNPs) along with a mortise-tenon joint hypothesis. This article tested the affinity difference between two epitope peptide sequences such as: LGEYGFQNALIVR (S1), DAFLGSFLYEYSR (S2) and one non-epitope peptide sequence as: FDEHVKLVNELTEF (S3). With the photoluminescent amino acid residues, the fluorescence quenching method based on the nanometal surface energy transfer (NSET) principle was able to study the thermodynamics of the current binding system. The binding constants (Ka) were determined and followed the order as: Ka-S1>Ka-S2>>Ka-S3. Moreover, Hill constants indicated that cooperativity only presented in the interactions of AP-AuNP with either S1 or S2, but not for S3. Moreover, gel electrophoresis, surface plasmon resonance, atomic force microscopy and three dimensional fluorescence microscopy were all also used to comprehensively analyse the binding interaction mechanism. These results further provided useful information to better understand the mortise-tenon joint, which might find applications to nanofabrication and biomedicine.

Study on physisorption between G-actin and amphiphilic polymer-coated gold nanoparticles.

Physisorptions occurs everywhere and constantly in living organisms and between nanomaterials and biomolecules. In this study, one of the most important proteins, G-actin, was selected to investigate its bio-nano physisorption with a model nanoparticle coated with a amphiphilic polymer. Using a photoluminescence quenching method, both the binding constant and the Hill constant were determined as 1.79×107 M-1 and 0.84, respectively. Thermodynamic calculations proved that such a physisorption was a spontaneous procedure. The physisorption-mediated protein-nanoparticle conjugates were robust enough to resist gel electrophoresis, and protein conformation was kept intact, as revealed using circular dichroism. This conjugate might be a promising candidate for nanofabrication or could play a significant role in actin-related bioactivities.

Allosteric properties of Geobacillus maltogenic amylase.

Maltogenic amylases (MAases, EC 3.2.1.133) have been gotten much attention due to their various applications in industry and commercial processes. MAases belong to subfamily 20 of glycoside hydrolase family 13 (NPase or CDases subfamily) and they have important differences with other members of the family. This enzyme consists of two subunits which form two active sites in the dimer form by binding of the central domain of each subunit to the N domain of the next one (domain-swapping dimeric structure). Allosterism is a possible way of regulating enzymatic activity and no evidence has been found regarding to the cooperativity and correlation between MAases subunits, therefore in this study the allosteric behavior of MAases from a native strain (Geobacillus sp. Gh6) was investigated. Unlike other members of α-amylase family, MAases showed positive cooperativity between their subunits and the enzyme exhibited sigmoidal nature towards all three cyclodextrin (CD) substrates with a Hill constant (nH) value equal to 2, 1.6 and 1.1 for α-CD, β-CD and γ-CD, respectively. On further analysis, the effect of glucose and maltose as MAases allosteric effectors in the presence of β-CD substrate showed that these two effectors had a biphasic effect; while they stimulated the enzyme activity at low concentrations (with a decrease in Hill constant), these metabolites acted as allosteric inhibitors at higher concentrations. Due to the key role of MAases in carbohydrate metabolization, an efficient regulating system for this enzyme is required. In this experiment, for the first time the allosteric properties of MAases were observed and investigated.

Genetically encoded Ca(2+) indicators; expanded affinity range, color hue and compatibility with optogenetics.

Fluorescent protein-based indicators are invaluable tools for functional imaging of living cells and organisms. Genetically encoded calcium indicators (GECIs) such as derivatives of yellow cameleons (YCs) and GCaMPs/pericams (Miyawaki et al., 1997; Nakai et al., 2001; Nagai et al., 2001) are a highly advanced class of indicators. Continued efforts for improvement of the performance of GECIs have resulted in brighter indicators with better photo-stability and expanded dynamic range, thus improving the sensitivity of detection. Fine-tuning of other properties, including Ca2+ affinity and Hill constant, have also contributed to increase the detectability of Ca2+ dynamics. Emerging optogenetic technology has forced the spectrally compatible GECI color variants. In this opinion, we highlight the recent development of GECIs including photo-switchable Ca2+ indicators and bioluminescence-based Ca2+ indicator, mainly invented in our group, focusing especially on the parameters determining their performance in order to provide a guideline for the selection of appropriate GECI for a given experiment.

Effect of particle size on conformation and enzymatic activity of EcoRI adsorbed on CdS nanoparticles

Better understanding of the interaction between nanoparticles (NPs) and protein is the basis for biological and biomedical applications of NPs. Water-soluble fluorescent CdS NPs have been widely used in the biological and biomedical fields and the study on effect of CdS NPs size on conformation and enzymatic activity of protein might be very important in its application. In this work, the interaction of CdS NPs with different size with type II restriction endonuclease (EcoRI) were investigated by atomic force microscopy, transmission electron microscopy, UV–vis spectroscopy, fluorescence quenching method, CD spectra, laser scanning confocal microscopy and gel electrophoresis. It was found that the equilibrium constant (kD) as well as the cooperativity degree of CdS NPs-EcoRI binding (Hill constant, n) strongly depended on the CdS NPs size. The different curvature of CdS NPs surface could result in different changes of EcoRI conformation. The gel electrophoresis indicated that the decrease in α-helix content more or less affected the activity of EcoRI.

Riboswitch Control of Aminoglycoside Antibiotic Resistance

The majority of riboswitches are regulatory RNAs that regulate gene expression by binding small-molecule metabolites. Here we report the discovery of an aminoglycoside-binding riboswitch that is widely distributed among antibiotic-resistant bacterial pathogens. This riboswitch is present in the leader RNA of the resistance genes that encode the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes that confer resistance to aminoglycoside antibiotics through modification of the drugs. We show that expression of the AAC and AAD resistance genes is regulated by aminoglycoside binding to a secondary structure in their 5' leader RNA. Reporter gene expression, direct measurements of drug RNA binding, chemical probing, and UV crosslinking combined with mutational analysis demonstrate that the leader RNA functions as an aminoglycoside-sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycosides antibiotic resistance.

Magnetic field effect on the binding of HSA with amlodipine and propranolol

The objective of this work is to study the effect of magnetic field (52mT) on the interaction between two drugs, amlodipine and propranolol, with human serum albumin (HSA) in physiological buffer (pH 7.4) making use of UV absorption and fluorescence and circular dichroism (CD) spectroscopy. The binding constants (K), binding capacities (g) and Hill constants (nH) of the reactions were determined. The results showed that the binding parameters are different in the absence and presence of the magnetic field. The CD spectroscopy revealed that amlodipine induces an increase and propranolol a decrease of α-helix content of HSA. In this case, the application of magnetic field, also, brings about a small change in the CD spectra. The binding distance, r, between donor (HSA) and acceptors (amlodipine and propranolol) was obtained according to Förster theory of non-radiation energy transfer. These results suggest that the magnetic field affects mostly the function of HSA such as drug binding, which plays a key role in drug distribution in the body, and slightly on the structure of HSA.

Role of Binding in Mycoplasma mobile and Mycoplasma pneumoniae Gliding Analyzed through Inhibition by Synthesized Sialylated Compounds

Mycoplasmas, which have been shown to be the causative pathogens in recent human pneumonia epidemics, bind to solid surfaces and glide in the direction of the membrane protrusion at a pole. During gliding, the legs of the mycoplasma catch, pull, and release sialylated oligosaccharides fixed on a solid surface. Sialylated oligosaccharides are major structures on animal cell surfaces and are sometimes targeted by pathogens, such as influenza virus. In the present study, we analyzed the inhibitory effects of 16 chemically synthesized sialylated compounds on the gliding and binding of Mycoplasma mobile and Mycoplasma pneumoniae and concluded the following. (i) The recognition of sialylated oligosaccharide by mycoplasma legs proceeds in a "lock-and-key" fashion, with the binding affinity dependent on structural differences among the sialylated compounds examined. (ii) The binding of the leg and the sialylated oligosaccharide is cooperative, with Hill constants ranging from 2 to 3. (iii) Mycoplasma legs may generate a drag force after a stroke, because the gliding speed decreased and pivoting motion occurred more frequently when the number of working legs was reduced by the addition of free sialylated compounds.