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1.
《MABS-AUSTIN》2013,5(4):497-508
The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a KD of 0.9 nM and koff of 6.6x10?4 s?1, and to selectively inhibit chymase activity with an IC50 value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.  相似文献   

2.
Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K tam-ch-15  = 8.7 (±0.5)×103 M−1, K tam-ch-100  = 5.9 (±0.4)×105 M−1, K tam-ch-200  = 2.4 (±0.4)×105 M−1 and K hydroxytam-ch-15  = 2.6(±0.3)×104 M−1, K hydroxytam – ch-100  = 5.2 (±0.7)×106 M−1 and K hydroxytam-ch-200  = 5.1 (±0.5)×105 M−1, K endox-ch-15  = 4.1 (±0.4)×103 M−1, K endox-ch-100  = 1.2 (±0.3)×106 M−1 and K endox-ch-200  = 4.7 (±0.5)×105 M−1 with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH2 residues with drug OH and NH2 groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.  相似文献   

3.
Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) with catalase were observed to be 2.27×106 M−1 and 1.66×106 M−1, respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC50 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition.  相似文献   

4.
Spontaneous clearance of acute hepatitis C virus (HCV) infection is associated with single nucleotide polymorphisms (SNPs) on the MHC class II. We fine-mapped the MHC region in European (n = 1,600; 594 HCV clearance/1,006 HCV persistence) and African (n = 1,869; 340 HCV clearance/1,529 HCV persistence) ancestry individuals and evaluated HCV peptide binding affinity of classical alleles. In both populations, HLA-DQβ1Leu26 (p valueMeta = 1.24 × 10−14) located in pocket 4 was negatively associated with HCV spontaneous clearance and HLA-DQβ1Pro55 (p valueMeta = 8.23 × 10−11) located in the peptide binding region was positively associated, independently of HLA-DQβ1Leu26. These two amino acids are not in linkage disequilibrium (r2 < 0.1) and explain the SNPs and classical allele associations represented by rs2647011, rs9274711, HLA-DQB103:01, and HLA-DRB101:01. Additionally, HCV persistence classical alleles tagged by HLA-DQβ1Leu26 had fewer HCV binding epitopes and lower predicted binding affinities compared to clearance alleles (geometric mean of combined IC50 nM of persistence versus clearance; 2,321 nM versus 761.7 nM, p value = 1.35 × 10−38). In summary, MHC class II fine-mapping revealed key amino acids in HLA-DQβ1 explaining allelic and SNP associations with HCV outcomes. This mechanistic advance in understanding of natural recovery and immunogenetics of HCV might set the stage for much needed enhancement and design of vaccine to promote spontaneous clearance of HCV infection.  相似文献   

5.
Studies in human populations and mouse models of disease have linked the common leptin receptor Q223R mutation to obesity, multiple forms of cancer, adverse drug reactions, and susceptibility to enteric and respiratory infections. Contradictory results cast doubt on the phenotypic consequences of this variant. We set out to determine whether the Q223R substitution affects leptin binding kinetics using surface plasmon resonance (SPR), a technique that allows sensitive real-time monitoring of protein-protein interactions. We measured the binding and dissociation rate constants for leptin to the extracellular domain of WT and Q223R murine leptin receptors expressed as Fc-fusion proteins and found that the mutant receptor does not significantly differ in kinetics of leptin binding from the WT leptin receptor. (WT: ka 1.76×106±0.193×106 M−1 s−1, kd 1.21×10−4±0.707×10−4 s−1, KD 6.47×10−11±3.30×10−11 M; Q223R: ka 1.75×106±0.0245×106 M−1 s−1, kd 1.47×10−4±0.0505×10−4 s−1, KD 8.43×10−11±0.407×10−11 M). Our results support earlier findings that differences in affinity and kinetics of leptin binding are unlikely to explain mechanistically the phenotypes that have been linked to this common genetic variant. Future studies will seek to elucidate the mechanism by which this mutation influences susceptibility to metabolic, infectious, and malignant pathologies.  相似文献   

6.
Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. The recent crystallization of ASIC1a identified potential binding sites for Cl in the extracellular domain that are highly conserved between ASIC isoforms. However, the significance of Cl binding is unknown. We investigated the effect of Cl substitution on heterologously expressed ASIC1a current and H+-gated currents from hippocampal neurons recorded by whole-cell patch clamp. Replacement of extracellular Cl with the impermeable and inert anion methanesulfonate (MeSO3) caused ASIC1a currents to desensitize at a faster rate and attenuated tachyphylaxis. However, peak current amplitude, pH sensitivity, and selectivity were unchanged. Other anions, including Br, I, and thiocyanate, also altered the kinetics of desensitization and tachyphylaxis. Mutation of the residues that form the Cl-binding site in ASIC1a abolished the modulatory effects of anions. The results of anion substitution on native ASIC channels in hippocampal neurons mirrored those in heterologously expressed ASIC1a and altered acid-induced neuronal death. Anion modulation of ASICs provides new insight into channel gating and may prove important in pathological brain conditions associated with changes in pH and Cl.  相似文献   

7.
We tested a number of inhibitory monovalent anions for their primary site of action on photosystem II(PSII) in chloroplasts. We find that the inhibitory effects of F, HCO2, NO2, NO3, and CH3CO2 are all reversed by addition of a high concentration of HCO3. This class of anions competitively inhibits H14CO3 binding to PSII. All of those anions tested reduced H14CO3 binding more in the light than in the dark. We conclude that the primary inhibitory site of action of a number of monovalent anions is at the HCO3 binding site(s) on the PSII complex. The carbonic anhydrase inhibitor gold cyanide, and also azide, inhibit PSII but at a site other than the HCO3 binding site. We suggest that the unique ability of HCO3 to reverse the effects of inhibitory anions reflects its singular ability to act as a proton donor/acceptor at the anion binding site. A similar role has been proposed for non-substrate-bound HCO3 on carbonic anhydrase by Yeagle et al. (1975 Proc Natl Acad Sci USA 72: 454-458).  相似文献   

8.
9.
GltPh from Pyrococcus horikoshii is a homotrimeric Na+-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na+ and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na+ binds with low affinity to the apoprotein (Kd 120 mm), with a particularly low kon value (5.1 m−1s−1). At least two sodium ions bind before aspartate. The binding of Na+ requires a very high activation energy (Ea 106.8 kJ mol−1) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na+ concentration present. Binding of aspartate was not observed in the absence of Na+, whereas in the presence of high Na+ concentrations (above the Kd for Na+) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (kon of 1.4 × 105 m−1s−1), with low Ea and Q10 values (42.6 kJ mol−1 and 1.8, respectively). We conclude that Na+ binding is most likely the rate-limiting step for substrate binding.  相似文献   

10.
Nitric oxide (NO) and superoxide (O2 ) are important cardiac signaling molecules that regulate myocyte contraction. For appropriate regulation, NO and O2 .− must exist at defined levels. Unfortunately, the NO and O2 .− levels are altered in many cardiomyopathies (heart failure, ischemia, hypertrophy, etc.) leading to contractile dysfunction and adverse remodeling. Hence, rescuing the nitroso-redox levels is a potential therapeutic strategy. Nitrone spin traps have been shown to scavenge O2 .− while releasing NO as a reaction byproduct; and we synthesized a novel, cell permeable nitrone, 2–2–3,4-dihydro-2H-pyrrole 1-oxide (EMEPO). We hypothesized that EMEPO would improve contractile function in myocytes with altered nitroso-redox levels. Ventricular myocytes were isolated from wildtype (C57Bl/6) and NOS1 knockout (NOS1−/−) mice, a known model of NO/O2 .− imbalance, and incubated with EMEPO. EMEPO significantly reduced O2 .− (lucigenin-enhanced chemiluminescence) and elevated NO (DAF-FM diacetate) levels in NOS1−/− myocytes. Furthermore, EMEPO increased NOS1−/− myocyte basal contraction (Ca2+ transients, Fluo-4AM; shortening, video-edge detection), the force-frequency response and the contractile response to β-adrenergic stimulation. EMEPO had no effect in wildtype myocytes. EMEPO also increased ryanodine receptor activity (sarcoplasmic reticulum Ca2+ leak/load relationship) and phospholamban Serine16 phosphorylation (Western blot). We also repeated our functional experiments in a canine post-myocardial infarction model and observed similar results to those seen in NOS1−/− myocytes. In conclusion, EMEPO improved contractile function in myocytes experiencing an imbalance of their nitroso-redox levels. The concurrent restoration of NO and O2 .− levels may have therapeutic potential in the treatment of various cardiomyopathies.  相似文献   

11.
Nucleic acids exist in a dynamic equilibrium with a number of molecules that constantly interact with them and regulate the cellular activities. The inherent nature of the structure and conformational integrity of these macromolecules can lead to altered biological activity through proper targeting of nucleic acids binding ligands or drug molecules. We studied the interaction of naturally occurring methylxanthines such as theophylline, theobromine and caffeine with DNA, using UV absorption and Fourier transform infrared (FTIR) spectroscopic methods, and especially monitored their binding affinity in the presence of Mg2+ and during helix-coil transitions of DNA by temperature (Tm) or pH melting profiles. The study indicates that all these molecules effectively bind to DNA in a dose dependent manner. The overall binding constants of DNA-theophylline = 3.5×103 M−1, DNA-theobromine = 1.1×103 M−1, and DNA-Caffeine = 3.8×103 M−1. On the other hand Tm/pH melting profiles showed 24–35% of enhanced binding activity of methylxanthines during helix-coil transitions of DNA rather than to its native double helical structure. The FTIR analysis divulged that theophylline, theobromine and caffeine interact with all the base pairs of DNA (A-T; G-C) and phosphate group through hydrogen bond (H-bond) interaction. In the presence of Mg2+, methylxanthines altered the structure of DNA from B to A-family. However, the B-family structure of DNA remained unaltered in DNA-methylxanthines complexes or in the absence of Mg2+. The spectral analyses indicated the order of binding affinity as “caffeine≥theophylline>theobromine” to the native double helical DNA, and “theophylline≥theobromine>caffeine to the denatured form of DNA and in the presence of divalent metal ions.  相似文献   

12.
A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 μg ml−1). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml−1) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 μg ml−1). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 μg ml−1) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.  相似文献   

13.
In maize chloroplasts, the ratio of HCO3 (anion) binding sites to high-affinity atrazine binding sites is unity. In the dark, atrazine noncompetitively inhibits the binding of half of the HCO3 to the photosystem II (PSII) complexes. The inhibition of binding saturates at 5 micromolar atrazine, little inhibition is seen at 0.5 micromolar atrazine, although the high-affinity herbicide binding sites are nearly filled at this concentration. This means that HCO3 and atrazine interact noncompetitively at a specific low-affinity herbicide binding site that exists on a portion of the PSII complexes. Light abolishes the inhibitory effects of atrazine on HCO3 binding. Based on the assumption that there is one high-affinity atrazine binding site per PSII complex, we conclude that there is also only one binding site for HCO3 with a dissociation constant near 80 micromolar. The location of the HCO3 binding site, and the low-affinity atrazine binding site, is not known.  相似文献   

14.
F1-ATPase is a rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α3β3-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the β-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F1 rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F1 molecules that carried a bead duplex on the γ-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 × 107 M−1s−1. A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F1 produced the same torque of ~40 pN·nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.  相似文献   

15.
Here, we report on a significant effect of substitutions on the binding affinity of a series of 2-amino-1,8-naphthyridines, i.e., 2-amino-1,8-naphthyridine (AND), 2-amino-7-methyl-1,8-naphthyridine (AMND), 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) and 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), all of which can bind to cytosine opposite an AP site in DNA duplexes. Fluorescence titration experiments show that the binding affinity for cytosine is effectively enhanced by the introduction of methyl groups to the naphthyridine ring, and the 1:1 binding constant (106 M−1) follows in the order of AND (0.30) < AMND (2.7) < ADMND (6.1) < ATMND (19) in solutions containing 110 mM Na+ (pH 7.0, at 20°C). The thermodynamic parameters obtained by isothermal titration calorimetry experiments indicate that the introduction of methyl groups effectively reduces the loss of binding entropy, which is indeed responsible for the increase in the binding affinity. The heat capacity change (ΔCp), as determined from temperature dependence of the binding enthalpy, is found to be significantly different between AND (−161 cal/mol K) and ATMND (−217 cal/mol K). The hydrophobic contribution appears to be a key force to explain the observed effect of substitutions on the binding affinity when the observed binding free energy (ΔGobs) is dissected into its component terms.  相似文献   

16.
17.
Biogenic polyamines are found to modulate protein synthesis at different levels. This effect may be explained by the ability of polyamines to bind and influence the secondary structure of tRNA, mRNA, and rRNA. We report the interaction between tRNA and the three biogenic polyamines putrescine, spermidine, spermine, and cobalt(III)hexamine at physiological conditions, using FTIR spectroscopy, capillary electrophoresis, and molecular modeling. The results indicated that tRNA was stabilized at low biogenic polyamine concentration, as a consequence of polyamine interaction with the backbone phosphate group. The main tRNA reactive sites for biogenic polyamine at low concentration were guanine-N7/O6, uracil-O2/O4, adenine-N3, and 2′OH of the ribose. At high polyamine concentration, the interaction involves guanine-N7/O6, adenine-N7, uracil-O2 reactive sites, and the backbone phosphate group. The participation of the polycation primary amino group, in the interaction and the presence of the hydrophobic contact, are also shown. The binding affinity of biogenic polyamine to tRNA molecule was in the order of spermine > spermidine > putrescine with KSpm = 8.7 × 105 M−1, KSpd = 6.1 × 105 M−1, and KPut = 1.0 × 105 M−1, which correlates with their positively charged amino group content. Hill analysis showed positive cooperativity for the biogenic polyamines and negative cooperativity for cobalt-hexamine. Cobalt(III)hexamine contains high- and low-affinity sites in tRNA with K1 = 3.2 × 105 M−1 and K2 = 1.7 × 105 M−1, that have been attributed to the interactions with guanine-N7 sites and the backbone PO2 group, respectively. This mechanism of tRNA binding could explain the condensation phenomenon observed at high Co(III) content, as previously shown in the Co(III)–DNA complexes.  相似文献   

18.
Voltage-gated Cl channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I and related anions. Extracellular and intracellular I exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br) and polyatomic (SCN, NO3 , CH3SO3 ) anions. In addition, I block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined K D values for I block in three distinct kinetic states and found that binding of I to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.  相似文献   

19.
Effects of NO2, ClO3, and ClO2 on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO3 influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m−3 NO2 fully induced NO3 transport but failed to induce NRA. Similar pretreatments with ClO3 and ClO2 induced neither NO3 transport nor NRA. Net ClO3 uptake was induced by NO3 but not by ClO3 itself, indicating that NO3 and ClO3 transport occur via the NO3 carrier. At the uptake step, NO2 and ClO2 strongly inhibited NO3 influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO3 proved to be a weak inhibitor of NO3 influx (Ki = 16 mol m−3) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO3 analogs as screening agents for the isolation of mutants defective in NO3 transport.  相似文献   

20.
Homann PH 《Plant physiology》1988,88(1):194-199
To further our understanding of the role of Cl and certain other monovalent anions in the oxygen evolving photosystem II of chloroplasts, dissociating and stabilizing anion effects on the extrinsic 17 and 23 kilodalton polypeptides of the photosynthetic water oxidizing complex were investigated. It was found that (a) the dissociation of the two polypeptides in Cl free media of pH ≈ 7 was enhanced by millimolar concentrations of the divalent anion SO42− and also by divalent cations like Mg2+ and Ca2+; (b) the dissociation was opposed by relatively low concentrations of monovalent anions with an order of effectiveness Cl = Br > NO3 > F > ClO4; (c) at molar concentrations, SO42− stabilized the binding of the 23 kilodalton polypeptide, while Cl and Br became dissociating agents, in agreement with studies by Blough and Sauer (1984 Biochim Biophys Acta 767: 377-381); (d) the binding of the polypeptides was strengthened at room temperature relative to 0°C, indicating an involvement of hydrophobic forces. It is suggested that a specific binding of Cl, or certain substitutes, organizes the protein surfaces and/or the adjacent water layers in the water oxidizing complex in a way that not only stabilizes its assembly, but is essential for the catalytic mechanism as well. Binding of, or charge screening by, divalent ions interferes with this process. At high salt concentrations, all these effects are overridden by “lyotropic” actions of the solutes that affect the integrity of the water oxidizing protein complex by stabilizing or disrupting critical hydrophobic domains.  相似文献   

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