AFRRI Reports, Second Quarter 1994

DTIC Science & Technology

1994-08-01

the antrum wete immediately placed in sterile 0.9% NaCl, kept on ice, coded, and then prepared for culture, smears, and urease assay by homogeniza...high urease specific activity (>1 |J.mol- min-1 ■ mg protein-1) plus high-affinity substrate binding (Mi- chaelis constant [K^\\ < 1 mmol/L),27 in at...031, respectively), and the characteristic bacterial growth with high-activity product.on of a urease with tight substrate binding " was found in

An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

2016-06-13

ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface inmore » which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.« less

Effect of the dilution rate on microbial competition: r-strategist can win over k-strategist at low substrate concentration.

PubMed

Winkler, Mari-K H; Boets, Pieter; Hahne, Birk; Goethals, Peter; Volcke, Eveline I P

2017-01-01

The conditions present in both in vitro and in vivo ecosystems determine the microbial population harbouring it. One commonly accepted theory is that a species with a high substrate affinity and low growth rate (k-strategist) will win the competition against a second species with a lower substrate affinity and higher growth rate (r-strategist) if both species are subjected to low substrate concentrations. In this study two nitrite oxidizing bacteria (NOB), Nitrospira defluvii (k-strategist) and Nitrobacter vulgaris (r-strategist), were cultivated in a continuous reactor systems. The minimal hydraulic retention time (HRT) required for maintaining the slower growing Nitrospira was first determined. A reactor containing Nitrobacter was set to the same HRT and Nitrospira was injected to evaluate the effect of the dilution rate on the competition between both species. By following the microbial population dynamics with qPCR analysis, it was shown that not only the substrate affinity drives the competition between k- and r-strategists but also the dilution rate. Experimental data and numerical simulations both revealed that the washout of Nitrobacter was significantly delayed at dilution rates close to the μmax of Nitrospira. The competition could be even reverted towards Nitrobacter (r-strategist) despite of low nitrite concentrations and dilution rates lower than the μmax of Nitrospira.

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter▿

PubMed Central

Aktas, Meriyem; Jost, Kathinka A.; Fritz, Christiane; Narberhaus, Franz

2011-01-01

Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called “Venus flytrap mechanism” of substrate binding. PMID:21803998

Computational study of β-N-acetylhexosaminidase from Talaromyces flavus, a glycosidase with high substrate flexibility.

PubMed

Kulik, Natallia; Slámová, Kristýna; Ettrich, Rüdiger; Křen, Vladimír

2015-01-28

β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest. Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model. The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.

Specificity of hammerhead ribozyme cleavage.

PubMed Central

Hertel, K J; Herschlag, D; Uhlenbeck, O C

1996-01-01

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths. Images PMID:8670879

Differential Uptake Mechanisms of Fluorescent Substrates into Stem-Cell-Derived Serotonergic Neurons.

PubMed

Matthaeus, Friederike; Schloss, Patrick; Lau, Thorsten

2015-12-16

The actions of the neurotransmitters serotonin, dopamine, and norepinephrine are partly terminated by diffusion and in part by their uptake into neurons via the selective, high-affinity transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET), respectively. There is also growing evidence that all three monoamines are taken up into neurons by low-affinity, high-capacity organic cation transporters (OCT) and the plasma membrane monoamine transporter (PMAT). Pharmacological characterization of these low-affinity recombinant transporter proteins in heterologous expression systems has revealed that they are not antagonized by classical inhibitors of SERT, DAT, or NET but that decynium-22 (D22) antagonizes OCT3 and PMAT, whereas corticosterone and progesterone selectively inhibit OCT3. Here, we show that SERT, PMAT, and OCT3, but not OCT1 and OCT2, are coexpressed in murine stem cell-derived serotonergic neurons. Using selective antagonists, we provide evidence that uptake of the fluorescent substrates FFN511, ASP+, and 5-HT into stem cell-derived serotonergic neurons is mediated differentially by these transporters and also involves an as yet unknown transport mechanism.

Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins.

PubMed

Marinelli, Fabrizio; Kuhlmann, Sonja I; Grell, Ernst; Kunte, Hans-Jörg; Ziegler, Christine; Faraldo-Gómez, José D

2011-12-06

Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na(+)-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581(T), and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former.

High-Affinity Accumulation of Chloroquine by Mouse Erythrocytes Infected with Plasmodium berghei

PubMed Central

Fitch, Coy D.; Yunis, Norman G.; Chevli, Rekha; Gonzalez, Yolanda

1974-01-01

Washed erythrocytes infected with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) P. berghei were used in model systems in vitro to study the accumulation of chloroquine with high affinity. The CS model could achieve distribution ratios (chloroquine in cells: chloroquine in medium) of 100 in the absence of substrate. 200—300 in the presence of 10 mM pyruvate or lactate, and over 600 in the presence of 1 mM glucose or glycerol. In comparable studies of the CR model, the distribution ratios were 100 in the absence of substrate and 300 or less in the presence of glucose or glycerol. The presence of lactate stimulated chloroquine accumulation in the CR model, whereas the presence of pyruvate did not. Lactate production from glucose and glycerol was undiminished in the CR model, and ATP concentrations were higher than in the CS model. Cold, iodoacetate, 2,4-dinitrophenol, or decreasing pH inhibited chloroquine accumulation in both models. These findings demonstrate substrate involvement in the accumulation of chloroquine with high affinity. In studies of the CS model, certain compounds competitively inhibited chloroquine accumulation, while others did not. This finding is attributable to a specific receptor that imposes structural constraints on the process of accumulation. For chloroquine analogues, the position and length of the side chain, the terminal nitrogen atom of the side chain, and the nitrogen atom in the quinoline ring are important determinants of binding to this receptor. PMID:4600044

Self-Powered Wireless Affinity-Based Biosensor Based on Integration of Paper-Based Microfluidics and Self-Assembled RFID Antennas.

PubMed

Yuan, Mingquan; Alocilja, Evangelyn C; Chakrabartty, Shantanu

2016-08-01

This paper presents a wireless, self-powered, affinity-based biosensor based on the integration of paper-based microfluidics with our previously reported method for self-assembling radio-frequency (RF) antennas. At the core of the proposed approach is a silver-enhancement technique that grows portions of a RF antenna in regions where target antigens hybridize with target specific affinity probes. The hybridization regions are defined by a network of nitrocellulose based microfluidic channels which implement a self-powered approach to sample the reagent and control its flow and mixing. The integration substrate for the biosensor has been constructed using polyethylene and the patterning of the antenna on the substrate has been achieved using a low-cost ink-jet printing technique. The substrate has been integrated with passive radio-frequency identification (RFID) tags to demonstrate that the resulting sensor-tag can be used for continuous monitoring in a food supply-chain where direct measurement of analytes is typically considered to be impractical. We validate the proof-of-concept operation of the proposed sensor-tag using IgG as a model analyte and using a 915 MHz Ultra-high-frequency (UHF) RFID tagging technology.

Nickel(II) Inhibits Tet-Mediated 5-Methylcytosine Oxidation by High Affinity Displacement of the Cofactor Iron(II).

PubMed

Yin, Ruichuan; Mo, Jiezhen; Dai, Jiayin; Wang, Hailin

2017-06-16

Ten-eleven translocation (Tet) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that regulate the dynamics of DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine (5mC). To exert physiologically important functions, redox-active iron chelated in the catalytic center of Tet proteins directly involves the oxidation of the multiple substrates. To understand the function and interaction network of Tet dioxygenases, it is interesting to obtain high affinity and a specific inhibitor. Surprisingly, here we found that natural Ni(II) ion can bind to the Fe(II)-chelating motif (HXD) with an affinity of 7.5-fold as high as Fe(II). Consistently, we further found that Ni(II) ion can displace the cofactor Fe(II) of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity with an estimated IC 50 of 1.2 μM. Essentially, Ni(II) can be used as a high affinity and selective inhibitor to explore the function and dynamics of Tet proteins.

Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity

PubMed Central

Jansen, Mickel L. A.; Daran-Lapujade, Pascale; de Winde, Johannes H.; Piper, Matthew D. W.; Pronk, Jack T.

2004-01-01

Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport. PMID:15066785

Na+ Interactions with the Neutral Amino Acid Transporter ASCT1*

PubMed Central

Scopelliti, Amanda J.; Heinzelmann, Germano; Kuyucak, Serdar; Ryan, Renae M.; Vandenberg, Robert J.

2014-01-01

The alanine, serine, cysteine transporters (ASCTs) belong to the solute carrier family 1A (SLC1A), which also includes the excitatory amino acid transporters (EAATs) and the prokaryotic aspartate transporter GltPh. Acidic amino acid transport by the EAATs is coupled to the co-transport of three Na+ ions and one proton, and the counter-transport of one K+ ion. In contrast, neutral amino acid exchange by the ASCTs does not require protons or the counter-transport of K+ ions and the number of Na+ ions required is not well established. One property common to SLC1A family members is a substrate-activated anion conductance. We have investigated the number and location of Na+ ions required by ASCT1 by mutating residues in ASCT1 that correspond to residues in the EAATs and GltPh that are involved in Na+ binding. Mutations to all three proposed Na+ sites influence the binding of substrate and/or Na+, or the rate of substrate exchange. A G422S mutation near the Na2 site reduced Na+ affinity, without affecting the rate of exchange. D467T and D467A mutations in the Na1 site reduce Na+ and substrate affinity and also the rate of substrate exchange. T124A and D380A mutations in the Na3 site selectively reduce the affinity for Na+ and the rate of substrate exchange without affecting substrate affinity. In many of the mutants that reduce the rate of substrate transport the amplitudes of the substrate-activated anion conductances are not substantially affected indicating altered ion dependence for channel activation compared with substrate exchange. PMID:24808181

Why do receptor–ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

DOE PAGES

Gao, Zhiwen; Gao, Yanfei

2016-05-14

We report that cell adhesion often exhibits the clustering of the receptor–ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation formore » the clustering/assembling of the receptor–ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor–ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Finally, cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.« less

Why do receptor–ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Zhiwen; Gao, Yanfei

We report that cell adhesion often exhibits the clustering of the receptor–ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation formore » the clustering/assembling of the receptor–ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor–ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Finally, cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.« less

Surface Effects and Challenges for Application of Piezoelectric Langasite Substrates in Surface Acoustic Wave Devices Caused by High Temperature Annealing under High Vacuum.

PubMed

Seifert, Marietta; Rane, Gayatri K; Kirbus, Benjamin; Menzel, Siegfried B; Gemming, Thomas

2015-12-19

Substrate materials that are high-temperature stable are essential for sensor devices which are applied at high temperatures. Although langasite is suggested as such a material, severe O and Ga diffusion into an O-affine deposited film was observed during annealing at high temperatures under vacuum conditions, leading to a damage of the metallization as well as a change of the properties of the substrate and finally to a failure of the device. Therefore, annealing of bare LGS (La 3 Ga 5 SiO 14 ) substrates at 800 ∘ C under high vacuum conditions is performed to analyze whether this pretreatment improves the suitability and stability of this material for high temperature applications in vacuum. To reveal the influence of the pretreatment on the subsequently deposited metallization, RuAl thin films are used as they are known to oxidize on LGS at high temperatures. A local study of the pretreated and metallized substrates using transmission electron microscopy reveals strong modification of the substrate surface. Micro cracks are visible. The composition of the substrate is strongly altered at those regions. Severe challenges for the application of LGS substrates under high-temperature vacuum conditions arise from these substrate damages, revealing that the pretreatment does not improve the applicability.

Enhanced Cycling Stability of Sulfur Electrodes through Effective Binding of Pyridine-Functionalized Polymer

DOE PAGES

Tsao, Yuchi; Chen, Zheng; Rondeau-Gagne, Simon; ...

2017-09-20

Porous carbons have previously been widely used as host materials for sulfur (S) electrodes because of their high conductivity and high surface area. However, they generally lack strong chemical affinity to stabilize polysulfide species. Therefore, conducting polymers have been employed to stabilize S electrodes. Integrating conducting polymers with high-surface-area carbons can create a new materials platform and synergize their functions. However, the previously used conducting polymers were often insoluble, and coating them uniformly from solution onto a nonpolar carbon substrate is a challenge. Here, we report that solution-processable isoindigo-based polymers incorporating polar substituents provide critical features: the conjugated backbone providesmore » good conductivity; functional pyridine groups provide high affinity to polysulfide species; and they possess high solubility in organic solvents. Here, these lead to effective coating on various carbonaceous substrates to provide highly stable sulfur electrodes. Importantly, the electrodes exhibit good capacity retention (80% over 300 cycles) at sulfur mass loading of 3.2 mg/cm 2, which significantly surpasses the performance of others reported in polymer-enabled sulfur cathodes.« less

The position of an arginine residue influences substrate affinity and K+ coupling in the human glutamate transporter, EAAT1.

PubMed

Ryan, Renae M; Kortt, Nicholas C; Sirivanta, Tan; Vandenberg, Robert J

2010-07-01

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and extracellular glutamate levels are controlled by a family of transporters known as excitatory amino acid transporters (EAATs). The EAATs transport glutamate and aspartate with similar micromolar affinities and this transport is coupled to the movement of Na(+), K(+), and H(+). The crystal structure of a prokaryotic homologue of the EAATs, aspartate transporter from Pyrococcus horokoshii (Glt(Ph)), has yielded important insights into the architecture of this transporter family. Glt(Ph) is a Na(+)-dependent transporter that has significantly higher affinity for aspartate over glutamate and is not coupled to H(+) or K(+). The highly conserved carboxy-terminal domains of the EAATs and Glt(Ph) contain the substrate and ion binding sites, however, there are a couple of striking differences in this region that we have investigated to better understand the transport mechanism. An arginine residue is in close proximity to the substrate binding site of both Glt(Ph) and the EAATs, but is located in transmembrane domain (TM) 8 in the EAATs and hairpin loop 1 (HP1) of Glt(Ph). Here we report that the position of this arginine residue can explain some of the functional differences observed between the EAATs and Glt(Ph). Moving the arginine residue from TM8 to HP1 in EAAT1 results in a transporter that has significantly increased affinity for both glutamate and aspartate and is K(+) independent. Conversely, moving the arginine residue from HP1 to TM8 in Glt(Ph) results in a transporter that has reduced affinity for aspartate.

Loop engineering reveals the importance of active-site-decorating loops and gating residue in substrate affinity modulation of arginine deiminase (an anti-tumor enzyme).

PubMed

Cheng, Feng; Yang, Jianhua; Bocola, Marco; Schwaneberg, Ulrich; Zhu, Leilei

2018-05-05

Protein engineering of enzyme loop regions is an effective strategy to improve enzymatic properties. Previous studies that aimed to boost the activity of PpADI (an arginine deiminase from Pseudomonas plecoglossicida) under physiological conditions yielded several significantly improved variants that harbor substitutions predominantly located in active-site-decorating loops. A multi-site saturation mutagenesis at four positions in loop 1 (37, 38, 42, and 43) and three positions in loop 4 (402, 403, and 404) was performed to elucidate the importance of these loops in modulating the substrate affinity of PpADI. The identified "best" variant (M6-L1-4) showed a decreased S 0.5 ('K M ') of 0.48 mM compared with the parent M6 (0.81 mM). Subsequently, a rational design to recombine beneficial substitutions within loops 1 and 4 yielded variant L6 with a substantially decreased S 0.5 value (0.17 mM). A comprehensive simulation analysis resulted in a conclusion that high loop flexibility (especially the gating residue Arg400) is beneficial for substrate affinity due to less efficient blocking of the active site. Copyright © 2018 Elsevier Inc. All rights reserved.

Kinetics of N-Utilization By Natural Phytoplankton Assemblages During Upwelling Events At The NW Iberian Shelf

NASA Astrophysics Data System (ADS)

Brion, N.; Elskens, M.; Dehairs, F.; Baeyens, W.

2003-04-01

The concentration-dependent uptakes of nitrate, ammonium and the effect of ammo-nium on the f-ratio were surveyed in surface waters of the NW Iberian shelf during June 1997, July 1998 and September 1999. Because relationships between rates and substrate concentrations were quite variable, ranging from linear to convex shaped curves, they were fitted to rational functions. Stepwize regression analysis yielded subsequent model equations with reasonable statistical properties which allowed describing all but all a few cases. Differentiating these equations with respect to the concentration gave the slope of the tangent to the curve, i.e., the variation in rate expected for a given perturbation of the ambient substrate concentration. The initial slope value was then used as an index to gauge the "affinity" of the plankton community for the nitrogen substrate utilization. In June 1997, the situation at the Iberian shelf showed no upwelling except near Cape Finistère. Overall, the phytoplankton community displayed a high "affinity" for both nitrate and ammonium and low f-ratio values, which is indicative of a re-generated production regime. High ammonium regeneration rates supported furthermore these observations. It was also demonstrated that the new production rates is only marginally sensitive to changes of the ambient nitrate and/or ammonium concentrations. This indicates that the production regime is rather stable throughout. Only at Cape Finistère, nitrate concentrations were high reflecting the onset of an upwelling event. In this zone, the phytoplankton community, taking advantage of its high affinity for nitrate enhanced both total N-uptake rate and f-ratio. In July 1998, the situation evolved towards an extension to the south of the upwelling event starting at Cape Finistère. In this southern zone of the upwelling the phytoplankton community displayed generally a lower affinity for nitrate (but not for ammonium) than in 1997. In spite of this lower affinity, nitrate uptake rate was dominant resulting in f-ratio values greater than 0.5, a characteristic of a new production regime. The new production rate is only marginally sensitive to increases of the ambient nitrate, but is drastically inhibited by small increases of the ambient ammonium. The situation of September 1999 was very close to that observed in July 1998, with higher nitrate concentrations in the coastal northern part of the sampling area dominated by upwelling.

Malathion-induced inhibition of human plasma cholinesterase studied by the fluorescence spectroscopy method

NASA Astrophysics Data System (ADS)

Pavelkić, V. M.; Krinulović, K. S.; Savić, J. Z.; Ilić, M. A.

2008-05-01

The in vitro effect of technical grade malathion was assessed via the kinetic parameters of human plasma butyrylcholinesterase (BChE) using N-methylindoxyl acetate as a substrate for BChE. An inhibitor kinetics study demonstrated the existence of a biphasic inhibition curve, indicating high-and low-affinity binding sites of malathion. The IC 50 values as calculated from the experimental inhibition curves were 1.33 × 10-9 and 1.48 × 10-5 M for the high-and low-affinity binding sites, respectively; Hill’s analysis gave 1.29 × 10-9 and 1.38 × 10-6 M. The Cornish-Bowden plots and their secondary plots indicated that the nature of inhibition was of mixed type with the predominant competitive character of both affinity binding sites.

Promotion of pro-osteogenic responses by a bioactive ceramic coating.

PubMed

Aniket; Young, Amy; Marriott, Ian; El-Ghannam, Ahmed

2012-12-01

The objective of this study was to analyze the responses of bone-forming osteoblasts to Ti-6Al-4V implant material coated with silica-calcium phosphate nanocomposite (SCPC50). Osteoblast differentiation at the interface with SCPC50-coated Ti-6Al-4V was correlated to the adsorption of high amount of serum proteins, high surface affinity to fibronectin, Ca uptake from and P and Si release into the medium. SCPC50-coated Ti-6Al-4V adsorbed significantly more serum protein (p < 0.05) than control uncoated substrates. Moreover, Western blot analysis showed that the SCPC50 coating had a high affinity for serum fibronectin. Protein conformation analyses by FTIR showed that the ratio of the area under the peak for amide I/amide II bands was significantly higher (p < 0.05) on the surface of SCPC50-coated substrates than that on the surface of the control uncoated substrates. Moreover, ICP - OES analyses indicated that SCPC50-coated substrates withdrew Ca ions from, and released P and Si ions into, the tissue culture medium, respectively. In conjunction with the favorable protein adsorption and modifications in medium composition, MC3T3-E1 osteoblast-like cells attached to SCPC50-coated substrates expressed 10-fold higher level of mRNA encoding osteocalcin and had significantly higher production of osteopontin and osteocalcin proteins than cells attached to the uncoated Ti-6A1-4V substrates. In addition, osteoblast-like cells attached to the SCPC50-coated substrates produced significantly lower levels of the inflammatory and osteoclastogenic cytokines, IL-6, IL-12p40, and RANKL than those attached to uncoated Ti-6Al-4V substrates. These results suggest that SCPC50 coating could enhance bone integration with orthopedic and maxillofacial implants while minimizing the induction of inflammatory bone cell responses. Copyright © 2012 Wiley Periodicals, Inc.

High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

PubMed Central

Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

2016-01-01

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

Facile fabrication of Ag dendrite-integrated anodic aluminum oxide membrane as effective three-dimensional SERS substrate

NASA Astrophysics Data System (ADS)

Zhang, Cong-yun; Lu, Ya; Zhao, Bin; Hao, Yao-wu; Liu, Ya-qing

2016-07-01

A novel surface enhanced Raman scattering (SERS)-active substrate has been successfully developed, where Ag-dendrites are assembled on the surface and embedded in the channels of anodic aluminum oxide (AAO) membrane, via electrodeposition in AgNO3/PVP aqueous system. Reaction conditions were systematically investigated to attain the best Raman enhancement. The growth mechanism of Ag dendritic nanostructures has been proposed. The Ag dendrite-integrated AAO membrane with unique hierarchical structures exhibits high SERS activity for detecting rhodamine 6G with a detection limit as low as 1 × 10-11 M. Furthermore, the three-dimensional (3D) substrates display a good reproducibility with the average intensity variations at the major Raman peak less than 12%. Most importantly, the 3D SERS substrates without any surface modification show an outstanding SERS response for the molecules with weak affinity for noble metal surfaces. The potential application for the detection of polycyclic aromatic hydrocarbons (PAHs) was evaluated with fluoranthene as Raman target molecule and a sensitive SERS detection with a limit down to 10-8 M was reached. The 3D SERS-active substrate shows promising potential for rapid detection of trace organic pollutants even weak affinity molecules in the environment.

Functional expression, production, and biochemical characterization of a laccase using yeast surface display technology.

PubMed

Bertrand, Brandt; Trejo-Hernández, María R; Morales-Guzmán, Daniel; Caspeta, Luis; Suárez Rodríguez, Ramón; Martínez-Morales, Fernando

2016-12-01

A Trametes versicolor laccase was functionally expressed on the membrane surface of Saccharomyces cerevisiae EBY100. Laccase expression was increased 6.57-fold by medium optimization and surpassed production by the native strain. Maximal laccase and biomass production reached 19 735 ± 1719 Ug -1 and 6.22 ± 0.53 gL -1 respectively, after 2 d of culture. Optimum oxidization of all substrates by laccase was observed at pH 3. Laccase showed high affinity towards substrates used with Km (mM) and Vmax (μmol min -1 ) values of 0.57 ± 0.0047 and 24.55 ± 0.64, 1.52 ± 0.52 and 9.25 ± 1.78, and 2.67 ± 0.12 and 11.26 ± 0.75, were reported for ABTS, 2, 6-DMP and GUA, respectively. EDTA and NaN 3 displayed none competitive inhibition towards laccase activity. The optimum temperature for activity was 50 °C; however, the enzyme was stable over a wide range of temperatures (25-70 °C). The biologically immobilized laccase showed high reusability towards phenolic substrates and low reusability with non-phenolic substrates. High affinity for a diversity phenolic compounds and great ethanol tolerance substantiates this laccase/yeast biocatalyst potential for application in the production of bioethanol. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

PubMed

Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

2016-10-01

Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. Copyright © 2016 Elsevier B.V. All rights reserved.

Elucidation of the factors affecting the oxidative activity of Acremonium sp. HI-25 ascorbate oxidase by an electrochemical approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murata, Kenichi; Nakamura, Nobuhumi; Ohno, Hiroyuki

Steady-state kinetics of Acremonium sp. HI-25 ascorbate oxidase toward p-hydroquinone derivatives have been examined by using an electrochemical analysis based on the theory of steady-state bioelectrocatalysis. The electrochemical technique has enabled one to examine the influence of electronic and chemical properties of substrates on the activity. It was proven that the oxidative activity of ascorbate oxidase was dominated by the highly selective substrate-binding affinity based on electrostatic interaction beyond the one-electron redox potential difference between ascorbate oxidase's type 1 copper site and substrate.

Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle

PubMed Central

Kits, K. Dimitri; Sedlacek, Christopher J.; Lebedeva, Elena V.; Han, Ping; Bulaev, Alexandr; Pjevac, Petra; Daebeler, Anne; Romano, Stefano; Albertsen, Mads; Stein, Lisa Y.; Daims, Holger; Wagner, Michael

2017-01-01

Summary paragraph Nitrification, the oxidation of ammonia (NH3) via nitrite (NO2-) to nitrate (NO3-), is a key process of the biogeochemical nitrogen cycle. For decades, ammonia and nitrite oxidation were thought to be separately catalyzed by ammonia-oxidizing bacteria (AOB) and archaea (AOA), and by nitrite-oxidizing bacteria (NOB). The recent discovery of complete ammonia oxidizers (comammox) in the NOB genus Nitrospira1,2, which alone convert ammonia to nitrate, raised questions about the ecological niches where comammox Nitrospira successfully compete with canonical nitrifiers. Here we isolated the first pure culture of a comammox bacterium, Nitrospira inopinata, and show that it is adapted to slow growth in oligotrophic and dynamic habitats based on a high affinity for ammonia, low maximum rate of ammonia oxidation, high growth yield compared to canonical nitrifiers, and genomic potential for alternative metabolisms. The nitrification kinetics of four AOA from soil and hot springs were determined for comparison. Their surprisingly poor substrate affinities and lower growth yields reveal that, in contrast to earlier assumptions, not all AOA are most competitive in strongly oligotrophic environments and that N. inopinata has the highest substrate affinity of all analyzed ammonia oxidizer isolates except the marine AOA Nitrosopumilus maritimus SCM13. These results suggest a role of comammox organisms for nitrification under oligotrophic and dynamic conditions. PMID:28847001

Substrate specificity of the violaxanthin de-epoxidase of the primitive green alga Mantoniella squamata (Prasinophyceae).

PubMed

Goss, Reimund

2003-09-01

The substrate specificity of the enzyme violaxanthin de-epoxidase (VDE) of the primitive green alga Mantoniella squamata (Prasinophyceae) was tested in in vitro enzyme assays employing the following xanthophyll mono-epoxides: antheraxanthin (Ax), diadinoxanthin (Ddx), lutein-epoxide (LE), cryptoxanthin-epoxide (CxE), 9- cis neoxanthin (cNx), all- trans neoxanthin (Nx), and xanthophyll di-epoxides: 9- cis violaxanthin (cVx), all- trans violaxanthin (Vx), cryptoxanthin-di-epoxide (CxDE). The data presented in this study show that the VDE of M. squamata not only exhibits a low affinity for the mono-epoxide Ax, as has been reported by R. Frommolt et al. (2001, Planta 213:446-456), but has a reduced substrate affinity for the mono-epoxides Ddx, LE, CxE, and Nx as well. On the other hand, xanthophylls with a second epoxy-group (Vx, CxDE) can be de-epoxidized with a higher efficiency. Such a preference for xanthophyll di-epoxides cannot be observed for the higher-plant VDE, where, in general, no marked differences in the pigment de-epoxidation rates between xanthophyll mono- and di-epoxides are visible. Despite this substantial difference between the VDEs of M. squamata and S. oleracea there are also features common to both enzymes. Neither VDE is able to convert xanthophylls with a 9- cis configuration in the acyclic polyene chain and both rely on substrates in the all- trans configuration. Both enzymes furthermore exhibit a dependence of enzyme activity on the polarity of the substrate. Highly polar (Nx) or non-polar (CxE) xanthophylls are de-epoxidized with greatly reduced rates in comparison to substrates with an intermediate polarity (Vx, Ax, LE, Ddx). This dependence on substrate polarity becomes more obvious when the higher-plant VDE is examined, as the substrate affinity of the VDE of M. squamata is more strongly influenced by the existence or absence of a second epoxy-group. In summary, the data presented in this study underline the fact that different VDEs, although in general catalyzing the same reaction sequence, are functionally diverse.

Biphasic association of T7 RNA polymerase and a nucleotide analogue, cibacron blue as a model to understand the role of initiating nucleotide in the mechanism of enzyme action.

PubMed

Pai, Sudipta; Das, Mili; Banerjee, Rahul; Dasgupta, Dipak

2011-08-01

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. Here we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, its effect on the function of the enzyme and the probable mode of binding of the dye. We have used difference absorption spectroscopy and isothermal titration calorimetry to show that the dye binds T7 RNAP in a biphasic manner. The first phase of the binding is characterized by inactivation of the enzyme. The second binding site overlaps with the common substrate-binding site of the enzyme. We have carried out docking experiment to map the binding site of the dye in the promoter bound protein. Competitive displacement of the dye from the high affinity site by labeled GTP and isothermal titration calorimetry of high affinity GTP bound enzyme with the dye suggests a strong correlation between the high affinity dye binding and the high affinity GTP binding in T7 RNAP reported earlier from our laboratory.

Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability.

PubMed

Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G; Ludwig, Roland

2018-02-02

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa , which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

PubMed

Singh, Appu Kumar; Ekka, Mary Krishna; Kaushik, Abhishek; Pandya, Vaibhav; Singh, Ravi P; Banerjee, Shrijita; Mittal, Monica; Singh, Vijay; Kumaran, S

2017-09-19

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (10 4 -10 6 ) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters

PubMed Central

Diallinas, George

2014-01-01

Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action. PMID:25309439

Substrate specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa.

PubMed

Wylie, J L; Worobec, E A

1993-07-01

Specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa was examined. At a concentration of [14C]glucose near the Vmax of the system, inhibition by maltose, galactose, and xylose was detected. This inhibition is similar to that detected in earlier in vivo studies and correlates with the known specificity of OprB, a glucose-specific porin of P. aeruginosa. At a level of [14C]glucose 100 times lower, only unlabelled glucose inhibited uptake to any extent. This matches the known in vitro specificity of the periplasmic glucose binding protein. These findings were used to explain the discrepancy between earlier in vivo and in vitro results reported in the literature.

Properties of Fructan:Fructan 1-Fructosyltransferases from Chicory and Globe Thistle, Two Asteracean Plants Storing Greatly Different Types of Inulin1

PubMed Central

Vergauwen, Rudy; Van Laere, André; Van den Ende, Wim

2003-01-01

Remarkably, within the Asteraceae, a species-specific fructan pattern can be observed. Some species such as artichoke (Cynara scolymus) and globe thistle (Echinops ritro) store fructans with a considerably higher degree of polymerization than the one observed in chicory (Cichorium intybus) and Jerusalem artichoke (Helianthus tuberosus). Fructan:fructan 1-fructosyltransferase (1-FFT) is the enzyme responsible for chain elongation of inulin-type fructans. 1-FFTs were purified from chicory and globe thistle. A comparison revealed that chicory 1-FFT has a high affinity for sucrose (Suc), fructose (Fru), and 1-kestose as acceptor substrate. This makes redistribution of Fru moieties from large to small fructans very likely during the period of active fructan synthesis in the root when import and concentration of Suc can be expected to be high. In globe thistle, this problem is avoided by the very low affinity of 1-FFT for Suc, Fru, and 1-kestose and the higher affinity for inulin as acceptor substrate. Therefore, the 1-kestose formed by Suc:Suc 1-fructosyltransferase is preferentially used for elongation of inulin molecules, explaining why inulins with a much higher degree of polymerization accumulate in roots of globe thistle. Inulin patterns obtained in vitro from 1-kestose and the purified 1-FFTs from both species closely resemble the in vivo inulin patterns. Therefore, we conclude that the species-specific fructan pattern within the Asteraceae can be explained by the different characteristics of their respective 1-FFTs. Although 1-FFT and bacterial levansucrases clearly differ in their ability to use Suc as a donor substrate, a kinetic analysis suggests that 1-FFT also works via a ping-pong mechanism. PMID:12970504

Efficient RNA pseudouridylation by eukaryotic H/ACA ribonucleoproteins requires high affinity binding and correct positioning of guide RNA

PubMed Central

Caton, Evan A; Kelly, Erin K; Kamalampeta, Rajashekhar

2018-01-01

Abstract H/ACA ribonucleoproteins (H/ACA RNPs) are responsible for introducing many pseudouridines into RNAs, but are also involved in other cellular functions. Utilizing a purified and reconstituted yeast H/ACA RNP system that is active in pseudouridine formation under physiological conditions, we describe here the quantitative characterization of H/ACA RNP formation and function. This analysis reveals a surprisingly tight interaction of H/ACA guide RNA with the Cbf5p–Nop10p–Gar1p trimeric protein complex whereas Nhp2p binds comparably weakly to H/ACA guide RNA. Substrate RNA is bound to H/ACA RNPs with nanomolar affinity which correlates with the GC content in the guide-substrate RNA base pairing. Both Nhp2p and the conserved Box ACA element in guide RNA are required for efficient pseudouridine formation, but not for guide RNA or substrate RNA binding. These results suggest that Nhp2p and the Box ACA motif indirectly facilitate loading of the substrate RNA in the catalytic site of Cbf5p by correctly positioning the upper and lower parts of the H/ACA guide RNA on the H/ACA proteins. In summary, this study provides detailed insight into the molecular mechanism of H/ACA RNPs. PMID:29177505

Endoprotease profiling with double-tagged peptide substrates: a new diagnostic approach in oncology.

PubMed

Peccerella, Teresa; Lukan, Nadine; Hofheinz, Ralf; Schadendorf, Dirk; Kostrezewa, Markus; Neumaier, Michael; Findeisen, Peter

2010-02-01

The measurement of disease-related proteolytic activity in complex biological matrices like serum is of emerging interest to improve the diagnosis of malignant diseases. We developed a mass spectrometry (MS)-based functional proteomic profiling approach that tracks degradation of artificial endoprotease substrates in serum specimens. The synthetic reporter peptides that are cleaved by tumor-associated endopeptidases were systematically optimized with regard to flanking affinity tags, linkers, and stabilizing elements. Serum specimens were incubated with reporter peptides under standardized conditions and the peptides subsequently extracted with affinity chromatography before MS. In a pilot study an optimized reporter peptide with the cleavage motif WKPYDAADL was added to serum specimens from colorectal tumor patients (n = 50) and healthy controls (n = 50). This reporter peptide comprised a known cleavage site for the cysteine-endopeptidase "cancer procoagulant." Serial affinity chromatography using biotin- and 6xHis tags was superior to the single affinity enrichment using only 6xHis tags. Furthermore, protease-resistant stop elements ensured signal accumulation after prolonged incubation. In contrast, signals from reporter peptides without stop elements vanished completely after prolonged incubation owing to their total degradation. Reporter-peptide spiking showed good reproducibility, and the difference in proteolytic activity between serum specimens from cancer patients and controls was highly significant (P < 0.001). The introduction of a few structural key elements (affinity tags, linkers, d-amino acids) into synthetic reporter peptides increases the diagnostic sensitivity for MS-based protease profiling of serum specimens. This new approach might lead to functional MS-based protease profiling for improved disease classification.

The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin TransporterⓈ

PubMed Central

Sarker, Subhodeep; Weissensteiner, René; Steiner, Ilka; Sitte, Harald H.; Ecker, Gerhard F.; Freissmuth, Michael; Sucic, Sonja

2015-01-01

The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuTAa) has been used as a model for mammalian Na+/Cl−-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuTAa liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERTY95F mutation, which greatly reduced the affinity for [3H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [3H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP+, paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants. PMID:20829432

Molecular recognition of pre-tRNA by Arabidopsis protein-only Ribonuclease P.

PubMed

Klemm, Bradley P; Karasik, Agnes; Kaitany, Kipchumba J; Shanmuganathan, Aranganathan; Henley, Matthew J; Thelen, Adam Z; Dewar, Allison J L; Jackson, Nathaniel D; Koutmos, Markos; Fierke, Carol A

2017-12-01

Protein-only ribonuclease P (PRORP) is an enzyme responsible for catalyzing the 5' end maturation of precursor transfer ribonucleic acids (pre-tRNAs) encoded by various cellular compartments in many eukaryotes. PRORPs from plants act as single-subunit enzymes and have been used as a model system for analyzing the function of the metazoan PRORP nuclease subunit, which requires two additional proteins for efficient catalysis. There are currently few molecular details known about the PRORP-pre-tRNA complex. Here, we characterize the determinants of substrate recognition by the single subunit Arabidopsis thaliana PRORP1 and PRORP2 using kinetic and thermodynamic experiments. The salt dependence of binding affinity suggests 4-5 contacts with backbone phosphodiester bonds on substrates, including a single phosphodiester contact with the pre-tRNA 5' leader, consistent with prior reports of short leader requirements. PRORPs contain an N-terminal pentatricopeptide repeat (PPR) domain, truncation of which results in a >30-fold decrease in substrate affinity. While most PPR-containing proteins have been implicated in single-stranded sequence-specific RNA recognition, we find that the PPR motifs of PRORPs recognize pre-tRNA substrates differently. Notably, the PPR domain residues most important for substrate binding in PRORPs do not correspond to positions involved in base recognition in other PPR proteins. Several of these residues are highly conserved in PRORPs from algae, plants, and metazoans, suggesting a conserved strategy for substrate recognition by the PRORP PPR domain. Furthermore, there is no evidence for sequence-specific interactions. This work clarifies molecular determinants of PRORP-substrate recognition and provides a new predictive model for the PRORP-substrate complex. © 2017 Klemm et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

PubMed Central

Ingram-Smith, Cheryl; Smith, Kerry S.

2007-01-01

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1. PMID:17350930

Cholinergic Receptor Substrates of Neuronal Plasticity and Learning

DTIC Science & Technology

1992-01-29

cortical binding of 3H- oxotremorine (OXO), a ligand having high affinity for M2 muscarinic receptors, are described in a manuscript by Vogt, Gabriel...of 1H Oxotremorine co-incubated with Pirenzepine (OXO-M/PZ) throughout the course of training in three thalamic nudei. -L As in the case of training

Systematic Localization and Identification of SUMOylation Substrates in Knock-In Mice Expressing Affinity-Tagged SUMO1.

PubMed

Tirard, Marilyn; Brose, Nils

2016-01-01

Protein SUMOylation is a posttranslational protein modification that is emerging as a key regulatory process in neurobiology. To date, however, SUMOylation in vivo has only been studied cursorily. Knock-in mice expressing His6-HA-SUMO1 from the Sumo1 locus allow for the highly specific localization and identification of endogenous SUMO1 substrates under physiological and pathophysiological conditions. By making use of the HA-tag and using wild-type mice for highly stringent negative control samples, SUMO1 targets can be specifically localized in and purified from cultured mouse nerve cells and mouse tissues.

Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2.

PubMed

Usami, Yuki; Uemura, Satsohi; Mochizuki, Takahiro; Morita, Asami; Shishido, Fumi; Inokuchi, Jin-ichi; Abe, Fumiyoshi

2014-07-01

Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition. Copyright © 2014 Elsevier B.V. All rights reserved.

Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters

PubMed Central

Ho, Cheng-Hsun; Frommer, Wolf B

2014-01-01

To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4, and 5 into fluorescence activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactome screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research. DOI: http://dx.doi.org/10.7554/eLife.01917.001 PMID:24623305

Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

PubMed

Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

2014-12-19

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure of Transmembrane Domain of Lysosome-associated Membrane Protein Type 2a (LAMP-2A) Reveals Key Features for Substrate Specificity in Chaperone-mediated Autophagy*

PubMed Central

Rout, Ashok K.; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

2014-01-01

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation. PMID:25342746

A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

PubMed Central

Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

2015-01-01

Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

Low-Temperature (10°C) Anaerobic Digestion of Dilute Dairy Wastewater in an EGSB Bioreactor: Microbial Community Structure, Population Dynamics, and Kinetics of Methanogenic Populations

PubMed Central

Cysneiros, Denise; O'Flaherty, Vincent

2013-01-01

The feasibility of anaerobic digestion of dairy wastewater at 10°C was investigated in a high height : diameter ratio EGSB reactor. Stable performance was observed at an applied organic loading rate (OLR) of 0.5–2 kg COD m−3 d−1 with chemical oxygen demand (COD) removal efficiencies above 85%. When applied OLR increased to values above 2 kg COD m−3 d−1, biotreatment efficiency deteriorated, with methanogenesis being the rate-limiting step. The bioreactor recovered quickly (3 days) after reduction of the OLR. qPCR results showed a reduction in the abundance of hydrogenotrophic methanogenic Methanomicrobiales and Methanobacteriales throughout the steady state period followed by a sharp increase in their numbers (111-fold) after the load shock. Specific methanogenic activity and maximum substrate utilising rate (A max) of the biomass at the end of trial indicated increased activity and preference towards hydrogenotrophic methanogenesis, which correlated well with the increased abundance of hydrogenotrophic methanogens. Acetoclastic Methanosaeta spp. remained at stable levels throughout the trial. However, increased apparent half-saturation constant (K m) at the end of the trial indicated a decrease in the specific substrate affinity for acetate of the sludge, suggesting that Methanosaeta spp., which have high substrate affinity, started to be outcompeted in the reactor. PMID:24089597

Low-temperature (10°C) anaerobic digestion of dilute dairy wastewater in an EGSB bioreactor: microbial community structure, population dynamics, and kinetics of methanogenic populations.

PubMed

Bialek, Katarzyna; Cysneiros, Denise; O'Flaherty, Vincent

2013-01-01

The feasibility of anaerobic digestion of dairy wastewater at 10°C was investigated in a high height : diameter ratio EGSB reactor. Stable performance was observed at an applied organic loading rate (OLR) of 0.5-2 kg COD m(-3) d(-1) with chemical oxygen demand (COD) removal efficiencies above 85%. When applied OLR increased to values above 2 kg COD m(-3) d(-1), biotreatment efficiency deteriorated, with methanogenesis being the rate-limiting step. The bioreactor recovered quickly (3 days) after reduction of the OLR. qPCR results showed a reduction in the abundance of hydrogenotrophic methanogenic Methanomicrobiales and Methanobacteriales throughout the steady state period followed by a sharp increase in their numbers (111-fold) after the load shock. Specific methanogenic activity and maximum substrate utilising rate (A(max)) of the biomass at the end of trial indicated increased activity and preference towards hydrogenotrophic methanogenesis, which correlated well with the increased abundance of hydrogenotrophic methanogens. Acetoclastic Methanosaeta spp. remained at stable levels throughout the trial. However, increased apparent half-saturation constant (K(m)) at the end of the trial indicated a decrease in the specific substrate affinity for acetate of the sludge, suggesting that Methanosaeta spp., which have high substrate affinity, started to be outcompeted in the reactor.

Impact of M36I polymorphism on the interaction of HIV-1 protease with its substrates: insights from molecular dynamics

PubMed Central

2014-01-01

Background Over the last decades, a vast structural knowledge has been gathered on the HIV-1 protease (PR). Noticeably, most of the studies focused the B-subtype, which has the highest prevalence in developed countries. Accordingly, currently available anti-HIV drugs target this subtype, with considerable benefits for the corresponding patients. However, in developing countries, there is a wide variety of HIV-1 subtypes carrying PR polymorphisms related to reduced drug susceptibility. The non-active site mutation, M36I, is the most frequent polymorphism, and is considered as a non-B subtype marker. Yet, the structural impact of this substitution on the PR structure and on the interaction with natural substrates remains poorly documented. Results Herein, we used molecular dynamics simulations to investigate the role of this polymorphism on the interaction of PR with six of its natural cleavage-sites substrates. Free energy analyses by MMPB/SA calculations showed an affinity decrease of M36I-PR for the majority of its substrates. The only exceptions were the RT-RH, with equivalent affinity, and the RH-IN, for which an increased affinity was found. Furthermore, molecular simulations suggest that, unlike other peptides, RH-IN induced larger structural fluctuations in the wild-type enzyme than in the M36I variant. Conclusions With multiple approaches and analyses we identified structural and dynamical determinants associated with the changes found in the binding affinity of the M36I variant. This mutation influences the flexibility of both PR and its complexed substrate. The observed impact of M36I, suggest that combination with other non-B subtype polymorphisms, could lead to major effects on the interaction with the 12 known cleavage sites, which should impact the virion maturation. PMID:25573486

Discovery of the ergothioneine transporter

PubMed Central

Gründemann, Dirk; Harlfinger, Stephanie; Golz, Stefan; Geerts, Andreas; Lazar, Andreas; Berkels, Reinhard; Jung, Norma; Rubbert, Andrea; Schömig, Edgar

2005-01-01

Variants of the SLC22A4 gene are associated with susceptibility to rheumatoid arthritis and Crohn's disease. SLC22A4 codes for an integral membrane protein, OCTN1, that has been presumed to carry organic cations like tetraethylammonium across the plasma membrane. Here, we show that the key substrate of this transporter is in fact ergothioneine (ET). Human OCTN1 was expressed in 293 cells. A substrate lead, stachydrine (alias proline betaine), was identified by liquid chromatography MS difference shading, a new substrate search strategy. Analysis of transport efficiency of stachydrine-related solutes, affinity, and Na+ dependence indicates that the physiological substrate is ET. Efficiency of transport of ET was as high as 195 μl per min per mg of protein. By contrast, the carnitine transporter OCTN2 from rat did not transport ET at all. Because ET is transported >100 times more efficiently than tetraethylammonium and carnitine, we propose the functional name ETT (ET transporter) instead of OCTN1. ET, all of which is absorbed from food, is an intracellular antioxidant with metal ion affinity. Its particular purpose is unresolved. Cells with expression of ETT accumulate ET to high levels and avidly retain it. By contrast, cells lacking ETT do not accumulate ET, because their plasma membrane is virtually impermeable for this compound. The real-time PCR expression profile of human ETT, with strong expression in CD71+ cells, is consistent with a pivotal function of ET in erythrocytes. Moreover, prominent expression of ETT in monocytes and SLC22A4 polymorphism associations suggest a protective role of ET in chronic inflammatory disorders. PMID:15795384

Substrate affinity of photosensitizers derived from chlorophyll-a: The ABCG2 transporter affects the phototoxic response of side population stem cell-like cancer cells to photodynamic therapy

PubMed Central

Morgan, Janet; Jackson, Jennifer D.; Zheng, Xiang; Pandey, Suresh K.; Pandey, Ravindra K.

2010-01-01

Photosensitizers (PS) synthesized with the aim of optimizing photodynamic therapy (PDT) of tumors do not always fulfill their potential when tested in vitro and in vivo in different tumor models. The ATP-dependent transporter ABCG2 a multi-drug resistant pump expressed at variable levels in cancerous cells, can bind and efflux a wide range of structurally different classes of compounds including several PS used pre-clinically and clinically such as porphyrins and chlorins. ABCG2 may lower intracellular levels of substrate PS below the threshold for cell death in tumors treated by PDT, leaving resistant cells to re-populate the tumor. To determine some of the structural factors that affect substrate affinity of PS for ABCG2, we used an ABCG2 expressing cell line (HEK 293 482R) and its non-expressing counterpart, and tyrosine kinase ABCG2 inhibitors in a simple flow cytometric assay to identify PS effluxed by the ABCG2 pump. We tested a series of conjugates of substrate PS with different groups attached at different positions on the tetrapyrrole macrocycle to examine whether a change in affinity for the pump occurred and whether such changes depended on the position or the structure/type of the attached group. PS without substitutions including pyropheophorbides and purpurinimides were generally substrates for ABCG2, but carbohydrate groups conjugated at positions 8, 12, 13 and 17 but not at position 3 abrogated ABCG2 affinity regardless of structure or linking moiety. At position 3, affinity was retained with the addition of iodobenzene, alkyl chains and monosaccharides, but not with disaccharides. This suggests that structural characteristics at position 3 may offer important contributions to requirements for binding to ABCG2. We examined several tumor cell lines for ABCG2 activity, and found that although some cell lines had negligible ABCG2 activity in bulk, they contained a small ABCG2-expressing side population (SP) thought to contain cells which are responsible for initiating tumor regrowth. We examined the relevance of the SP to PDT resistance with ABCG2 substrates in vitro and in vivo in the murine mammary tumor 4T1. We show for the first time in vivo that the substrate PS HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a) but not the non-substrate PS HPPH-Gal (a galactose conjugate of HPPH) selectively preserved the SP which was primarily responsible for regrowth in vitro. The SP could be targeted by addition of imatinib mesylate, a tyrosine kinase inhibitor which inhibits the ATPase activity of ABCG2, and prevents efflux of substrates. A PDT resistant SP may be responsible for recurrences observed both pre-clinically and clinically. To prevent ABCG2 mediated resistance, choosing non-substrate PS or administering an ABCG2 inhibitor alongside a substrate PS might be advantageous when treating ABCG2 expressing tumors with PDT. PMID:20684544

Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease

PubMed Central

Altman, Michael D.; Nalivaika, Ellen A.; Prabu-Jeyabalan, Moses; Schiffer, Celia A.; Tidor, Bruce

2009-01-01

Drug resistance in HIV-1 protease, a barrier to effective treatment, is generally caused by mutations in the enzyme that disrupt inhibitor binding but still allow for substrate processing. Structural studies with mutant, inactive enzyme, have provided detailed information regarding how the substrates bind to the protease yet avoid resistance mutations; insights obtained inform the development of next generation therapeutics. Although structures have been obtained of complexes between substrate peptide and inactivated (D25N) protease, thermodynamic studies of peptide binding have been challenging due to low affinity. Peptides that bind tighter to the inactivated protease than the natural substrates would be valuable for thermodynamic studies as well as to explore whether the structural envelope observed for substrate peptides is a function of weak binding. Here, two computational methods — namely, charge optimization and protein design — were applied to identify peptide sequences predicted to have higher binding affinity to the inactivated protease, starting from an RT–RH derived substrate peptide. Of the candidate designed peptides, three were tested for binding with isothermal titration calorimetry, with one, containing a single threonine to valine substitution, measured to have more than a ten-fold improvement over the tightest binding natural substrate. Crystal structures were also obtained for the same three designed peptide complexes; they show good agreement with computational prediction. Thermodynamic studies show that binding is entropically driven, more so for designed affinity enhanced variants than for the starting substrate. Structural studies show strong similarities between natural and tighter-binding designed peptide complexes, which may have implications in understanding the molecular mechanisms of drug resistance in HIV-1 protease. PMID:17729291

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hall, Justin; Brault, Amy; Vincent, Fabien

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a highmore » affinity (K D = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.« less

Active site-directed double mutants of dihydrofolate reductase.

PubMed

Ercikan-Abali, E A; Mineishi, S; Tong, Y; Nakahara, S; Waltham, M C; Banerjee, D; Chen, W; Sadelain, M; Bertino, J R

1996-09-15

Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22-Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

PubMed Central

Livingston, Alison L.; O’Shea, Valerie L.; Kim, Taewoo; Kool, Eric T.; David, Sheila S.

2009-01-01

Escherchia coli MutY plays an important role in preventing mutations associated with the oxidative lesion 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG) in DNA by excising adenines from OG:A mismatches as the first step of base excision repair. To determine the importance of specific steps in the base pair recognition and base removal process of MutY, we have evaluated the effects of modifications of the OG:A substrate on the kinetics of base removal, mismatch affinity and repair to G:C in an Escherchia coli-based assay. Surprisingly, adenine modification was tolerated in the cellular assay, while modification of OG results in minimal cellular repair. High affinity for the mismatch and efficient base removal require the presence of OG. Taken together, these results suggest that the presence of OG is a critical feature for MutY to locate OG:A mismatches and select the appropriate adenines for excision to initiate repair in vivo prior to replication. PMID:18026095

Carrier-mediated γ-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers.

PubMed

Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

2012-06-01

The aim of the present study was to investigate the transport of γ-aminobutyric acid (GABA) across the basolateral membrane of intestinal cells. The proton-coupled amino acid transporter, hPAT1, mediates the influx of GABA and GABA mimetic drug substances such as vigabatrin and gaboxadol and the anticancer prodrug δ-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least two transporters seem to be involved in the basolateral transport of GABA. The basolateral uptake consisted of a high-affinity system with a K(m) of 290 μM and V(max) of 75 pmol cm(-2) min(-1) and a low affinity system with a K(m) of approximately 64 mM and V(max) of 1.6 nmol cm(-2) min(-1). The high-affinity transporter is Na(+) and Cl(-) dependent. The substrate specificity of the high-affinity transporter was further studied and Gly-Sar, Leucine, gaboxadol, sarcosine, lysine, betaine, 5-hydroxythryptophan, proline and glycine reduced the GABA uptake to approximately 44-70% of the GABA uptake in the absence of inhibitor. Other substances such as β-alanine, GABA, 5-aminovaleric acid, taurine and δ-aminolevulinic acid reduced the basolateral GABA uptake to 6-25% of the uptake in the absence of inhibitor. Our results indicate that the distance between the charged amino- and acid-groups is particular important for inhibition of basolateral GABA uptake. Thus, there seems to be a partial substrate overlap between the basolateral GABA transporter and hPAT1, which may prove important for understanding drug interactions at the level of intestinal transport. Copyright © 2012 Elsevier B.V. All rights reserved.

Rhodamine Inhibitors of P-glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity

PubMed Central

Gannon, Michael K.; Holt, Jason J.; Bennett, Stephanie M.; Wetzel, Bryan R.; Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.; Sawada, Geri A.; Higgins, J. William; Tombline, Gregory; Raub, Thomas J.; Detty, Michael R.

2012-01-01

We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His10, for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave KM of 0.087 μM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC50’s of ~2 μM in MDCKII-MDR1 cells. PMID:19402665

A Triazole-Containing Metal-Organic Framework as a Highly Effective and Substrate Size-Dependent Catalyst for CO2 Conversion.

PubMed

Li, Pei-Zhou; Wang, Xiao-Jun; Liu, Jia; Lim, Jie Sheng; Zou, Ruqiang; Zhao, Yanli

2016-02-24

A highly porous metal-organic framework (MOF) incorporating both exposed metal sites and nitrogen-rich triazole groups was successfully constructed via solvothermal assembly of a clicked octcarboxylate ligand and Cu(II) ions, which presents a high affinity toward CO2 molecules clearly verified by gas adsorption and Raman spectral detection. The constructed MOF featuring CO2-adsorbing property and exposed Lewis-acid metal sites could serve as an excellent catalyst for CO2-based chemical fixation. Catalytic activity of the MOF was confirmed by remarkably high efficiency on CO2 cycloaddition with small epoxides. When extending the substrates to larger ones, its activity showed a sharp decrease. These observations reveal that MOF-catalyzed CO2 cycloaddition of small substrates was carried out within the framework, while large ones cannot easily enter into the porous framework for catalytic reactions. Thus, the synthesized MOF exhibits high catalytic selectivity to different substrates on account of the confinement of the pore diameter. The high efficiency and size-dependent selectivity toward small epoxides on catalytic CO2 cycloaddition make this MOF a promising heterogeneous catalyst for carbon fixation.

Knock-Down of a Tonoplast Localized Low-Affinity Nitrate Transporter OsNPF7.2 Affects Rice Growth under High Nitrate Supply

PubMed Central

Hu, Rui; Qiu, Diyang; Chen, Yi; Miller, Anthony J.; Fan, Xiaorong; Pan, Xiaoping; Zhang, Mingyong

2016-01-01

The large nitrate transporter 1/peptide transporter family (NPF) has been shown to transport diverse substrates, including nitrate, amino acids, peptides, phytohormones, and glucosinolates. However, the rice (Oryza sativa) root-specific family member OsNPF7.2 has not been functionally characterized. Here, our data show that OsNPF7.2 is a tonoplast localized low-affinity nitrate transporter, that affects rice growth under high nitrate supply. Expression analysis showed that OsNPF7.2 was mainly expressed in the elongation and maturation zones of roots, especially in the root sclerenchyma, cortex and stele. It was also induced by high concentrations of nitrate. Subcellular localization analysis showed that OsNPF7.2 was localized on the tonoplast of large and small vacuoles. Heterologous expression in Xenopus laevis oocytes suggested that OsNPF7.2 was a low-affinity nitrate transporter. Knock-down of OsNPF7.2 retarded rice growth under high concentrations of nitrate. Therefore, we deduce that OsNPF7.2 plays a role in intracellular allocation of nitrate in roots, and thus influences rice growth under high nitrate supply. PMID:27826301

Nanotextured polymer substrates show enhanced cancer cell isolation and cell culture

NASA Astrophysics Data System (ADS)

Islam, Muhymin; Sajid, Adeel; Arif Iftakher Mahmood, M.; Motasim Bellah, Mohammad; Allen, Peter B.; Kim, Young-Tae; Iqbal, Samir M.

2015-06-01

Detection of circulating tumor cells (CTCs) in the early stages of cancer is a great challenge because of their exceedingly small concentration. There are only a few approaches sensitive enough to differentiate tumor cells from the plethora of other cells in a sample like blood. In order to detect CTCs, several antibodies and aptamers have already shown high affinity. Nanotexture can be used to mimic basement membrane to further enhance this affinity. This article reports an approach to fabricate nanotextured polydimethylsiloxane (PDMS) substrates using micro reactive ion etching (micro-RIE). Three recipes were used to prepare nanotextured PDMS using oxygen and carbon tetrafluoride. Micro-RIE provided better control on surface properties. Nanotexturing improved the affinity of PDMS surfaces to capture cancer cells using surface immobilized aptamers against cell membrane overexpressed with epidermal growth factor receptors. In all cases, nanotexture of PDMS increased the effective surface area by creating nanoscale roughness on the surface. Nanotexture also enhanced the growth rate of cultured cells compared to plain surfaces. A comparison among the three nanotextured surfaces demonstrated an almost linear relationship between the surface roughness and density of captured tumor cells. The nanotextured PDMS mimicked biophysical environments for cells to grow faster. This can have many implications in microfluidic platforms used for cell handling.

Occupancy of the Zinc-binding Site by Transition Metals Decreases the Substrate Affinity of the Human Dopamine Transporter by an Allosteric Mechanism*

PubMed Central

Li, Yang; Mayer, Felix P.; Hasenhuetl, Peter S.; Burtscher, Verena; Schicker, Klaus; Sitte, Harald H.; Freissmuth, Michael; Sandtner, Walter

2017-01-01

The human dopamine transporter (DAT) has a tetrahedral Zn2+-binding site. Zn2+-binding sites are also recognized by other first-row transition metals. Excessive accumulation of manganese or of copper can lead to parkinsonism because of dopamine deficiency. Accordingly, we examined the effect of Mn2+, Co2+, Ni2+, and Cu2+ on transport-associated currents through DAT and DAT-H193K, a mutant with a disrupted Zn2+-binding site. All transition metals except Mn2+ modulated the transport cycle of wild-type DAT with affinities in the low micromolar range. In this concentration range, they were devoid of any action on DAT-H193K. The active transition metals reduced the affinity of DAT for dopamine. The affinity shift was most pronounced for Cu2+, followed by Ni2+ and Zn2+ (= Co2+). The extent of the affinity shift and the reciprocal effect of substrate on metal affinity accounted for the different modes of action: Ni2+ and Cu2+ uniformly stimulated and inhibited, respectively, the substrate-induced steady-state currents through DAT. In contrast, Zn2+ elicited biphasic effects on transport, i.e. stimulation at 1 μm and inhibition at 10 μm. A kinetic model that posited preferential binding of transition metal ions to the outward-facing apo state of DAT and a reciprocal interaction of dopamine and transition metals recapitulated all experimental findings. Allosteric activation of DAT via the Zn2+-binding site may be of interest to restore transport in loss-of-function mutants. PMID:28096460

Regulation of Nitrate Transport in Citrus Rootstocks Depending on Nitrogen Availability

PubMed Central

Cerezo, Miguel; Camañes, Gemma; Flors, Víctor; Primo-Millo, Eduardo

2007-01-01

Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO3−. The Vmax for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks. Additionally, we studied the regulation of root NO3− uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO3− and down-regulated by the N status and by NO3− itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status. The use of various metabolic uncouplers or inhibitors indicated that NO3− net uptake mediated by iHATS and LATS was an active transport system in both rootstocks. PMID:19516998

Substrate growth dynamics and biomineralization of an Ediacaran encrusting poriferan.

PubMed

Wood, Rachel; Penny, Amelia

2018-01-10

The ability to encrust in order to secure and maintain growth on a substrate is a key competitive innovation in benthic metazoans. Here we describe the substrate growth dynamics, mode of biomineralization and possible affinity of Namapoikia rietoogensis , a large (up to 1 m), robustly skeletal, and modular Ediacaran metazoan which encrusted the walls of synsedimentary fissures within microbial-metazoan reefs. Namapoikia formed laminar or domal morphologies with an internal structure of open tubules and transverse elements, and had a very plastic, non-deterministic growth form which could encrust both fully lithified surfaces as well as living microbial substrates, the latter via modified skeletal holdfasts. Namapoikia shows complex growth interactions and substrate competition with contemporary living microbialites and thrombolites, including the production of plate-like dissepiments in response to microbial overgrowth which served to elevate soft tissue above the microbial surface. Namapoikia could also recover from partial mortality due to microbial fouling. We infer initial skeletal growth to have propagated via the rapid formation of an organic scaffold via a basal pinacoderm prior to calcification. This is likely an ancient mode of biomineralization with similarities to the living calcified demosponge Vaceletia. Namapoikia also shows inferred skeletal growth banding which, combined with its large size, implies notable individual longevity. In sum, Namapoikia was a large, relatively long-lived Ediacaran clonal skeletal metazoan that propagated via an organic scaffold prior to calcification, enabling rapid, effective and dynamic substrate occupation and competition in cryptic reef settings. The open tubular internal structure, highly flexible, non-deterministic skeletal organization, and inferred style of biomineralization of Namapoikia places probable affinity within total-group poriferans. © 2018 The Author(s).

Protein Allostery and Conformational Dynamics.

PubMed

Guo, Jingjing; Zhou, Huan-Xiang

2016-06-08

The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodynamic properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and molecular dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, for example, the relationship between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.

The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains.

PubMed

English, Charles A; Sherman, Woody; Meng, Wenli; Gierasch, Lila M

2017-09-08

Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

PubMed Central

Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

2017-01-01

Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

PubMed

Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

2010-01-01

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

Anion exchange pathways for Cl sup minus transport in rabbit renal microvillus membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karniski, L.P.; Aronson, P.S.

1987-09-01

The authors evaluated the mechanisms of chloride transport in microvillus membrane vesicles isolated from the rabbit renal cortex. The presence of Cl-formate exchange was confirmed. Outward gradients of oxaloacetate, HCO{sub 3}, acetate, lactate, succinate, sulfate, and p-aminohippurate (PAH) stimulated the rate of Cl uptake minimally or not at all. However, an outward gradient of oxalate stimulated Cl uptake by 70%, and an outward Cl gradient induced uphill oxalate uptake, indicting Cl-oxalate exchange. Moreover, an outward formate gradient induced uphill oxalate uptake, indicating formate-oxalate exchange. Studies of inhibitor and substrate specificity indicated the probably operation of at least two separate anionmore » exchangers in mediating Cl transport. The Cl-formate exchanger accepted Cl and formate as substrates, had little or no affinity for oxalate, was sensitive to inhibition by furosemide, and was less sensitive to inhibition by 4,4{prime}-diisothiocyanostilbene-2,2{prime}-disulfonic acid (DIDS). The Cl (formate)-oxalate exchanger also accepted Cl and formate as substrates but had high affinity for oxalate, was highly sensitive to inhibition by DIDS, and was less sensitive to inhibition by furosemide. The Cl-formate exchanger was electroneutral, whereas the Cl (formate)-oxalate exchanger was electrogenic. They conclude that at least separate anion exchangers mediating Cl transport are present on the luminal membrane of the rabbit proximal tubule cell. These exchangers may play important roles in mediating transtubular Cl and oxalate transport in this nephron segment.« less

Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2.

PubMed

Carland, Jane E; Thomas, Michael; Mostyn, Shannon N; Subramanian, Nandhitha; O'Mara, Megan L; Ryan, Renae M; Vandenberg, Robert J

2018-03-21

Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuT Aa , and more recently the Drosophila dopamine transporter and the human serotonin transporter. However, there is disputed evidence that the bacterial leucine transporter, LeuT Aa , contains two substrate binding sites that work cooperatively in the mechanism of transport, with the binding of a second substrate being required for the release of the substrate from the primary site. An alternate proposal is that there may be low affinity binding sites that serve to direct the flow of substrates to the primary site. We have used a combination of molecular dynamics simulations of substrate interactions with a homology model of GlyT2, together with radiolabeled amino acid uptake assays and electrophysiological analysis of wild-type and mutant transporters, to provide evidence that substrate selectivity of GlyT2 is determined entirely by the primary substrate binding site and, furthermore, if a secondary site exists then it is a low affinity nonselective amino acid binding site.

Determination of alkaline phosphatase based on affinity adsorption solid-substrate room temperature phosphorimetry using rhodamine 6G-dibromoluciferin luminescent nanoparticle to label lectin and prediction of diseases.

PubMed

Liu, Jia-Ming; Liu, Zhen-Bo; Hu, Li-Xiang; He, Hang-Xia; Yang, Min-Lan; Zhou, Ping; Chen, Xin-Hua; Zheng, Min-Min; Zeng, Xiao-Yi; Xu, Yue-Long

2006-10-15

In the presence of heavy atom perturber LiAc, the silicon dioxide nanoparticle containing rhodamine 6G (R) and dibromoluciferin (D) (R-D-SiO(2)) can emit strong and stable solid-substrate room temperature phosphorescence signal of R (lambda(ex)/lambda(em)=481/648 nm) and D (lambda(ex)/lambda(em)=457/622 nm) on the surface of acetyl cellulose membrane (ACM). R-D-SiO(2) is used to label triticum vulgare lectin (WGA). Then two types of affinity adsorption reactions, R-D-SiO(2)-WGA- alkaline phosphatase (ALP) (direct method) and WGA-ALP-WGA-R-D-SiO(2) (sandwich method), are carried out on ACM. The conditions and the analytical characteristics for the determination of ALP using affinity adsorption solid-substrate room temperature phosphorimetry (AA-SS-RTP) were studied. For a 0.40-microl drop of sample, results show that the detection limits of the sandwich method are 0.16 ag spot(-1)(457/622 nm) and 0.17 ag spot(-1)(481/648 nm), and the detection limits of the direct method are 0.41 ag spot(-1) (457/622 nm) and 0.44 ag spot(-1) (481/648 nm). The contents of ALP in human serum correlated well with those obtained by enzyme-linked immunoassay. This study shows that AA-SS-RTP whether by the sandwich method or the direct method, can combine very well the characteristics of both high sensitivity of SS-RTP and specificity of the immunoreaction. Simultaneously, whether the phosphorescence excitation/emission wavelength of either R or D in R-D-SiO(2) is chosen to determine ALP, this can promote the agility and widen the adaptability of AA-SS-RTP.

Cooperative effects for CYP2E1 differ between styrene and its metabolites

PubMed Central

Hartman, Jessica H.; Boysen, Gunnar; Miller, Grover P.

2014-01-01

Cooperative interactions are frequently observed in the metabolism of drugs and pollutants by cytochrome P450s; nevertheless, the molecular determinants for cooperativity remain elusive. Previously, we demonstrated that steady-state styrene metabolism by CYP2E1 exhibits positive cooperativity.We hypothesized that styrene metabolites have lower affinity than styrene toward CYP2E1 and limited ability to induce cooperative effects during metabolism. To test the hypothesis, we determined the potency and mechanism of inhibition for styrene and its metabolites toward oxidation of 4-nitrophenol using CYP2E1 Supersomes® and human liver microsomes.Styrene inhibited the reaction through a mixed cooperative mechanism with high affinity for the catalytic site (67 μM) and lower affinity for the cooperative site (1100 μM), while increasing substrate turnover at high concentrations. Styrene oxide and 4-vinylphenol possessed similar affinity for CYP2E1. Styrene oxide behaved cooperatively like styrene, but 4-vinylphenol decreased turnover at high concentrations. Styrene glycol was a very poor competitive inhibitor. Among all compounds, there was a positive correlation with binding and hydrophobicity.Taken together, these findings for CYP2E1 further validate contributions of cooperative mechanisms to metabolic processes, demonstrate the role of molecular structure on those mechanisms and underscore the potential for heterotropic cooperative effects between different compounds. PMID:23327532

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klein, M.; Canoll, P.D.; Musacchio, J.M.

1991-01-01

The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drugmore » has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.« less

Determination of trace glucose and forecast of human diseases by affinity adsorption solid substrate room temperature phosphorimetry based on Triticum valgaris lectin labeled with 4.0-generation dendrimers

NASA Astrophysics Data System (ADS)

Li, Zhiming; Zhu, Guohui; Liu, Jiaming; Lu, Qiaomei; Yang, Minlan; Wu, Hong; Shi, Xiumei; Chen, Xinhua

2007-08-01

A new phosphorescence labeling reagent Triton-100X-4.0G-D (4.0G-D refers to 4.0-generation dendrimers) was found. Quantitative specific affinity adsorption (AA) reaction between Triton-100X-4.0G-D-WGA and glucose (G) was carried out on the surface of nitrocellulose membrane (NCM), and the Δ Ip of the product of AA reaction was linear correlation to the content of G. Based on the facts above, a new method for the determination of trace G was established by WGA labeled with Triton-100X-4.0G-D affinity adsorption solid substrate room temperature phosphorimetry (Triton-100X-4.0G-D-WGA-AA-SS-RTP). This research showed that AA-SS-RTP for either direct method or sandwich method could combine very well the characteristics of both the high sensitivity of SS-RTP and the specificity of the AA reaction. Detection limits (LD) were 0.24 fg spot -1 for direct method and 0.18 fg spot -1 for sandwich method, indicating both of them were of high sensitivity. The method has been applied to the determination of the content of G in human serum, and the results were coincided with those obtained by glucose oxidize enzyme method. It can also be applied to forecast accurately some human diseases, such as primary hepatic carcinoma, cirrhosis, acute and chronic hepatitis, transfer hepatocellular, etc. Meanwhile, the mechanism for the determination of G with AA-SS-RTP was discussed.

A Cyclic Peptidic Serine Protease Inhibitor: Increasing Affinity by Increasing Peptide Flexibility

PubMed Central

Jiang, Longguang; Paaske, Berit; Kromann-Hansen, Tobias; Jensen, Jan K.; Sørensen, Hans Peter; Liu, Zhuo; Nielsen, Jakob T.; Christensen, Anni; Hosseini, Masood; Sørensen, Kasper K.; Nielsen, Niels Christian; Jensen, Knud J.; Huang, Mingdong; Andreasen, Peter A.

2014-01-01

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden. PMID:25545505

Application of a coupled enzyme assay to characterize nicotinamide riboside kinases.

PubMed

Dölle, Christian; Ziegler, Mathias

2009-02-15

The recently identified nicotinamide riboside kinases (Nrks) constitute a distinct pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. Here we present the combination of an established optical adenosine triphosphatase (ATPase) test, the pyruvate kinase/lactate dehydrogenase system, with the Nrk-catalyzed reaction to determine kinetic properties of these enzymes, in particular affinities for ATP. The assay allows variation of both nucleoside and phosphate donor substrates, thereby providing major advantages for the characterization of these enzymes. We confirm previously established kinetic parameters and identify differences in substrate selectivity between the two human Nrk isoforms. The proposed assay is inexpensive and may be applied for high-throughput screening.

Energetic, Structural, and Antimicrobial Analyses of [beta]-Lactam Side Chain Recognition by [beta]-Lactamases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caselli, E.; Powers, R.A.; Blaszczak, L.C.

2010-03-05

Penicillins and cephalosporins are among the most widely used and successful antibiotics. The emergence of resistance to these {beta}-lactams, most often through bacterial expression of {beta}-lactamases, threatens public health. To understand how {beta}-lactamases recognize their substrates, it would be helpful to know their binding energies. Unfortunately, these have been difficult to measure because {beta}-lactams form covalent adducts with {beta}-lactamases. This has complicated functional analyses and inhibitor design. To investigate the contribution to interaction energy of the key amide (R1) side chain of {beta}-lactam antibiotics, eight acylglycineboronic acids that bear the side chains of characteristic penicillins and cephalosporins, as well asmore » four other analogs, were synthesized. These transition-state analogs form reversible adducts with serine {beta}-lactamases. Therefore, binding energies can be calculated directly from K{sub i} values. The K{sub i} values measured span four orders of magnitude against the Group I {beta}-lactamase AmpC and three orders of magnitude against the Group II {beta}-lactamase TEM-1. The acylglycineboronic acids have K{sub i} values as low as 20 nM against AmpC and as low as 390 nM against TEM-1. The inhibitors showed little activity against serine proteases, such as chymotrypsin. R1 side chains characteristic of {beta}-lactam inhibitors did not have better affinity for AmpC than did side chains characteristic of {beta}-lactam substrates. Two of the inhibitors reversed the resistance of pathogenic bacteria to {beta}-lactams in cell culture. Structures of two inhibitors in their complexes with AmpC were determined by X-ray crystallography to 1.90 {angstrom} and 1.75 {angstrom} resolution; these structures suggest interactions that are important to the affinity of the inhibitors. Acylglycineboronic acids allow us to begin to dissect interaction energies between {beta}-lactam side chains and {beta}-lactamases. Surprisingly, there is little correlation between the affinity contributed by R1 side chains and their occurrence in {beta}-lactam inhibitors or {beta}-lactam substrates of serine {beta}-lactamases. Nevertheless, presented in acylglycineboronic acids, these side chains can lead to inhibitors with high affinities and specificities. The structures of their complexes with AmpC give a molecular context to their affinities and may guide the design of anti-resistance compounds in this series.« less

Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter.

PubMed

Schmitt, Kyle C; Mamidyala, Sreeman; Biswas, Swati; Dutta, Aloke K; Reith, Maarten E A

2010-03-01

Bivalent ligands--compounds incorporating two receptor-interacting moieties linked by a flexible chain--often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric 'heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [(3)H] 2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and beta-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N-linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N--previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors--indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the 'multivalent ligand' strategy.

Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niessen, Markus; Jaschinski, Frank; Item, Flurin

2007-02-15

Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the {beta}-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH andmore » PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission.« less

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System.

PubMed

Pliotas, Christos; Grayer, Samuel C; Ekkerman, Silvia; Chan, Anthony K N; Healy, Jess; Marius, Phedra; Bartlett, Wendy; Khan, Amjad; Cortopassi, Wilian A; Chandler, Shane A; Rasmussen, Tim; Benesch, Justin L P; Paton, Robert S; Claridge, Timothy D W; Miller, Samantha; Booth, Ian R; Naismith, James H; Conway, Stuart J

2017-08-15

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

Allosteric Inhibition via R-state Destabilization in ATP Sulfurylase from Penicillium chrysogenum

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacRae, I. J.

2002-01-01

The structure of the cooperative hexameric enzyme ATP sulfurylase from Penicillium chrysogenum bound to its allosteric inhibitor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), was determined to 2.6 {angstrom} resolution. This structure represents the low substrate-affinity T-state conformation of the enzyme. Comparison with the high substrate-affinity R-state structure reveals that a large rotational rearrangement of domains occurs as a result of the R-to-T transition. The rearrangement is accompanied by the 17 {angstrom} movement of a 10-residue loop out of the active site region, resulting in an open, product release-like structure of the catalytic domain. Binding of PAPS is proposed to induce the allosteric transition bymore » destabilizing an R-state-specific salt linkage between Asp 111 in an N-terminal domain of one subunit and Arg 515 in the allosteric domain of a trans-triad subunit. Disrupting this salt linkage by site-directed mutagenesis induces cooperative inhibition behavior in the absence of an allosteric effector, confirming the role of these two residues.« less

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System

PubMed Central

2017-01-01

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme–substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding. PMID:28656748

Biochemical profiling in silico--predicting substrate specificities of large enzyme families.

PubMed

Tyagi, Sadhna; Pleiss, Juergen

2006-06-25

A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules.

[Characterization of a recombinant aminopeptidase Lmo1711 from Listeria monocytogenes].

PubMed

He, Zhan; Wang, Hang; Han, Xiao; Ma, Tiantian; Hang, Yi; Yu, Huifei; Wei, Fangfang; Sun, Jing; Yang, Yongchun; Cheng, Changyong; Song, Houhui

2018-05-25

We aimed to obtain the recombinant aminopeptidase encoded by Listeria monocytogenes (L. monocytogenes) gene lmo1711, and characterized the enzyme. First, the amino acid sequences of Lmo1711 from L. monocytogenes EGD-e and its homologues in other microbial species were aligned and the putative active sites were analyzed. The putative model of Lmo1711 was constructed through the SWISS-MODEL Workspace. Then, the plasmid pET30a-Lmo1711 was constructed and transformed into E. coli for expression of the recombinant Lmo1711. The his-tagged soluble protein was purified using the nickel-chelated affinity column chromatography. With the amino acid-p-nitroaniline as the substrate, Lmo1711 hydrolyzed the substrate to free p-nitroaniline monomers, whose absorbance measured at 405 nm reflected the aminopeptidase activity. The specificity of Lmo1711 to substrates was then examined by changing various substrates, and the effect of metal ions on the catalytic efficiency of this enzyme was further determined. Based on the bioinformatics data, Lmo1711 is a member of the M29 family aminopeptidases, containing a highly conserved catalytic motif (Glu-Glu-His-Tyr-His-Asp) with typical structure arrangements of the peptidase family. The recombinant Lmo1711 with a size of about 49.3 kDa exhibited aminopeptidase activity and had a selectivity to the substrates, with the highest degree of affinity for leucine-p-nitroaniline. Interestingly, the enzymatic activity of Lmo1711 can be activated by Cd²⁺, Zn²⁺, and is strongly stimulated by Co²⁺. We here, for the first time demonstrate that L. monocytogenes lmo1711 encodes a cobalt-activated aminopeptidase of M29 family.

Two amino acid residues confer different binding affinities of Abelson family kinase SRC homology 2 domains for phosphorylated cortactin.

PubMed

Gifford, Stacey M; Liu, Weizhi; Mader, Christopher C; Halo, Tiffany L; Machida, Kazuya; Boggon, Titus J; Koleske, Anthony J

2014-07-11

The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular and thermodynamic mechanisms of the chloride-dependent human angiotensin-I-converting enzyme (ACE).

PubMed

Yates, Christopher J; Masuyer, Geoffrey; Schwager, Sylva L U; Akif, Mohd; Sturrock, Edward D; Acharya, K Ravi

2014-01-17

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg(522). Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu(403)-Lys(118) salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains.

Molecular and Thermodynamic Mechanisms of the Chloride-dependent Human Angiotensin-I-converting Enzyme (ACE)*

PubMed Central

Yates, Christopher J.; Masuyer, Geoffrey; Schwager, Sylva L. U.; Akif, Mohd; Sturrock, Edward D.; Acharya, K. Ravi

2014-01-01

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg522. Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu403-Lys118 salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains. PMID:24297181

Enzymatic properties of separated isozymes of the Na,K-ATPase. Substrate affinities, kinetic cooperativity, and ion transport stoichiometry.

PubMed

Sweadner, K J

1985-09-25

There are two isozymes of the Na,K-ATPase, which can be purified separately from rat renal medulla and brainstem axolemma. Here the basic kinetic properties of the two Na,K-ATPases have been compared in conditions permitting enzyme turnover. The two isozymes are half-maximally activated at different concentrations of ATP, the axolemma Na,K-ATPase having the higher affinity. They are half-maximally activated by Na+ and K+ at very similar concentrations but show differences in cooperativity toward Na+. The affinities of both isozymes for ATP and Na+ are affected in a qualitatively similar way by variations in the concentration of K+. Both isozymes transport 22Na+ and 42K+ in a ratio close to 3:2 in artificial lipid vesicles. The two isozymes differ most strikingly in the inhibition of ATPase activity by ouabain. The axolemma Na,K-ATPase has a high affinity for ouabain with positive cooperativity, while the renal medulla Na,K-ATPase has a lower affinity with negative cooperativity. It is likely that the cooperativity differences are due to kinetic effects, reflecting different rates of conformation transitions during enzyme turnover. The functional result of the contrasting cooperativities is that the difference in sensitivity to ouabain is amplified.

RNase One Gene Isolation, Expression, and Affinity Purification Models Research Experimental Progression and Culminates with Guided Inquiry-Based Experiments

ERIC Educational Resources Information Center

Bailey, Cheryl P.

2009-01-01

This new biochemistry laboratory course moves through a progression of experiments that generates a platform for guided inquiry-based experiments. RNase One gene is isolated from prokaryotic genomic DNA, expressed as a tagged protein, affinity purified, and tested for activity and substrate specificity. Student pairs present detailed explanations…

Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake

PubMed Central

2012-01-01

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na+-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline KM with no change in Vmax. As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling. PMID:23077721

Nonoisotopic assay for the presynaptic choline transporter reveals capacity for allosteric modulation of choline uptake.

PubMed

Ruggiero, Alicia M; Wright, Jane; Ferguson, Shawn M; Lewis, Michelle; Emerson, Katie S; Iwamoto, Hideki; Ivy, Michael T; Holmstrand, Ericka C; Ennis, Elizabeth A; Weaver, C David; Blakely, Randy D

2012-10-17

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling.

Effect of pretreatment severity in continuous steam explosion on enzymatic conversion of wheat straw: Evidence from kinetic analysis of hydrolysis time courses.

PubMed

Monschein, Mareike; Nidetzky, Bernd

2016-01-01

Focusing on continuous steam explosion, the influence of pretreatment severity due to varied acid loading on hydrolysis of wheat straw by Trichoderma reesei cellulases was investigated based on kinetic evaluation of the saccharification of each pretreated substrate. Using semi-empirical descriptors of the hydrolysis time course, key characteristics of saccharification efficiency were captured in a quantifiable fashion. Not only hydrolysis rates per se, but also the transition point of their bi-phasic decline was crucial for high saccharification degree. After 48h the highest saccharification was achieved for substrate pretreated at relatively low severity (1.2% acid). Higher severity increased enzyme binding to wheat straw, but reduced the specific hydrolysis rates. Higher affinity of the lignocellulosic material for cellulases does not necessarily result in increased saccharification, probably because of lignin modifications occurring at high pretreatment severities. At comparable severity, continuous pretreatment produced a substrate more susceptible to enzymatic hydrolysis than the batch process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Non-Invasive Assessment of Liver Function

PubMed Central

Helmke, Steve; Colmenero, Jordi; Everson, Gregory T.

2015-01-01

Purpose of review It is our opinion that there is an unmet need in Hepatology for a minimally- or noninvasive test of liver function and physiology. Quantitative liver function tests (QLFTs) define the severity and prognosis of liver disease by measuring the clearance of substrates whose uptake or metabolism is dependent upon liver perfusion or hepatocyte function. Substrates with high affinity hepatic transporters exhibit high “first-pass” hepatic extraction and their clearance measures hepatic perfusion. In contrast, substrates metabolized by the liver have low first-pass extraction and their clearance measures specific drug metabolizing pathways. Recent Findings We highlight one QLFT, the dual cholate test, and introduce the concept of a disease severity index (DSI) linked to clinical outcome that quantifies the simultaneous processes of hepatocyte uptake, clearance from the systemic circulation, clearance from the portal circulation, and portal-systemic shunting. Summary It is our opinion that dual cholate is a relevant test for defining disease severity, monitoring the natural course of disease progression, and quantifying the response to therapy. PMID:25714706

Impact of x-Linkable Polymer Blends on Phase Morphology and Adhesion

NASA Astrophysics Data System (ADS)

Liu, Chun; Wan, Grace; Keene, Ellen; Harris, Joseph; Zhang, Sipei; Anderson, Stephanie; Li Pi Shan, Colin

Adhesion to dissimilar substrate is highly important to multiple industrial applications such as automotive adhesives, food packaging, transportation etc. Adhesive design has to include components that are affinity to both substrates, e.g. high surface energy polar and low surface non-polar substrates. Typically, these adhesive components are thermodynamically incompatible with each other, leading to macrophase separation and thus adhesive failure. By using functional adhesive components plus some additives, the adhesive can be in-situ cross-linked to prevent the macrophase separation with controlled phase morphology. Herein, we present the study on a cross-linkable adhesive formulation consisting of acrylic emulsion and polyolefin aqueous dispersion with additives for enhancing cross-linking and controlled phase morphologies. Contact angle measurement and ATR-IR spectroscopy are used to characterize the properties of adhesive surface. DMA is used to study the mechanical property of adhesive before and after cross-linking. The detailed phase morphologies are revealed by AFM, SEM and TEM. The resulting adhesive morphologies are correlated with the adhesive performance to establish structure-property relationship.

Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.

PubMed

Pedroso, Marcelo M; Ely, Fernanda; Carpenter, Margaret C; Mitić, Nataša; Gahan, Lawrence R; Ollis, David L; Wilcox, Dean E; Schenk, Gerhard

2017-07-05

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its β site in the absence of a substrate. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and in the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the β site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the β site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first- and second-coordination sphere residues at the β site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second-sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.

Biochemical properties of Na+/K(+)-ATPase in axonal growth cone particles isolated from fetal rat brain.

PubMed

Mercado, R; Hernández, J

1994-08-01

Axonal growth cones (AGC) isolated from fetal rat brain have an important specific activity of N+/K(+)-ATPase. Kinetic assays of the enzyme in AGC showed that Km values for ATP or K+ are similar to those reported for the adult brain enzyme. For Na+ the affinity (Km) was lower. Vmax for the three substrates was several times lower in AGC as compared to the adult value. We also observed two apparent inhibition constants of Na+/K(+)-ATPase by ouabain, one of low affinity, possibly corresponding to the alpha 1 isoform and another of high affinity which is different to that described for the alpha 2 isoform of the enzyme. These results support an important role for the sodium pump in the maintainance of volume and cationic balance in neuronal differentiating structures. The functional differences observed also suggest that the enzymatic complex of Na+/K(+)-ATPase in AGC is in a transitional state towards the adult configuration.

Asymmetric ring structure of Vps4 required for ESCRT-III disassembly

NASA Astrophysics Data System (ADS)

Caillat, Christophe; Macheboeuf, Pauline; Wu, Yuanfei; McCarthy, Andrew A.; Boeri-Erba, Elisabetta; Effantin, Gregory; Göttlinger, Heinrich G.; Weissenhorn, Winfried; Renesto, Patricia

2015-12-01

The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.

Optimality of affine control system of several species in competition on a sequential batch reactor

NASA Astrophysics Data System (ADS)

Rodríguez, J. C.; Ramírez, H.; Gajardo, P.; Rapaport, A.

2014-09-01

In this paper, we analyse the optimality of affine control system of several species in competition for a single substrate on a sequential batch reactor, with the objective being to reach a given (low) level of the substrate. We allow controls to be bounded measurable functions of time plus possible impulses. A suitable modification of the dynamics leads to a slightly different optimal control problem, without impulsive controls, for which we apply different optimality conditions derived from Pontryagin principle and the Hamilton-Jacobi-Bellman equation. We thus characterise the singular trajectories of our problem as the extremal trajectories keeping the substrate at a constant level. We also establish conditions for which an immediate one impulse (IOI) strategy is optimal. Some numerical experiences are then included in order to illustrate our study and show that those conditions are also necessary to ensure the optimality of the IOI strategy.

Enzymatic mechanisms of soil-carbon response to temperature on Mt. Kilimanjaro

NASA Astrophysics Data System (ADS)

Blagodatskaya, Evgenia; Blagodatskiy, Sergey; Kuzyakov, Yakov

2016-04-01

Short-term acceleration of soil organic matter (SOM) decomposition by increasing temperature contradicts the acclimation observed in long-term studies. We used the unique altitudinal gradient (from colline tropical zone to subalpine zone) on Mt. Kilimanjaro to demonstrate the mechanisms of short- and long-term acclimation of extra- and intracellular enzymes that decompose polymers (cellulose, chitin, phytate) and oxidize monomers (14C-glucose). Basing on Michaelis-Menten kinetics we determined the enzymes affinity to substrate (Km) and mineralization potential of heterotrophic microorganisms (Vmax) 1) for three hydrolytic enzymes: β-1,4-glucosidase, N-acetyl- β -D-glucosaminidase and phosphatase by the application of fluorogenically labeled substrates and 2) for mineralization of 14C-labeled glucose by substrate-dependent respiratory response. Here we show that the amount of available substrate is responsible for temperature sensitivity of hydrolysis of polymers in soil, whereas monomers oxidation to CO2 does not depend on substrate amount and is mainly temperature governed. We also found that substrate affinity of enzymes (which is usually decreases with the temperature) differently responded to warming for the process of depolymerisation versus monomers oxidation. We suggest the mechanism to temperature acclimation based on different temperature sensitivity of enzymes kinetics for hydrolysis of polymers and for monomers oxidation

Quantitative framework for ordered degradation of APC/C substrates.

PubMed

Lu, Dan; Girard, Juliet R; Li, Weihan; Mizrak, Arda; Morgan, David O

2015-11-16

During cell-cycle progression, substrates of a single master regulatory enzyme can be modified in a specific order. Here, we used experimental and computational approaches to dissect the quantitative mechanisms underlying the ordered degradation of the substrates of the ubiquitin ligase APC/C(Cdc20), a key regulator of chromosome segregation in mitosis. We show experimentally that the rate of catalysis varies with different substrates of APC/C(Cdc20). Using a computational model based on multi-step ubiquitination, we then show how changes in the interaction between a single substrate and APC/C(Cdc20) can alter the timing of degradation onset relative to APC/C(Cdc20) activation, while ensuring a fast degradation rate. Degradation timing and dynamics depend on substrate affinity for the enzyme as well as the catalytic rate at which the substrate is modified. When two substrates share the same pool of APC/C(Cdc20), their relative enzyme affinities and rates of catalysis influence the partitioning of APC/C(Cdc20) among substrates, resulting in substrate competition. Depending on how APC/C(Cdc20) is partitioned among its substrates, competition can have minor or major effects on the degradation of certain substrates. We show experimentally that increased expression of the early APC/C(Cdc20) substrate Clb5 does not delay the degradation of the later substrate securin, arguing against a role for competition with Clb5 in establishing securin degradation timing. The degradation timing of APC/C(Cdc20) substrates depends on the multi-step nature of ubiquitination, differences in substrate-APC/C(Cdc20) interactions, and competition among substrates. Our studies provide a conceptual framework for understanding how ordered modification can be established among substrates of the same regulatory enzyme, and facilitate our understanding of how precise temporal control is achieved by a small number of master regulators to ensure a successful cell division cycle.

THE EFFECTS OF TYPE II BINDING ON METABOLIC STABILITY AND BINDING AFFINITY IN CYTOCHROME P450 CYP3A4

PubMed Central

Peng, Chi-Chi; Pearson, Josh T.; Rock, Dan A.; Joswig-Jones, Carolyn A.; Jones, Jeffrey P.

2010-01-01

One goal in drug design is to decrease clearance due to metabolism. It has been suggested that a compound’s metabolic stability can be increased by incorporation of a sp2 nitrogen into an aromatic ring. Nitrogen incorporation is hypothesized to increase metabolic stability by coordination of nitrogen to the heme iron (termed type II binding). However, questions regarding binding affinity, metabolic stability, and how metabolism of type II binders occurs remain unanswered. Herein, we use pyridinyl quinoline-4-carboxamide analogs to answer these questions. We show that type II binding can have a profound influence on binding affinity for CYP3A4, and the difference in binding affinity can be as high as 1,200 fold. We also find that type II binding compounds can be extensively metabolized, which is not consistent with the dead-end complex kinetic model assumed for type II binders. Two alternate kinetic mechanisms are presented to explain the results. The first involves a rapid equilibrium between the type II bound substrate and a metabolically oriented binding mode. The second involves direct reduction of the nitrogen-coordinated heme followed by oxygen binding. PMID:20346909

Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein.

PubMed

Ben Halima, Saoussen; Mishra, Sabyashachi; Raja, K Muruga Poopathi; Willem, Michael; Baici, Antonio; Simons, Kai; Brüstle, Oliver; Koch, Philipp; Haass, Christian; Caflisch, Amedeo; Rajendran, Lawrence

2016-03-08

Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD). AD is characterized by toxic β-amyloid (Aβ) peptides produced by β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP). β-secretase inhibitors reduce Aβ levels, but mechanism-based side effects arise because they also inhibit β-cleavage of non-amyloid substrates like Neuregulin. We report that β-secretase has a higher affinity for Neuregulin than it does for APP. Kinetic studies demonstrate that the affinities and catalytic efficiencies of β-secretase are higher toward non-amyloid substrates than toward APP. We show that non-amyloid substrates are processed by β-secretase in an endocytosis-independent manner. Exploiting this compartmentalization of substrates, we specifically target the endosomal β-secretase by an endosomally targeted β-secretase inhibitor, which blocked cleavage of APP but not non-amyloid substrates in many cell systems, including induced pluripotent stem cell (iPSC)-derived neurons. β-secretase inhibitors can be designed to specifically inhibit the Alzheimer process, enhancing their potential as AD therapeutics without undesired side effects. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Delineation of xenobiotic substrate sites in rat glutathione S-transferase M1-1

PubMed Central

Hearne, Jennifer L.; Colman, Roberta F.

2005-01-01

Glutathione S-transferases catalyze the conjugation of glutathione with endogenous and exogenous xenobiotics. Hu and Colman (1995) proposed that there are two distinct substrate sites in rat GST M1-1, a 1-chloro-2,4-dintrobenzene (CDNB) substrate site located in the vicinity of tyrosine-115, and a monobromobimane (mBBr) substrate site. To determine whether the mBBr substrate site is distinguishable from the CDNB substrate site, we tested S-(hydroxyethyl)bimane, a nonreactive derivative of mBBr, for its ability to compete kinetically with the substrates. We find that S-(hydroxyethyl)bimane is a competitive inhibitor (KI = 0.36 μM) when mBBr is used as substrate, but not when CDNB is used as substrate, demonstrating that these two sites are distinct. Using site-directed mutagenesis, we have localized the mBBr substrate site to an area midway through α-helix 4 (residues 90–114) and have identified residues that are important in the enzymatic reaction. Substitution of alanine at positions along α-helix 4 reveals that mutations at positions 103, 104, and 109 exhibit a greater perturbation of the enzymatic reaction with mBBr than with CDNB as substrate. Various other substitutions at positions 103 and 104 reveal that a hydrophobic residue is necessary at each of these positions to maintain optimal affinity of the enzyme for mBBr and preserve the secondary structure of the enzyme. Substitutions at position 109 indicate that this residue is important in the enzyme’s affinity for mBBr but has a minimal effect on Vmax. These results demonstrate that the promiscuity of rat GST M1-1 is in part due to at least two distinct substrate sites. PMID:16195544

Delineation of xenobiotic substrate sites in rat glutathione S-transferase M1-1.

PubMed

Hearne, Jennifer L; Colman, Roberta F

2005-10-01

Glutathione S-transferases catalyze the conjugation of glutathione with endogenous and exogenous xenobiotics. Hu and Colman (1995) proposed that there are two distinct substrate sites in rat GST M1-1, a 1-chloro-2,4-dintrobenzene (CDNB) substrate site located in the vicinity of tyrosine-115, and a monobromobimane (mBBr) substrate site. To determine whether the mBBr substrate site is distinguishable from the CDNB substrate site, we tested S-(hydroxyethyl)bimane, a nonreactive derivative of mBBr, for its ability to compete kinetically with the substrates. We find that S-(hydroxyethyl)bimane is a competitive inhibitor (K(I) = 0.36 microM) when mBBr is used as substrate, but not when CDNB is used as substrate, demonstrating that these two sites are distinct. Using site-directed mutagenesis, we have localized the mBBr substrate site to an area midway through alpha-helix 4 (residues 90-114) and have identified residues that are important in the enzymatic reaction. Substitution of alanine at positions along alpha-helix 4 reveals that mutations at positions 103, 104, and 109 exhibit a greater perturbation of the enzymatic reaction with mBBr than with CDNB as substrate. Various other substitutions at positions 103 and 104 reveal that a hydrophobic residue is necessary at each of these positions to maintain optimal affinity of the enzyme for mBBr and preserve the secondary structure of the enzyme. Substitutions at position 109 indicate that this residue is important in the enzyme's affinity for mBBr but has a minimal effect on Vmax. These results demonstrate that the promiscuity of rat GST M1-1 is in part due to at least two distinct substrate sites.

Combining affinity proteomics and network context to identify new phosphatase substrates and adapters in growth pathways

PubMed Central

Sacco, Francesca; Boldt, Karsten; Calderone, Alberto; Panni, Simona; Paoluzi, Serena; Castagnoli, Luisa; Ueffing, Marius; Cesareni, Gianni

2014-01-01

Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI3K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26, respectively. PMID:24847354

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Gyanendra; Swaminathan, Subramanyam

Botulinum Neurotoxins are the most poisonous of all toxins with lethal dose in nanogram quantities. They are also potential biological warfare and bioterrorism agents due to their high toxicity and ease of preparation. On the other hand BoNTs are also being increasingly used for therapeutic and cosmetic purposes, and with that the chances of accidental overdose are increasing. And despite the potential damage they could cause to human health, there are no post-intoxication drugs available so far. But progress is being made in this direction. The crystal structures in native form and bound with substrate peptides have been determined, andmore » these are enabling structure-based drug discovery possible. High throughput assays have also been designed to speed up the screening progress. Substrate-based and small molecule inhibitors have been identified. But turning high affinity inhibitors into clinically viable drug candidates has remained a challenge. We discuss here the latest developments and the future challenges in drug discovery for Botulinum neurotoxins.« less

Enzyme Technology of Peroxidases: Immobilization, Chemical and Genetic Modification

NASA Astrophysics Data System (ADS)

Longoria, Adriana; Tinoco, Raunel; Torres, Eduardo

An overview of enzyme technology applied to peroxidases is made. Immobilization on organic, inorganic, and hybrid supports; chemical modification of amino acids and heme group; and genetic modification by site-directed and random mutagenesis are included. Different strategies that were carried out to improve peroxidase performance in terms of stability, selectivity, and catalytic activity are analyzed. Immobilization of peroxidases on inorganic and organic materials enhances the tolerance of peroxidases toward the conditions normally found in many industrial processes, such as the presence of an organic solvent and high temperature. In addition, it is shown that immobilization helps to increase the Total Turnover Number at levels high enough to justify the use of a peroxidase-based biocatalyst in a synthesis process. Chemical modification of peroxidases produces modified enzymes with higher thermostability and wider substrate variability. Finally, through mutagenesis approaches, it is possible to produce modified peroxidases capable of oxidizing nonnatural substrates with high catalytic activity and affinity.

Pharmacophore, QSAR, and binding mode studies of substrates of human cytochrome P450 2D6 (CYP2D6) using molecular docking and virtual mutations and an application to chinese herbal medicine screening.

PubMed

Mo, Sui-Lin; Liu, Wei-Feng; Li, Chun-Guang; Zhou, Zhi-Wei; Luo, Hai-Bin; Chew, Helen; Liang, Jun; Zhou, Shu-Feng

2012-07-01

The highly polymorphic human cytochrome P450 2D6 (CYP2D6) metabolizes about 25% of currently used drugs. In this study, we have explored the interaction of a large number of substrates (n = 120) with wild-type and mutated CYP2D6 by molecular docking using the CDOCKER module. Before we conducted the molecular docking and virtual mutations, the pharmacophore and QSAR models of CYP2D6 substrates were developed and validated. Finally, we explored the interaction of a traditional Chinese herbal formula, Fangjifuling decoction, with CYP2D6 by virtual screening. The optimized pharmacophore model derived from 20 substrates of CYP2D6 contained two hydrophobic features and one hydrogen bond acceptor feature, giving a relevance ratio of 76% when a validation set of substrates were tested. However, our QSAR models gave poor prediction of the binding affinity of substrates. Our docking study demonstrated that 117 out of 120 substrates could be docked into the active site of CYP2D6. Forty one out of 117 substrates (35.04%) formed hydrogen bonds with various active site residues of CYP2D6 and 53 (45.30%) substrates formed a strong π-π interaction with Phe120 (53/54), with only carvedilol showing π-π interaction with Phe483. The active site residues involving hydrogen bond formation with substrates included Leu213, Lys214, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, Phe483, and Phe484. Furthermore, the CDOCKER algorithm was further applied to study the impact of mutations of 28 active site residues (mostly non-conserved) of CYP2D6 on substrate binding modes using five probe substrates including bufuralol, debrisoquine, dextromethorphan, sparteine, and tramadol. All mutations of the residues examined altered the hydrogen bond formation and/or aromatic interactions, depending on the probe used in molecular docking. Apparent changes of the binding modes have been observed with the Glu216Asp and Asp301Glu mutants. Overall, 60 compounds out of 130 from Fangjifuling decoction matched our pharmacophore model for CYP2D6 substrates. Fifty four out of these 60 compounds could be docked into the active site of CYP2D6 and 24 of 54 compounds formed hydrogen bonds with Glu216, Asp301, Ser304, and Ala305 in CYP2D6. These results have provided further insights into the factors that determining the binding modes of substrates to CYP2D6. Screening of high-affinity ligands for CYP2D6 from herbal formula using computational models is a useful approach to identify potential herb-drug interactions.

Analysis of Structural Features Contributing to Weak Affinities of Ubiquitin/Protein Interactions.

PubMed

Cohen, Ariel; Rosenthal, Eran; Shifman, Julia M

2017-11-10

Ubiquitin is a small protein that enables one of the most common post-translational modifications, where the whole ubiquitin molecule is attached to various target proteins, forming mono- or polyubiquitin conjugations. As a prototypical multispecific protein, ubiquitin interacts non-covalently with a variety of proteins in the cell, including ubiquitin-modifying enzymes and ubiquitin receptors that recognize signals from ubiquitin-conjugated substrates. To enable recognition of multiple targets and to support fast dissociation from the ubiquitin modifying enzymes, ubiquitin/protein interactions are characterized with low affinities, frequently in the higher μM and lower mM range. To determine how structure encodes low binding affinity of ubiquitin/protein complexes, we analyzed structures of more than a hundred such complexes compiled in the Ubiquitin Structural Relational Database. We calculated various structure-based features of ubiquitin/protein binding interfaces and compared them to the same features of general protein-protein interactions (PPIs) with various functions and generally higher affinities. Our analysis shows that ubiquitin/protein binding interfaces on average do not differ in size and shape complementarity from interfaces of higher-affinity PPIs. However, they contain fewer favorable hydrogen bonds and more unfavorable hydrophobic/charge interactions. We further analyzed how binding interfaces change upon affinity maturation of ubiquitin toward its target proteins. We demonstrate that while different features are improved in different experiments, the majority of the evolved complexes exhibit better shape complementarity and hydrogen bond pattern compared to wild-type complexes. Our analysis helps to understand how low-affinity PPIs have evolved and how they could be converted into high-affinity PPIs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selection of a novel peptide aptamer with high affinity for TiO2-nanoparticle through a direct electroporation with TiO2-binding phage complexes.

PubMed

Inoue, Ippei; Ishikawa, Yasuaki; Uraoka, Yukiharu; Yamashita, Ichiro; Yasueda, Hisashi

2016-11-01

We have developed an easy and rapid screening method of peptide aptamers with high affinity for a target material TiO 2 using M13 phage-display and panning procedure. In a selection step, the phage-substrate complexes and Escherichia coli cells were directly applied by electric pulse for electroporation, without separating the objective phages from the TiO 2 nanoparticles. Using this simple and rapid method, we obtained a novel peptide aptamer (named ST-1 with the sequence AYPQKFNNNFMS) with highly strong binding activity for TiO 2 . A cage-shaped protein fused with both ST-1 and an available carbon nanotube-affinity peptide was designed and produced in E. coli. The multi-functional supraprotein could efficiently mineralize a titanium-compound around the surface of single-wall carbon nanotubes (SWNTs), indicating that the ST-1 is valuable in the fabrication of nano-composite materials with titanium-compounds. The structural analysis of ST-1 variants indicated the importance of the N-terminal region (as a motif of AXPQKX 6 S) of the aptamer in the TiO 2 -binding activity. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Characterizing Isozymes of Chlorite Dismutase for Water Treatment

PubMed Central

Mobilia, Kellen C.; Hutchison, Justin M.; Zilles, Julie L.

2017-01-01

This work investigated the potential for biocatalytic degradation of micropollutants, focusing on chlorine oxyanions as model contaminants, by mining biology to identify promising biocatalysts. Existing isozymes of chlorite dismutase (Cld) were characterized with respect to parameters relevant to this high volume, low-value product application: kinetic parameters, resistance to catalytic inactivation, and stability. Maximum reaction velocities (Vmax) were typically on the order of 104 μmol min-1 (μmol heme)-1. Substrate affinity (Km) values were on the order of 100 μM, except for the Cld from Candidatus Nitrospira defluvii (NdCld), which showed a significantly lower affinity for chlorite. NdCld also had the highest susceptibility to catalytic inactivation. In contrast, the Cld from Ideonella dechloratans was least susceptible to catalytic inactivation, with a maximum turnover number of approximately 150,000, more than sevenfold higher than other tested isozymes. Under non-reactive conditions, Cld was quite stable, retaining over 50% of activity after 30 days, and most samples retained activity even after 90–100 days. Overall, Cld from I. dechloratans was the most promising candidate for environmental applications, having high affinity and activity, a relatively low propensity for catalytic inactivation, and excellent stability. PMID:29312158

A chemical proteomics approach for global analysis of lysine monomethylome profiling.

PubMed

Wu, Zhixiang; Cheng, Zhongyi; Sun, Mingwei; Wan, Xuelian; Liu, Ping; He, Tieming; Tan, Minjia; Zhao, Yingming

2015-02-01

Methylation of lysine residues on histone proteins is known to play an important role in chromatin structure and function. However, non-histone protein substrates of this modification remain largely unknown. An effective approach for system-wide analysis of protein lysine methylation, particularly lysine monomethylation, is lacking. Here we describe a chemical proteomics approach for global screening for monomethyllysine substrates, involving chemical propionylation of monomethylated lysine, affinity enrichment of the modified monomethylated peptides, and HPLC/MS/MS analysis. Using this approach, we identified with high confidence 446 lysine monomethylation sites in 398 proteins, including three previously unknown histone monomethylation marks, representing the largest data set of protein lysine monomethylation described to date. Our data not only confirms previously discovered lysine methylation substrates in the nucleus and spliceosome, but also reveals new substrates associated with diverse biological processes. This method hence offers a powerful approach for dynamic study of protein lysine monomethylation under diverse cellular conditions and in human diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Classification of a Haemophilus influenzae ABC Transporter HI1470/71 through Its Cognate Molybdate Periplasmic Binding Protein, MolA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tirado-Lee, Leidamarie; Lee, Allen; Rees, Douglas C.

2014-10-02

molA (HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB{sub 2}C{sub 2} (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7 {angstrom} resolution, respectively. The MolA-binding protein binds molybdate and tungstate, but not other oxyanions such as sulfate and phosphate, making it the first class III molybdate-binding protein structurally solved. The {approx}100 {mu}M binding affinity for tungstate and molybdate is significantly lower than observed for the class II ModA molybdate-binding proteins that have nanomolar to low micromolar affinity for molybdate.more » The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus.« less

Effect of Pentacene-dielectric Affinity on Pentacene Thin Film Growth Morphology in Organic Field-effect Transistors

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Kim; M Jang; H Yang

2011-12-31

Organic field-effect transistors (OFETs) are fabricated by depositing a thin film of semiconductor on the functionalized surface of a SiO{sub 2} dielectric. The chemical and morphological structures of the interface between the semiconductor and the functionalized dielectric are critical for OFET performance. We have characterized the effect of the affinity between semiconductor and functionalized dielectric on the properties of the semiconductor-dielectric interface. The crystalline microstructure/nanostructure of the pentacene semiconductor layers, grown on a dielectric substrate that had been functionalized with either poly(4-vinyl pyridine) or polystyrene (to control hydrophobicity), and grown under a series of substrate temperatures and deposition rates, weremore » characterized by X-ray diffraction, photoemission spectroscopy, and atomic force microscopy. By comparing the morphological features of the semiconductor thin films with the device characteristics (field-effect mobility, threshold voltage, and hysteresis) of the OFET devices, the effect of affinity-driven properties on charge modulation, charge trapping, and charge carrier transport could be described.« less

Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors.

PubMed

Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

2016-07-18

Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process.

Enhanced protein adsorption and patterning on nanostructured latex-coated paper.

PubMed

Juvonen, Helka; Määttänen, Anni; Ihalainen, Petri; Viitala, Tapani; Sarfraz, Jawad; Peltonen, Jouko

2014-06-01

Specific interactions of extracellular matrix proteins with cells and their adhesion to the substrate are important for cell growth. A nanopatterned latex-coated paper substrate previously shown to be an excellent substrate for cell adhesion and 2D growth was studied for directed immobilization of proteins. The nanostructured latex surface was formed by short-wavelength IR irradiation of a two-component latex coating consisting of a hydrophilic film-forming styrene butadiene acrylonitrile copolymer and hydrophobic polystyrene particles. The hydrophobic regions of the IR-treated latex coating showed strong adhesion of bovine serum albumin (cell repelling protein), fibronectin (cell adhesive protein) and streptavidin. Opposite to the IR-treated surface, fibronectin and streptavidin had a poor affinity toward the untreated pristine latex coating. Detailed characterization of the physicochemical surface properties of the latex-coated substrates revealed that the observed differences in protein affinity were mainly due to the presence or absence of the protein repelling polar and charged surface groups. The protein adsorption was assisted by hydrophobic (dehydration) interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

Allosteric regulation of tryptophan synthase channeling: the internal aldimine probed by trans-3-indole-3'-acrylate binding.

PubMed

Casino, Patricia; Niks, Dimitri; Ngo, Huu; Pan, Peng; Brzovic, Peter; Blumenstein, Lars; Barends, Thomas Reinier; Schlichting, Ilme; Dunn, Michael F

2007-07-03

Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.

Characterization of a beta-glycosidase highly active on disaccharides and of a beta-galactosidase from Tenebrio molitor midgut lumen.

PubMed

Ferreira, Alexandre H P; Terra, Walter R; Ferreira, Clélia

2003-02-01

The midgut of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae has four beta-glycosidases. The properties of two of these enzymes (betaGly1 and betaGly2) have been described elsewhere. In this paper, the characterization of the other two glycosidases (betaGly3 and betaGly4) is described. BetaGly3 has one active site, hydrolyzes disaccharides, cellodextrins, synthetic substrates and beta-glucosides produced by plants. The enzyme is inhibited by amygdalin, cellotriose, cellotetraose and cellopentaose in high concentrations, probably due to transglycosylation. betaGly3 hydrolyzes beta 1,4-glycosidic linkages with a catalytic rate independent of the substrate polymerization degree (k(int)) of 11.9 s(-1). Its active site is formed by four subsites, where subsites +1 and -1 bind glucose residues with higher affinity than subsite +2. The main role of betaGly3 seems to be disaccharide hydrolysis. BetaGly4 is a beta-galactosidase, since it has highest activity against beta-galactosides. It can also hydrolyze fucosides, but not glucosides, and has Triton X-100 as a non-essential activator (K(a)=15 microM, pH 4.5). betaGly4 has two active sites that can hydrolyze p-nitrophenyl beta-galactoside (NPbetaGal). The one hydrolyzing NPbetaGal with more efficiency is also active against methylumbellipheryl beta-D-galactoside and lactose. The other active site hydrolyzes NPbetaFucoside and binds NPbetaGal weakly. BetaGly4 hydrolyzes hydrophobic substrates with high catalytical efficiency and is able to bind octyl-beta-thiogalactoside in its active site with high affinity. The betaGly4 physiological role is supposed to be the hydrolysis of galactolipids that are found in membranes from vegetal tissues. As the enzyme has a hydrophobic site where Triton X-100 can bind, it might be activated by membrane lipids, thus becoming fully active only at the surface of cell membranes.

Dynamic Seeding of Perfusing Human Umbilical Vein Endothelial Cells (HUVECs) onto Dual-Function Cell Adhesion Ligands: Arg-Gly-Asp (RGD)-Streptavidin and Biotinylated Fibronectin

PubMed Central

Anamelechi, Charles C.; Clermont, Edward C.; Novak, Matthew T.; Reichert, William M.

2014-01-01

Surfaces decorated with high affinity ligands can be used to facilitate rapid attachment of endothelial cells; however, standard equilibrium cell detachment studies are poorly suited for assessing these initial adhesion events. Here, a dynamic seeding and cell retention method was used to examine the initial attachment of perfusing human umbilical vein endothelial cells (HUVECs) to bare Teflon-AF substrates, substrates pre-adsorbed with fibronectin alone, or substrates co-pre-adsorbed with two dual-function cell-adhesion ligands: biotinylated fibronectin (bFN) and RGD-streptavidin mutant (RGD-SA). Cell attachment was evaluated as a function of cell trypsinization (integrin digestion), surface protein formulation, and cell perfusion rate. Surfaces co-pre-adsorbed with bFN and RGD-SA showed the highest density of attached cells after 8 min of perfusion and the highest percent retention when subjected to shear flow at 60 dynes/cm2 for 2 min. Surfaces with no ligand treatment showed the lowest cell attachment and retention under flow in all cases. HUVECs trypsinized with mild 0.025% trypsin/ethylenediaminetetraacetic acid (EDTA) showed greater cell adhesion after perfusion and higher percent retention after shear flow than those trypsinized using harsher 0.05% trypsin/EDTA. The preferential affinities of the two dual-function ligands for α5β1 and αvβ3 integrins were also examined by surface plasmon resonance (SPR) spectroscopy. The dynamic cell seeding studies confirmed that the dual-function ligand system promotes HUVEC adhesion and retention at short time points when tested using a perfusion assay. SPR studies showed that the two ligands exhibited equal affinity for both α5β1 and αvβ3 integrins but that the combined ligands bound more total integrins than the two ligands tested separately. PMID:19348476

Interactions between Cellulolytic Enzymes with Native, Autohydrolysis, and Technical Lignins and the Effect of a Polysorbate Amphiphile in Reducing Nonproductive Binding.

PubMed

Fritz, Consuelo; Ferrer, Ana; Salas, Carlos; Jameel, Hasan; Rojas, Orlando J

2015-12-14

Understanding enzyme-substrate interactions is critical in designing strategies for bioconversion of lignocellulosic biomass. In this study we monitored molecular events, in situ and in real time, including the adsorption and desorption of cellulolytic enzymes on lignins and cellulose, by using quartz crystal microgravimetry and surface plasmon resonance. The effect of a nonionic surface active molecule was also elucidated. Three lignin substrates relevant to the sugar platform in biorefinery efforts were considered, namely, hardwood autohydrolysis cellulolytic (HWAH), hardwood native cellulolytic (MPCEL), and nonwood native cellulolytic (WSCEL) lignin. In addition, Kraft lignins derived from softwoods (SWK) and hardwoods (HWK) were used as references. The results indicated a high affinity between the lignins with both, monocomponent and multicomponent enzymes. More importantly, the addition of nonionic surfactants at concentrations above their critical micelle concentration reduced remarkably (by over 90%) the nonproductive interactions between the cellulolytic enzymes and the lignins. This effect was hypothesized to be a consequence of the balance of hydrophobic and hydrogen bonding interactions. Moreover, the reduction of surface roughness and increased wettability of lignin surfaces upon surfactant treatment contributed to a lower affinity with the enzymes. Conformational changes of cellulases were observed upon their adsorption on lignin carrying preadsorbed surfactant. Weak electrostatic interactions were determined in aqueous media at pH between 4.8 and 5.5 for the native cellulolytic lignins (MPCEL and WSCEL), whereby a ∼20% reduction in the enzyme affinity was observed. This was mainly explained by electrostatic interactions (osmotic pressure effects) between charged lignins and cellulases. Noteworthy, adsorption of nonionic surfactants onto cellulose, in the form cellulose nanofibrils, did not affect its hydrolytic conversion. Overall, our results highlight the benefit of nonionic surfactant pretreatment to reduce nonproductive enzyme binding while maintaining the reactivity of the cellulosic substrate.

Purification and characterization of a rice class I chitinase, OsChia1b, produced in Esherichia coli.

PubMed

Mizuno, Ryoji; Itoh, Yoshikane; Nishizawa, Yoko; Kezuka, Yuichiro; Suzuki, Kazushi; Nonaka, Takamasa; Watanabe, Takeshi

2008-03-01

To determine the properties and structure of OsChia1b, a family 19 chitinase from Oryza sativa L. cv. Nipponbare (japonica ssp.), recombinant OsChia1b was produced in Esherichia coli cells and purified to homogeneity by chitin affinity column chromatography. OsChia1b was highly active against soluble chitinous substrate, but not against crystalline chitin, and clearly inhibited hyphal extension of Trichoderma reesei.

Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein.

PubMed

Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R

2006-11-28

We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.

A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula.

PubMed

Straub, Daniel; Ludewig, Uwe; Neuhäuser, Benjamin

2014-11-01

Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with Km AMT1;1 = 89 µM and Km AMT2;1 = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger (15)N-ammonium uptake than MtAMT2;1, but NH4 (+) currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent Km = 90 µM at -80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply.

Extracellular enzyme kinetics scale with resource availability

USGS Publications Warehouse

Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

2014-01-01

Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors.

PubMed

Swedberg, Joakim E; Harris, Jonathan M

2011-10-04

Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.

Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

PubMed Central

Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

2016-01-01

The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

Method and apparatus for detection of fluorescently labeled materials

DOEpatents

Stern, David; Fiekowsky, Peter

2004-05-25

Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.

Na+/substrate Coupling in the Multidrug Antiporter NorM Probed with a Spin-labeled Substrate

PubMed Central

Steed, P. Ryan; Stein, Richard A.; Mishra, Smriti; Goodman, Michael C.; Mchaourab, Hassane S.

2013-01-01

NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na+ gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM from Vibrio cholerae. It is therefore ideal to characterize mechanistically relevant binding interactions with NorM and to directly address the coupling of ion and drug binding. Fluorescence and EPR experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Na+. Using double electron-electron resonance (DEER) spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na+ did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na+ does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na+ gradient across the native membrane, govern Na+/drug coupling during antiport. PMID:23902581

Development of Substrate-Selective Probes for Affinity Pulldown of Histone Demethylases

PubMed Central

2015-01-01

JmjC-domain containing histone demethylases (JHDMs) play critical roles in many key cellular processes and have been implicated in multiple disease conditions. Each enzyme within this family is known to have a strict substrate scope, specifically the position of the lysine within the histone and its degree of methylation. While much progress has been made in determining the substrates of each enzyme, new methods with which to systematically profile each histone mark are greatly needed. Novel chemical tools have the potential to fill this role and, furthermore, can be used as probes to answer fundamental questions about these enzymes and serve as potential therapeutic leads. In this work, we first investigated three small-molecule probes differing in the degree of “methylation state” and their differential bindings to JHDM1A (an H3K36me1/2 demethylase) using a fluorescence polarization-based competition assay. We then applied this specificity toward the “methylation state” and combined it with specificity toward lysine position in the design and synthesis of a peptidic probe targeting H3K36me2 JHDMs. The probe is further functionalized with a benzophenone cross-linking moiety and a biotin for affinity purification. Results showed binding of the peptidic probe to JHDM1A and specific enrichment of this protein in the presence of its native histone substrates. Affinity purification pulldown experiments from nuclear lysate coupled with mass spectrometry revealed the capability of the probe to pull out and enrich JHDMs along with other epigenetic proteins and transcriptional regulators. PMID:25335116

Structure-Activity Relationships of Substituted Cathinones, with Transporter Binding, Uptake, and Release

PubMed Central

Wolfrum, Katherine M.; Reed, John F.; Kim, Sunyoung O.; Swanson, Tracy; Johnson, Robert A.; Janowsky, Aaron

2017-01-01

Synthetic cathinones are components of “bath salts” and have physical and psychologic side effects, including hypertension, paranoia, and hallucinations. Here, we report interactions of 20 “bath salt” components with human dopamine, serotonin, and norepinephrine transporters [human dopamine transporter (hDAT), human serotonin transporter (hSERT), and human norepinephrine transporter (hNET), respectively] heterologously expressed in human embryonic kidney 293 cells. Transporter inhibitors had nanomolar to micromolar affinities (Ki values) at radioligand binding sites, with relative affinities of hDAT>hNET>hSERT for α-pyrrolidinopropiophenone (α-PPP), α-pyrrolidinobutiophenone, α-pyrrolidinohexiophenone, 1-phenyl-2-(1-pyrrolidinyl)-1-heptanone, 3,4-methylenedioxy-α-pyrrolidinopropiophenone, 3,4-methylenedioxy-α-pyrrolidinobutiophenone, 4-methyl-α-pyrrolidinopropiophenone, α-pyrrolidinovalerophenone, 4-methoxy-α-pyrrolidinovalerophenone, α-pyrrolidinopentiothiophenone (alpha-PVT), and α-methylaminovalerophenone, and hDAT>hSERT>hNET for methylenedioxypentedrone. Increasing the α-carbon chain length increased the affinity and potency of the α-pyrrolidinophenones. Uptake inhibitors had relative potencies of hDAT>hNET>hSERT except α-PPP and α-PVT, which had highest potencies at hNET. They did not induce [3H]neurotransmitter release. Substrates can enter presynaptic neurons via transporters, and the substrates methamphetamine and 3,4-methylenedioxymethylamphetamine are neurotoxic. We determined that 3-fluoro-, 4-bromo-, 4-chloro-methcathinone, and 4-fluoroamphetamine were substrates at all three transporters; 5,6-methylenedioxy-2-aminoindane (MDAI) and 4-methylethcathinone (4-MEC) were substrates primarily at hSERT and hNET; and 3,4-methylenedioxy-N-ethylcathinone (ethylone) and 5-methoxy-methylone were substrates only at hSERT and induced [3H]neurotransmitter release. Significant correlations between potencies for inhibition of uptake and for inducing release were observed for these and additional substrates. The excellent correlation of efficacy at stimulating release versus Ki/IC50 ratios suggested thresholds of binding/uptake ratios above which compounds were likely to be substrates. Based on their potencies at hDAT, most of these compounds have potential for abuse and addiction. 4-Bromomethcathinone, 4-MEC, 5-methoxy-methylone, ethylone, and MDAI, which have higher potencies at hSERT than hDAT, may have empathogen psychoactivity. PMID:27799294

High-throughput screening of dye-ligands for chromatography.

PubMed

Kumar, Sunil; Punekar, Narayan S

2014-01-01

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen-bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. Choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices provide a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid-handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lay in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum.

PubMed

Liebau, Eva; De Maria, Francesca; Burmeister, Cora; Perbandt, Markus; Turella, Paola; Antonini, Giovanni; Federici, Giorgio; Giansanti, Francesco; Stella, Lorenzo; Lo Bello, Mario; Caccuri, Anna Maria; Ricci, Giorgio

2005-07-15

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Antibody Epitope of Human α-Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease.

PubMed

Kukacka, Zdenek; Iurascu, Marius; Lupu, Loredana; Rusche, Hendrik; Murphy, Mary; Altamore, Lorenzo; Borri, Fabio; Maeser, Stefan; Papini, Anna Maria; Hennermann, Julia; Przybylski, Michael

2018-05-08

α-Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α-galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α-galactosidase A is known as Fabry disease or Fabry-Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy-limiting, and eventually life-threatening complications of ERT. The present study focused on the epitope determination of human α-galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309-332) recognized by a human monoclonal anti-αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309-332), was synthesized by solid-phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (K D =39×10 -9  m), which is nearly identical to that of the full-length enzyme (K D =16×10 -9  m). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full-length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of Preferential Orientation of Lamellae in the Interfacial Region between a Block Copolymer-based Pressure-Sensitive Adhesive and a Solid Substrate on the Peel Strength.

PubMed

Shimokita, Keisuke; Saito, Itsuki; Yamamoto, Katsuhiro; Takenaka, Mikihito; Yamada, Norifumi L; Miyazaki, Tsukasa

2018-02-27

We have investigated the relationship between the peel strength of a block copolymer-based pressure-sensitive adhesive comprising of poly(methyl methacrylate) (PMMA) and poly(n-butyl acrylate) (PnBA) components from the substrate and the microdomain orientations in the interfacial region between the adhesive and the substrate. For the PMMA substrate, the PMMA component in the adhesive with a strong affinity for the substrate is attached to the surface of the substrate during an aging process of the sample at 140 °C. Next, the PMMA layer adjacent to the substrate surface is overlaid with a PnBA layer, which gets covalently connected, resulting in the horizontal alignment of the lamellae in the interfacial region. The peel strength of the adhesive substantially increases during aging at 140 °C, which takes the same time as the completion of the horizontally oriented lamellar structure. However, in the case of the polystyrene (PS) substrate, both the components in the adhesive repel the substrate, leading to the formation of the vertically oriented lamellar structure. As a result, the peel strength of the adhesive with respect to its PS substrate does not entirely increase on aging. It is suggested that the peel strength of the adhesive is highly correlated with the interfacial energy between the adhesive and substrate, which can be estimated from the microdomain orientation in the interfacial region.

Monitoring the chemical production of citrus-derived bioactive 5-demethylnobiletin using surface enhanced Raman spectroscopy

PubMed Central

Zheng, Jinkai; Fang, Xiang; Cao, Yong; Xiao, Hang; He, Lili

2013-01-01

To develop an accurate and convenient method for monitoring the production of citrus-derived bioactive 5-demethylnobiletin from demethylation reaction of nobiletin, we compared surface enhanced Raman spectroscopy (SERS) methods with a conventional HPLC method. Our results show that both the substrate-based and solution-based SERS methods correlated with HPLC method very well. The solution method produced lower root mean square error of calibration and higher correlation coefficient than the substrate method. The solution method utilized an ‘affinity chromatography’-like procedure to separate the reactant nobiletin from the product 5-demthylnobiletin based on their different binding affinity to the silver dendrites. The substrate method was found simpler and faster to collect the SERS ‘fingerprint’ spectra of the samples as no incubation between samples and silver was needed and only trace amount of samples were required. Our results demonstrated that the SERS methods were superior to HPLC method in conveniently and rapidly characterizing and quantifying 5-demethylnobiletin production. PMID:23885986

Preparation of molecularly imprinted polymers specific to glycoproteins, glycans and monosaccharides via boronate affinity controllable-oriented surface imprinting.

PubMed

Xing, Rongrong; Wang, Shuangshou; Bie, Zijun; He, Hui; Liu, Zhen

2017-05-01

Molecularly imprinted polymers (MIPs) are materials that are designed to be receptors for a template molecule (e.g., a protein). They are made by polymerizing the polymerizable reagents in the presence of the template; when the template is removed, the material can be used for many applications that would traditionally use antibodies. Thus, MIPs are biomimetic of antibodies and in this capacity have found wide applications, such as sensing, separation and diagnosis. However, many imprinting approaches are uncontrollable, and facile imprinting approaches widely applicable to a large variety of templates remain limited. We developed an approach called boronate affinity controllable-oriented surface imprinting, which allows for easy and efficient preparation of MIPs specific to glycoproteins, glycans and monosaccharides. This approach relies on immobilization of a template (glycoprotein, glycan or monosaccharide) on a boronic-acid-functionalized substrate through boronate affinity interaction, followed by self-polymerization of biocompatible monomer(s) to form an imprinting layer on the substrate with appropriate thickness. Imprinting in this approach is performed in a controllable manner, permitting the thickness of the imprinting layer to be fine-tuned according to the molecular size of the template by adjusting the imprinting time. This not only simplifies the imprinting procedure but also makes the approach widely applicable to a large range of sugar-containing biomolecules. MIPs prepared by this approach exhibit excellent binding properties and can be applied to complex real samples. The MIPs prepared by this protocol have been used in affinity separation, disease diagnosis and bioimaging. The entire protocol, including preparation, property characterization and performance evaluation, takes ∼3-8 d, depending on the type of substrate and template used.

Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction.

PubMed

Wang, Hualei; Sun, Huihui; Wei, Dongzhi

2013-02-18

A nitrilase-mediated pathway has significant advantages in the production of optically pure (R)-(-)-mandelic acid. However, unwanted byproduct, low enantioselectivity, and specific activity reduce its value in practical applications. An ideal nitrilase that can efficiently hydrolyze mandelonitrile to optically pure (R)-(-)-mandelic acid without the unwanted byproduct is needed. A novel nitrilase (BCJ2315) was discovered from Burkholderia cenocepacia J2315 through phylogeny-based enzymatic substrate specificity prediction (PESSP). This nitrilase is a mandelonitrile hydrolase that could efficiently hydrolyze mandelonitrile to (R)-(-)-mandelic acid, with a high enantiomeric excess of 98.4%. No byproduct was observed in this hydrolysis process. BCJ2315 showed the highest identity of 71% compared with other nitrilases in the amino acid sequence. BCJ2315 possessed the highest activity toward mandelonitrile and took mandelonitrile as the optimal substrate based on the analysis of substrate specificity. The kinetic parameters Vmax, Km, Kcat, and Kcat/Km toward mandelonitrile were 45.4 μmol/min/mg, 0.14 mM, 15.4 s(-1), and 1.1×10(5) M(-1)s(-1), respectively. The recombinant Escherichia coli M15/BCJ2315 had a strong substrate tolerance and could completely hydrolyze mandelonitrile (100 mM) with fewer amounts of wet cells (10 mg/ml) within 1 h. PESSP is an efficient method for discovering an ideal mandelonitrile hydrolase. BCJ2315 has high affinity and catalytic efficiency toward mandelonitrile. This nitrilase has great advantages in the production of optically pure (R)-(-)-mandelic acid because of its high activity and enantioselectivity, strong substrate tolerance, and having no unwanted byproduct. Thus, BCJ2315 has great potential in the practical production of optically pure (R)-(-)-mandelic acid in the industry.

The comprehensive study and the reduction of contact resistivity on the n-InGaAs M-I-S contact system with different inserted insulators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao, M.-H., E-mail: mhliaoa@ntu.edu.tw; Lien, C.

2015-05-15

Five different kinds of insulators including BaTiO{sub 3}, TiO{sub 2}, Al{sub 2}O{sub 3}, CdO and ZnO on the n-type InGaAs metal-insulator-semiconductor (M-I-S) ohmic contact structure are studied. The effect for the dielectric constant (ε) of inserted insulator and the conduction band offset (CBO) between an insulator and semiconductor substrate is analyzed by a unified M-I-S contact model. Based on the theoretical model and experimental data, we demonstrates that the inserted ZnO insulator with the high electron affinity and the low CBO (∼0.1 eV) to the InGaAs substrate results in ∼10 times contact resistivity reduction, even the ε of ZnO ismore » not pretty high (∼10)« less

Discovery of a Kelch-like ECH-associated protein 1-inhibitory tetrapeptide and its structural characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sogabe, Satoshi; Sakamoto, Kotaro; Kamada, Yusuke

Keap1 constitutively binds to the transcription factor Nrf2 to promote its degradation, resulting in negative modulation of genes involved in cellular protection against oxidative stress. Keap1 is increasingly recognized as an attractive target for treating diseases involving oxidative stress, including cancer, atherosclerosis, diabetes, arthritis, and neurodegeneration. We used phage-display peptide screening to identify a tetrapeptide showing moderate binding affinity, which inhibits the interaction between Nrf2 and Keap1. The tetrapeptide does not include an ETGE motif, which is a commonly found consensus sequence in known peptidic inhibitors. In addition to affinity parameters, IC{sub 50}, K{sub D}, and thermodynamic parameters, the crystalmore » structure of the complex was determined to elucidate the binding conformation. The binding interactions resemble those of known small-molecule inhibitors as opposed to those of substrates and peptidic inhibitors. Although the tetrapeptide's affinity is not very high, our results may help facilitate the designing of small-molecule inhibitors during lead generation in drug discovery. - Highlights: • Keap1 inhibitory tetrapeptide with moderate affinity was discovered. • Crystal structure of the complex showed the unique binding mode. • Structural information gives a valuable insight for design of therapeutic compounds.« less

Methods of making functionalized nanorods

DOEpatents

Gur, Ilan [San Francisco, CA; Milliron, Delia [Berkeley, CA; Alivisatos, A Paul [Oakland, CA; Liu, Haitao [Berkeley, CA

2012-01-10

A process for forming functionalized nanorods. The process includes providing a substrate, modifying the substrate by depositing a self-assembled monolayer of a bi-functional molecule on the substrate, wherein the monolayer is chosen such that one side of the bi-functional molecule binds to the substrate surface and the other side shows an independent affinity for binding to a nanocrystal surface, so as to form a modified substrate. The process further includes contacting the modified substrate with a solution containing nanocrystal colloids, forming a bound monolayer of nanocrystals on the substrate surface, depositing a polymer layer over the monolayer of nanocrystals to partially cover the monolayer of nanocrystals, so as to leave a layer of exposed nanocrystals, functionalizing the exposed nanocrystals, to form functionalized nanocrystals, and then releasing the functionalized nanocrystals from the substrate.

Proteolytic crosstalk in multi-protease networks

NASA Astrophysics Data System (ADS)

Ogle, Curtis T.; Mather, William H.

2016-04-01

Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis

PubMed Central

Goos, Sarah; Kämper, Jörg; Sauer, Norbert

2010-01-01

Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s) and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1) from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host. PMID:20161717

N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

PubMed

Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

2015-07-01

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

Proteomic validation of protease drug targets: pharmacoproteomics of matrix metalloproteinase inhibitor drugs using isotope-coded affinity tag labelling and tandem mass spectrometry.

PubMed

Butler, G S; Overall, C M

2007-01-01

We illustrate the use of quantitative proteomics, namely isotope-coded affinity tag labelling and tandem mass spectrometry, to assess the targets and effects of the blockade of matrix metalloproteinases by an inhibitor drug in a breast cancer cell culture system. Treatment of MT1-MMP-transfected MDA-MB-231 cells with AG3340 (Prinomastat) directly affected the processing a multitude of matrix metalloproteinase substrates, and indirectly altered the expression of an array of other proteins with diverse functions. Therefore, broad spectrum blockade of MMPs has wide-ranging biological consequences. In this human breast cancer cell line, secreted substrates accumulated uncleaved in the conditioned medium and plasma membrane protein substrates were retained on the cell surface, due to reduced processing and shedding of these proteins (cell surface receptors, growth factors and bioactive molecules) to the medium in the presence of the matrix metalloproteinase inhibitor. Hence, proteomic investigation of drug-perturbed cellular proteomes can identify new protease substrates and at the same time provides valuable information for target validation, drug efficacy and potential side effects prior to commitment to clinical trials.

Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors

PubMed Central

Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

2016-01-01

Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process. PMID:27438863

Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization.

PubMed

Tavladoraki, Paraskevi; Cervelli, Manuela; Antonangeli, Fabrizio; Minervini, Giovanni; Stano, Pasquale; Federico, Rodolfo; Mariottini, Paolo; Polticelli, Fabio

2011-04-01

Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure-function relationships, SMO enzyme-substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme-substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

Multifunctional Fe3O4@SiO2-Au Satellite Structured SERS Probe for Charge Selective Detection of Food Dyes.

PubMed

Sun, Zhenli; Du, Jingjing; Yan, Li; Chen, Shu; Yang, Zhilin; Jing, Chuanyong

2016-02-10

Nanofabrication of multifunctional surface-enhanced Raman scattering (SERS) substrates is strongly desirable but currently remains a challenge. The motivation of this study was to design such a substrate, a versatile core-satellite Fe3O4@SiO2-Au (FA) hetero-nanostructure, and demonstrate its use for charge-selective detection of food dye molecules as an exemplary application. Our experimental results and three-dimensional finite difference time domain (FDTD) simulation suggest that tuning the Au nanoparticle (NP) gap to sub-10 nm, which could be readily accomplished, substantially enhanced the Raman signals. Further layer-by-layer deposition of a charged polyelectrolyte on this magnetic SERS substrate induced active adsorption and selective detection of food dye molecules of opposite charge on the substrates. Molecular dynamics (MD) simulations suggest that the selective SERS enhancement could be attributed to the high affinity and close contact (within a 20 Å range) between the substrate and molecules. Density function theory (DFT) calculations confirm the charge transfer from food dye molecules to Au NPs via the polyelectrolytes. This multifunctional SERS platform provides easy separation and selective detection of charged molecules from complex chemical mixtures.

Classical Swine Fever Virus Glycoprotein Erns Is an Endoribonuclease with an Unusual Base Specificity

PubMed Central

Hausmann, Yvonne; Roman-Sosa, Gleyder; Thiel, Heinz-Jürgen; Rümenapf, Till

2004-01-01

The glycoprotein Erns of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for Erns. Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5′ of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that Erns is a highly active endoribonuclease (kcat/Km = 2 × 106 to 10 × 106 M−1 s−1) with a strong affinity to NpU containing single-stranded RNA substrates (Km = 85 to 260 nM). PMID:15113930

Novel Substrate-Based Inhibitors of Human Glutamate Carboxypeptidase II with Enhanced Lipophilicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plechanovová, Anna; Byun, Youngjoo; Alquicer, Glenda

2012-10-09

Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance ofmore » nonpolar interactions governing GCPII affinity toward novel substrates as well as formerly unnoticed plasticity of the S1' specificity pocket. On the basis of those data, we designed, synthesized, and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.« less

Recent developments with metalloprotease inhibitor class of drug candidates for Botulinum neurotoxins

DOE PAGES

Kumar, Gyanendra; Swaminathan, Subramanyam

2015-03-01

Botulinum Neurotoxins are the most poisonous of all toxins with lethal dose in nanogram quantities. They are also potential biological warfare and bioterrorism agents due to their high toxicity and ease of preparation. On the other hand BoNTs are also being increasingly used for therapeutic and cosmetic purposes, and with that the chances of accidental overdose are increasing. And despite the potential damage they could cause to human health, there are no post-intoxication drugs available so far. But progress is being made in this direction. The crystal structures in native form and bound with substrate peptides have been determined, andmore » these are enabling structure-based drug discovery possible. High throughput assays have also been designed to speed up the screening progress. Substrate-based and small molecule inhibitors have been identified. But turning high affinity inhibitors into clinically viable drug candidates has remained a challenge. We discuss here the latest developments and the future challenges in drug discovery for Botulinum neurotoxins.« less

Recent developments with metalloprotease inhibitor class of drug candidates for botulinum neurotoxins.

PubMed

Kumar, Gyanendra; Swaminathan, Subramanyam

2015-01-01

Botulinum Neurotoxins are the most poisonous of all toxins with lethal dose in nanogram quantities. They are potential biological warfare and bioterrorism agents due to their high toxicity and ease of preparation. On the other hand BoNTs are also being increasingly used for therapeutic and cosmetic purposes, and with that the chances of accidental overdose are increasing. And despite the potential damage they could cause to human health, there are no post-intoxication drugs available so far. But progress is being made in this direction. The crystal structures in native form and bound with substrate peptides have been determined, and these are enabling structure-based drug discovery possible. High throughput assays have also been designed to speed up the screening progress. Substrate-based and small molecule inhibitors have been identified. But turning high affinity inhibitors into clinically viable drug candidates has remained a challenge. We discuss here the latest developments and the future challenges in drug discovery for Botulinum neurotoxins.

In vitro P-glycoprotein activity does not completely explain in vivo efficacy of novel centrally effective oxime acetylcholinesterase reactivators.

PubMed

Dail, Mary Beth; Meek, Edward Caldwell; Chambers, Howard Wayne; Chambers, Janice Elaine

2018-05-03

Novel-substituted phenoxyalkyl pyridinium oxime acetylcholinesterase (AChE) reactivators (US patent 9,227,937) that showed convincing evidence of penetration into the brains of intact rats were developed by our laboratories. The oximes separated into three groups based on their levels of brain AChE reactivation following exposure of rats to the sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP). P-glycoprotein (P-gp) is a major blood-brain barrier (BBB) transporter and requires ATP for efflux. To determine if P-gp affinity screening could be used to reduce animal use, we measured in vitro oxime-stimulated ATPase activity to see if the in vivo reactivation efficacies related to the oximes' functions as P-gp substrates. High efficacy oximes were expected to be poor P-gp substrates, thus remaining in the brain longer. The high efficacy oximes (24-35% brain AChE reactivation) were worse P-gp substrates than the low efficacy oximes (0-7% brain AChE reactivation). However, the oxime group with medium in vivo reactivation of 10-17% were even worse P-gp substrates than the high efficacy group so their reactivation ability was not reflected by P-gp export. The results suggest that in vitro P-gp ATPase activity can remove the low efficacy oximes from in vivo testing, but is not sufficient to differentiate between the top two tiers.

Regulated release of ERdj3 from unfolded proteins by BiP

PubMed Central

Jin, Yi; Awad, Walid; Petrova, Kseniya; Hendershot, Linda M

2008-01-01

DnaJ proteins often bind to unfolded substrates and recruit their Hsp70 partners. This induces a conformational change in the Hsp70 that stabilizes its binding to substrate. By some unknown mechanism, the DnaJ protein is released. We examined the requirements for the release of ERdj3, a mammalian ER DnaJ, from substrates and found that BiP promoted the release of ERdj3 only in the presence of ATP. Mutations in ERdj3 or BiP that disrupted their interaction interrupted the release of ERdj3. BiP mutants that were defective in any step of the ATPase cycle were also unable to release ERdj3. These results demonstrate that a functional interaction between ERdj3 and BiP, including both a direct interaction and the ability to stimulate BiP's ATPase activity are required to release ERdj3 from substrate and support a model where ERdj3 must recruit BiP and stimulate its high-affinity association with the substrate through activation of ATP hydrolysis to trigger its own release from substrates. On the basis of similarities among DnaJs and Hsp70s, this is likely to be applicable to other Hsp70–DnaJ pairs. PMID:18923428

Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions.

PubMed

Silvas, Tania V; Hou, Shurong; Myint, Wazo; Nalivaika, Ellen; Somasundaran, Mohan; Kelch, Brian A; Matsuo, Hiroshi; Kurt Yilmaz, Nese; Schiffer, Celia A

2018-05-14

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A's involvement in mutation of endogenous or exogenous DNA.

The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation

PubMed Central

Senarathna, S M D K Ganga; Page-Sharp, Madhu; Crowe, Andrew

2016-01-01

The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10−6 cm/sec, followed by amodiaquine around 20 x 10−6 cm/sec; both mefloquine and artesunate were around 10 x 10−6 cm/sec. Methylene blue was between 2 and 6 x 10−6 cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine. PMID:27045516

Functional paper-based SERS substrate for rapid and sensitive detection of Sudan dyes in herbal medicine

NASA Astrophysics Data System (ADS)

Wu, Mianmian; Li, Pan; Zhu, Qingxia; Wu, Meiran; Li, Hao; Lu, Feng

2018-05-01

There has been an increasing demand for rapid and sensitive techniques for the identification of Sudan compounds that emerged as the most often illegally added fat-soluble dyes in herbal medicine. In this report, we have designed and fabricated a functionalized filter paper consisting of gold nanorods (GNRs) and mono-6-thio-cyclodextrin (HS-β-CD) as a surface-enhanced Raman spectroscopy (SERS) substrate, in which the GNR provides sufficient SERS enhancement, and the HS-β-CD with strong chemical affinity toward GNR provides the inclusion compound to capture hydrophobic molecules. Moreover, the CD-GNR were uniformly assembled on filter paper cellulose through the electrostatic adsorption and hydrogen bond, so that the CD-GNR paper-based SERS substrate (CD-GNR-paper) demonstrated higher sensitivity for the determination of Sudan III (0.1 μM) and Sudan IV (0.5 μM) than GNRs paper-based SERS substrate (GNR-paper), with high stability after the storage in the open air for 90 days. Importantly, CD-GNR-paper can effectively collect the Sudan dyes from illegally adulterated onto samples of Resina Draconis with a simple operation, further open up new exciting opportunity for SERS detection of more compounds illegally added with high sensitivity and fast signal responses.

Effects on transport of rapidly penetrating, competing substrates: activation and inhibition of the choline carrier in erythrocytes by imidazole.

PubMed

Devés, R; Krupka, R M

1987-01-01

The properties of the choline transport system are fundamentally altered in saline solution containing 5 mM imidazole buffer instead of 5 mM phosphate: (i) The system no longer exhibits accelerated exchange. (ii) Choline in the external compartment fails to increase the rate of inactivation of the carrier by N-ethylmaleimide. (iii) Depending on the relative concentrations of choline and imidazole, transport may be activated or inhibited. The maximum rates are increased more than fivefold by imidazole, but at moderate substrate concentrations activation is observed with low concentrations of imidazole and inhibition with high concentrations. (iv) The imidazole effect is asymmetric, there being a greater tendency to activate exit than entry. All this behavior is predicted by the carrier model if imidazole is a substrate of the choline carrier having a high maximum transport rate but a relatively low affinity, and if imidazole rapidly enters the cell by simple diffusion, so that it can add to carrier sites on both sides of the membrane. Addition at the cis side inhibits, and at the trans side activates. According to the carrier model, asymmetry is a necessary consequence of the potassium ion gradient in red cells, potassium ion being another substrate of the choline system.

Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes.

PubMed

Book, Adam J; Gladman, Nicholas P; Lee, Sang-Sook; Scalf, Mark; Smith, Lloyd M; Vierstra, Richard D

2010-08-13

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes*

PubMed Central

Book, Adam J.; Gladman, Nicholas P.; Lee, Sang-Sook; Scalf, Mark; Smith, Lloyd M.; Vierstra, Richard D.

2010-01-01

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis. PMID:20516081

Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA

PubMed Central

Shell, Steven M.; Hawkins, Edward K.; Tsai, Miaw-Sheue; Hlaing, Aye Su; Rizzo, Carmelo J.; Chazin, Walter J.

2013-01-01

The xeroderma pigmentosum complementation group C protein (XPC) serves as the primary initiating factor in the global genome nucleotide excision repair pathway (GG-NER). Recent reports suggest XPC also stimulates repair of oxidative lesions by base excision repair. However, whether XPC distinguishes among various types of DNA lesions remains unclear. Although the DNA binding properties of XPC have been studied by several groups, there is a lack of consensus over whether XPC discriminates between DNA damaged by lesions associated with NER activity versus those that are not. In this study we report a high-throughput fluorescence anisotropy assay used to measure the DNA binding affinity of XPC for a panel of DNA substrates containing a range of chemical lesions in a common sequence. Our results demonstrate that while XPC displays a preference for binding damaged DNA, the identity of the lesion has little effect on the binding affinity of XPC. Moreover, XPC was equally capable of binding to DNA substrates containing lesions not repaired by GG-NER. Our results support an indirect read-out model for sensing the presence of lesions by human XPC and suggest XPC may act as a general sensor of damaged DNA capable of recognizing DNA containing lesions not repaired by NER. PMID:24051049

Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities.

PubMed

Parisi, Mónica G; Moreno, Silvia; Fernández, Graciela

2008-04-01

A dual function protein was isolated from Allium sativum bulbs and was characterized. The protein had a molecular mass of 25-26 kDa under non-reducing conditions, whereas two polypeptide chains of 12.5+/-0.5 kDa were observed under reducing conditions. E-64 and leupeptin inhibited the proteolytic activity of the protein, which exhibited characteristics similar to cysteine peptidase. The enzyme exhibited substrate specificity and hydrolyzed natural substrates such as alpha-casein (K(m): 23.0 microM), azocasein, haemoglobin and gelatin. It also showed a high affinity for synthetic peptides such as Cbz-Ala-Arg-Arg-OMe-beta-Nam (K(m): 55.24 microM, k(cat): 0.92 s(-1)). The cysteine peptidase activity showed a remarkable stability after incubation at moderate temperatures (40-50 degrees C) over a pH range of 5.5-6.5. The N-terminus of the protein displayed a 100% sequence similarity to the sequences of a mannose-binding lectin isolated from garlic bulbs. Moreover, the purified protein was retained in the chromatographic column when Con-A Sepharose affinity chromatography was performed and the protein was able to agglutinate trypsin-treated rabbit red cells. Therefore, our results indicate the presence of an additional cysteine peptidase activity on a lectin previously described.

Biochemical Regulatory Features of Activation-Induced Cytidine Deaminase Remain Conserved from Lampreys to Humans

PubMed Central

King, Justin J.; Amemiya, Chris T.; Hsu, Ellen

2017-01-01

ABSTRACT Activation-induced cytidine deaminase (AID) is a genome-mutating enzyme that initiates class switch recombination and somatic hypermutation of antibodies in jawed vertebrates. We previously described the biochemical properties of human AID and found that it is an unusual enzyme in that it exhibits binding affinities for its substrate DNA and catalytic rates several orders of magnitude higher and lower, respectively, than a typical enzyme. Recently, we solved the functional structure of AID and demonstrated that these properties are due to nonspecific DNA binding on its surface, along with a catalytic pocket that predominantly assumes a closed conformation. Here we investigated the biochemical properties of AID from a sea lamprey, nurse shark, tetraodon, and coelacanth: representative species chosen because their lineages diverged at the earliest critical junctures in evolution of adaptive immunity. We found that these earliest-diverged AID orthologs are active cytidine deaminases that exhibit unique substrate specificities and thermosensitivities. Significant amino acid sequence divergence among these AID orthologs is predicted to manifest as notable structural differences. However, despite major differences in sequence specificities, thermosensitivities, and structural features, all orthologs share the unusually high DNA binding affinities and low catalytic rates. This absolute conservation is evidence for biological significance of these unique biochemical properties. PMID:28716949

Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

PubMed

Chufan, Eduardo E; Kapoor, Khyati; Ambudkar, Suresh V

2016-02-01

P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp. Published by Elsevier Inc.

Mapping the UDP-Glucuronic Acid Binding Site in UDP-Glucuronosyltransferase-1 A10 by Homology-based Modeling: Confirmation with Biochemical Evidence†

PubMed Central

Banerjee, Rajat; Pennington, Matthew W.; Garza, Amanda; Owens, Ida S.

2008-01-01

The UDP-glucuronosyltransferase (UGT) isozyme system is critical for protecting the body against endogenous and exogenous chemicals by linking glucuronic acid donated by UDP-glucuronic acid to a lipophilic acceptor substrate. UGTs convert metabolites, dietary constituents and environmental toxicants to highly excretable glucuronides. Because of difficulties associated with purifying endoplasmic reticulum-bound UGTs for structural studies, we carried out homology-based computer modeling to aid analysis. The search found structural homology in Escherichia coli UDP-galactose 4-epimerase. Consistent with predicted similarities involving the common UDP-moiety in substrates, UDP-glucose and UDP-hexanol amine caused competitive inhibition by Lineweaver-Burk plots. Among predicted binding sites N292, K314, K315 and K404 in UGT1A10, two informative sets of mutants K314R/Q/A/E /G and K404R/E had null activities or 2.7-fold higher/50% less activity, respectively. Scatchard analysis of binding data of affinity-ligand, 5-azido-uridine-[β-32P]-diphosphoglucuronic acid, to purified UGT1A10-His or UGT1A7-His revealed high and low affinity binding sites. 2-Nitro 5-thiocyanobenzoic acid-digested UGT1A10-His bound with radiolabeled affinity-ligand revealed an 11.3- and 14.3-kDa peptide associated with K314 and K404, respectively, in a discontinuous SDS-PAGE system. Similar treatment of 1A10His-K314A bound with the ligand lacked both peptides; 1A10-HisK404R- and 1A10-HisK404E showed 1.3-fold greater- and 50% less-label in the 14.3-kDa peptide, respectively, compared to 1A10-His without affecting the 11.3-kDa peptide. Scatchard analysis of binding data of affinity-ligand to 1A10His-K404R and -K404E showed a 6-fold reduction and a large increase in Kd, respectively. Our results indicate: K314 and K404 are required UDP-glcA binding sites in 1A10, that K404 controls activity and high affinity sites and that K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321--equivalent to K314-- in UGT2B15 and 2B17 and I321 in the inactive UGT8, which suggests UGT2B15 and 2B17 contain suboptimal activity. Hence our data strongly support UDPglcA binding to K314 and K404 in UGT1A10. PMID:18570380

Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors.

PubMed

Adamczewski, M; Paolini, R; Kinet, J P

1992-09-05

The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.

Differential 14-3-3 affinity capture reveals new downstream targets of phosphatidylinositol 3-kinase signaling.

PubMed

Dubois, Fanny; Vandermoere, Franck; Gernez, Aurélie; Murphy, Jane; Toth, Rachel; Chen, Shuai; Geraghty, Kathryn M; Morrice, Nick A; MacKintosh, Carol

2009-11-01

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d(0)/d(4)) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d(0)/d(4) values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d(0)/d(4) scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser(19) of ZNRF2 (RTRAYpS(19)GS), phospho-Ser(90) of SASH1 (RKRRVpS(90)QD), and phospho- Ser(493) of lipolysis-stimulated lipoprotein receptor (RPRARpS(493)LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.

Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling*

PubMed Central

Dubois, Fanny; Vandermoere, Franck; Gernez, Aurélie; Murphy, Jane; Toth, Rachel; Chen, Shuai; Geraghty, Kathryn M.; Morrice, Nick A.; MacKintosh, Carol

2009-01-01

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d0/d4) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d0/d4 values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d0/d4 scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser19 of ZNRF2 (RTRAYpS19GS), phospho-Ser90 of SASH1 (RKRRVpS90QD), and phospho- Ser493 of lipolysis-stimulated lipoprotein receptor (RPRARpS493LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways. PMID:19648646

Influence of substrate mineralogy on bacterial mineralization of calcium carbonate: implications for stone conservation.

PubMed

Rodriguez-Navarro, Carlos; Jroundi, Fadwa; Schiro, Mara; Ruiz-Agudo, Encarnación; González-Muñoz, María Teresa

2012-06-01

The influence of mineral substrate composition and structure on bacterial calcium carbonate productivity and polymorph selection was studied. Bacterial calcium carbonate precipitation occurred on calcitic (Iceland spar single crystals, marble, and porous limestone) and silicate (glass coverslips, porous sintered glass, and quartz sandstone) substrates following culturing in liquid medium (M-3P) inoculated with different types of bacteria (Myxococcus xanthus, Brevundimonas diminuta, and a carbonatogenic bacterial community isolated from porous calcarenite stone in a historical building) and direct application of sterile M-3P medium to limestone and sandstone with their own bacterial communities. Field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), powder X-ray diffraction (XRD), and 2-dimensional XRD (2D-XRD) analyses revealed that abundant highly oriented calcite crystals formed homoepitaxially on the calcitic substrates, irrespective of the bacterial type. Conversely, scattered spheroidal vaterite entombing bacterial cells formed on the silicate substrates. These results show that carbonate phase selection is not strain specific and that under equal culture conditions, the substrate type is the overruling factor for calcium carbonate polymorph selection. Furthermore, carbonate productivity is strongly dependent on the mineralogy of the substrate. Calcitic substrates offer a higher affinity for bacterial attachment than silicate substrates, thereby fostering bacterial growth and metabolic activity, resulting in higher production of calcium carbonate cement. Bacterial calcite grows coherently over the calcitic substrate and is therefore more chemically and mechanically stable than metastable vaterite, which formed incoherently on the silicate substrates. The implications of these results for technological applications of bacterial carbonatogenesis, including building stone conservation, are discussed.

Influence of Substrate Mineralogy on Bacterial Mineralization of Calcium Carbonate: Implications for Stone Conservation

PubMed Central

Jroundi, Fadwa; Schiro, Mara; Ruiz-Agudo, Encarnación; González-Muñoz, María Teresa

2012-01-01

The influence of mineral substrate composition and structure on bacterial calcium carbonate productivity and polymorph selection was studied. Bacterial calcium carbonate precipitation occurred on calcitic (Iceland spar single crystals, marble, and porous limestone) and silicate (glass coverslips, porous sintered glass, and quartz sandstone) substrates following culturing in liquid medium (M-3P) inoculated with different types of bacteria (Myxococcus xanthus, Brevundimonas diminuta, and a carbonatogenic bacterial community isolated from porous calcarenite stone in a historical building) and direct application of sterile M-3P medium to limestone and sandstone with their own bacterial communities. Field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), powder X-ray diffraction (XRD), and 2-dimensional XRD (2D-XRD) analyses revealed that abundant highly oriented calcite crystals formed homoepitaxially on the calcitic substrates, irrespective of the bacterial type. Conversely, scattered spheroidal vaterite entombing bacterial cells formed on the silicate substrates. These results show that carbonate phase selection is not strain specific and that under equal culture conditions, the substrate type is the overruling factor for calcium carbonate polymorph selection. Furthermore, carbonate productivity is strongly dependent on the mineralogy of the substrate. Calcitic substrates offer a higher affinity for bacterial attachment than silicate substrates, thereby fostering bacterial growth and metabolic activity, resulting in higher production of calcium carbonate cement. Bacterial calcite grows coherently over the calcitic substrate and is therefore more chemically and mechanically stable than metastable vaterite, which formed incoherently on the silicate substrates. The implications of these results for technological applications of bacterial carbonatogenesis, including building stone conservation, are discussed. PMID:22447589

Identification of cellular MMP substrates using quantitative proteomics: isotope-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantification (iTRAQ).

PubMed

Butler, Georgina S; Dean, Richard A; Morrison, Charlotte J; Overall, Christopher M

2010-01-01

Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cellular context. Here we describe the use of two protein labeling techniques, Isotope-Coded Affinity Tags (ICAT and Isobaric Tags for Relative and Absolute Quantification (iTRAQ), which we have used successfully to identify novel matrix metalloproteinase (MMP) substrates in cell culture systems (1-4). ICAT and iTRAQ can label proteins and protease cleavage products of secreted proteins, protein domains shed from the cell membrane or pericellular matrix of protease-transfected cells that have accumulated in conditioned medium, or cell surface proteins in membrane preparations; isotopically distinct labels are used for control cells. Tryptic digestion and tandem mass spectrometry of the generated fragments enable sequencing of differentially labeled but otherwise identical pooled peptides. The isotopic tag, which is unique for each label, identifies the peptides originating from each sample, for instance, protease-transfected or control cells, and comparison of the peak areas enables relative quantification of the peptide in each sample. Thus proteins present in altered amounts between protease-expressing and null cells are implicated as protease substrates and can be further validated as such.

Comparison of 6-mercaptopurine with 6-thioguanine for the analysis of thiopurine S-methyltransferase activity in human erythrocyte by LC-MS/MS.

PubMed

Mei, Shenghui; Li, Xindi; Gong, Xiaoqing; Zhang, Xiaoyi; Li, Xingang; Yang, Li; Zhu, Leting; Zhou, Heng; Liu, Yonghong; Zhou, Anna; Zhang, Xinghu; Zhao, Zhigang

2017-09-01

Thiopurines (TPDs) are first-line drugs in treating neuromyelitis optica spectrum disorders (NMOSD). Evaluation of thiopurine S-methyltransferase activity (TPMT), a major determinant of TPD toxicity, before TPD treatment using 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) as substrate was suggested. However, the equivalent of the two substrates in TPMT activity evaluation was unknown, and an alternative substrate was required in TPMT activity evaluation in patients who were already taking 6-MP or 6-TG. Before evaluating the agreement of 6-MP and 6-TG in TPMT activity measurement in patients with NMOSD, the affinity of the two substrates for the active center of TPMT should be established. A computer-based simulation indicated that 6-MP and 6-TG had similar affinities for the two active sites of TPMT. According to the guidelines, an LC-MS/MS method was developed and validated to evaluate the TPMT activity in human erythrocyte hemolysate using 6-MP or 6-TG as substrates via 1 h incubation at 37°C. The method was applied in 81 patients with NMOSD. Evaluated by Bland-Altman plot, 6-methylmercaptopurine and 6-methylthioguanine represented TPMT activities were in agreement with each other. Further studies are warranted to confirm the results. Copyright © 2017 John Wiley & Sons, Ltd.

Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU.

PubMed

Hoff, Kevin G; Ta, Dennis T; Tapley, Tim L; Silberg, Jonathan J; Vickery, Larry E

2002-07-26

Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20.

Ubiquitinated Proteome: Ready for Global?*

PubMed Central

Shi, Yi; Xu, Ping; Qin, Jun

2011-01-01

Ubiquitin (Ub) is a small and highly conserved protein that can covalently modify protein substrates. Ubiquitination is one of the major post-translational modifications that regulate a broad spectrum of cellular functions. The advancement of mass spectrometers as well as the development of new affinity purification tools has greatly expedited proteome-wide analysis of several post-translational modifications (e.g. phosphorylation, glycosylation, and acetylation). In contrast, large-scale profiling of lysine ubiquitination remains a challenge. Most recently, new Ub affinity reagents such as Ub remnant antibody and tandem Ub binding domains have been developed, allowing for relatively large-scale detection of several hundreds of lysine ubiquitination events in human cells. Here we review different strategies for the identification of ubiquitination site and discuss several issues associated with data analysis. We suggest that careful interpretation and orthogonal confirmation of MS spectra is necessary to minimize false positive assignments by automatic searching algorithms. PMID:21339389

Nonpeptide-Based Small-Molecule Probe for Fluorogenic and Chromogenic Detection of Chymotrypsin.

PubMed

Wu, Lei; Yang, Shu-Hou; Xiong, Hao; Yang, Jia-Qian; Guo, Jun; Yang, Wen-Chao; Yang, Guang-Fu

2017-03-21

We report herein a nonpeptide-based small-molecule probe for fluorogenic and chromogenic detection of chymotrypsin, as well as the primary application for this probe. This probe was rationally designed by mimicking the peptide substrate and optimized by adjusting the recognition group. The refined probe 2 exhibits good specificity toward chymotrypsin, producing about 25-fold higher enhancement in both the fluorescence intensity and absorbance upon the catalysis by chymotrypsin. Compared with the most widely used peptide substrate (AMC-FPAA-Suc) of chymotrypsin, probe 2 shows about 5-fold higher binding affinity and comparable catalytical efficiency against chymotrypsin. Furthermore, it was successfully applied for the inhibitor characterization. To the best of our knowledge, probe 2 is the first nonpeptide-based small-molecule probe for chymotrypsin, with the advantages of simple structure and high sensitivity compared to the widely used peptide-based substrates. This small-molecule probe is expected to be a useful molecular tool for drug discovery and chymotrypsin-related disease diagnosis.

Redirection of pyruvate flux toward desired metabolic pathways through substrate channeling between pyruvate kinase and pyruvate-converting enzymes in Saccharomyces cerevisiae.

PubMed

Kim, Sujin; Bae, Sang-Jeong; Hahn, Ji-Sook

2016-04-07

Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging.

Drug transport by reconstituted P-glycoprotein in proteoliposomes. Effect of substrates and modulators, and dependence on bilayer phase state.

PubMed

Lu, P; Liu, R; Sharom, F J

2001-03-01

The P-glycoprotein multidrug transporter (Pgp) is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Pgp is envisaged as a 'hydrophobic vacuum cleaner', and drugs are believed to gain access to the substrate binding sites from within the membrane, rather than from the aqueous phase. The intimate association of both Pgp and its substrates with the membrane suggests that its function may be regulated by the biophysical properties of the lipid bilayer. Using the high affinity fluorescent substrate tetramethylrosamine (TMR), we have monitored, in real time, transport in proteoliposomes containing reconstituted Pgp. The TMR concentration gradient generated by Pgp was collapsed by the addition of either the ATPase inhibitor, vanadate, or Pgp modulators. TMR transport by Pgp obeyed Michaelis--Menten kinetics with respect to both of its substrates. The Km for ATP was 0.48 mM, close to the K(m) for ATP hydrolysis, and the K(m) for TMR was 0.3 microM. TMR transport was inhibited in a concentration-dependent fashion by verapamil and cyclosporin A, and activated (probably by a positive allosteric effect) by the transport substrate colchicine. TMR transport by Pgp reconstituted into proteoliposomes composed of two synthetic phosphatidylcholines showed a highly unusual biphasic temperature dependence. The rate of TMR transport was relatively high in the rigid gel phase, reached a maximum at the melting temperature of the bilayer, and then decreased in the fluid liquid crystalline phase. This pattern of temperature dependence suggests that the rate of drug transport by Pgp may be dominated by partitioning of drug into the bilayer.

Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

PubMed Central

Ali, Shahin S.; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M.

2013-01-01

Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing. PMID:23382943

Influence of active site location on catalytic activity in de novo-designed zinc metalloenzymes.

PubMed

Zastrow, Melissa L; Pecoraro, Vincent L

2013-04-17

While metalloprotein design has now yielded a number of successful metal-bound and even catalytically active constructs, the question of where to put a metal site along a linear, repetitive sequence has not been thoroughly addressed. Often several possibilities in a given sequence may exist that would appear equivalent but may in fact differ for metal affinity, substrate access, or protein dynamics. We present a systematic variation of active site location for a hydrolytically active ZnHis3O site contained within a de novo-designed three-stranded coiled coil. We find that the maximal rate, substrate access, and metal-binding affinity are dependent on the selected position, while catalytic efficiency for p-nitrophenyl acetate hydrolysis can be retained regardless of the location of the active site. This achievement demonstrates how efficient, tailor-made enzymes which control rate, pKa, substrate and solvent access (and selectivity), and metal-binding affinity may be realized. These findings may be applied to the more advanced de novo design of constructs containing secondary interactions, such as hydrogen-bonding channels. We are now confident that changes to location for accommodating such channels can be achieved without location-dependent loss of catalytic efficiency. These findings bring us closer to our ultimate goal of incorporating the secondary interactions we believe will be necessary in order to improve both active site properties and the catalytic efficiency to be competitive with the native enzyme, carbonic anhydrase.

Structure and mechanisms of Escherichia coli aspartate transcarbamoylase.

PubMed

Lipscomb, William N; Kantrowitz, Evan R

2012-03-20

Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein. Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, L-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity. In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60 Å from the active site, inducing structural alterations that modulate catalytic activity. The delineation of the structure and function in this particular model system will help in understanding the molecular basis of cooperativity and allosteric regulation in other systems as well.

Kinetic characteristics of native γ-glutamylcysteine ligase in the aging housefly, Musca domestica L.☆

PubMed Central

Toroser, Dikran; Sohal, Rajindar S.

2010-01-01

The catalytic activity of γ-glutamylcysteine ligase (γ-GCL; EC 6.3.2.2) was compared between relatively young (4-day-old) and old (19-day-old) houseflies (Musca domestica) in order to understand the mechanism of putative deterioration of glutathione homeostasis during the aging process. Hanes–Woolf analyses ([S]/v vs [S]) indicated that γ-GCL had significantly higher affinities for its substrates in the young than in the old flies. The Km values in the young and old flies were, respectively, for glutamate 0.6 and 5.5 mM; for cysteine 0.3 and 4.6 mM; and for ATP 1.2 and 2.9 mM. Furthermore, young but not old flies exhibited substrate-dependent inhibition of γ-GCL activity at >5 mM cysteine indicating a loss of metabolic regulation during aging. The age-associated differences in the affinity of native γ-GCL towards its substrates suggest that de novo synthesis of glutathione would be relatively less efficient in the old houseflies. PMID:15596139

Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

PubMed

Holstein, Carly A; Chevalier, Aaron; Bennett, Steven; Anderson, Caitlin E; Keniston, Karen; Olsen, Cathryn; Li, Bing; Bales, Brian; Moore, David R; Fu, Elain; Baker, David; Yager, Paul

2016-02-01

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side.

PubMed

Lin, Kuan-Hung; Ali, Akbar; Rusere, Linah; Soumana, Djade I; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-15

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 μM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, with the aim of achieving better specificity and lower hydrophilicity than those of current scaffolds in the design of antiflaviviral inhibitors. Copyright © 2017 American Society for Microbiology.

Method of producing catalytic material for fabricating nanostructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seals, Roland D.; Menchhofer, Paul A.; Howe, Jane Y.

Methods of fabricating nano-catalysts are described. In some embodiments the nano-catalyst is formed from a powder-based substrate material and is some embodiments the nano-catalyst is formed from a solid-based substrate material. In some embodiments the substrate material may include metal, ceramic, or silicon or another metalloid. The nano-catalysts typically have metal nanoparticles disposed adjacent the surface of the substrate material. The methods typically include functionalizing the surface of the substrate material with a chelating agent, such as a chemical having dissociated carboxyl functional groups (--COO), that provides an enhanced affinity for metal ions. The functionalized substrate surface may then bemore » exposed to a chemical solution that contains metal ions. The metal ions are then bound to the substrate material and may then be reduced, such as by a stream of gas that includes hydrogen, to form metal nanoparticles adjacent the surface of the substrate.« less

Method of producing catalytic materials for fabricating nanostructures

DOEpatents

Seals, Roland D; Menchhofer, Paul A; Howe, Jane Y; Wang, Wei

2013-02-19

Methods of fabricating nano-catalysts are described. In some embodiments the nano-catalyst is formed from a powder-based substrate material and is some embodiments the nano-catalyst is formed from a solid-based substrate material. In some embodiments the substrate material may include metal, ceramic, or silicon or another metalloid. The nano-catalysts typically have metal nanoparticles disposed adjacent the surface of the substrate material. The methods typically include functionalizing the surface of the substrate material with a chelating agent, such as a chemical having dissociated carboxyl functional groups (--COO), that provides an enhanced affinity for metal ions. The functionalized substrate surface may then be exposed to a chemical solution that contains metal ions. The metal ions are then bound to the substrate material and may then be reduced, such as by a stream of gas that includes hydrogen, to form metal nanoparticles adjacent the surface of the substrate.

Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

DOEpatents

Maupin-Furlow, Julie A [Gainesville, FL; Talarico, Lee Ann [Gainesville, FL; Raj, Krishnan Chandra [Tamil Nadu, IN; Ingram, Lonnie O [Gainesville, FL

2008-02-05

The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells.

Morphology manipulation of M13 bacteriophage template for nanostructure assembly

NASA Astrophysics Data System (ADS)

Ngo-Duc, Tam-Triet; Zaman, Mohammed S.; Moon, Chung-Hee; Haberer, Elaine D.

2014-08-01

A gold-binding M13 bacteriophage was used as a model system to explore templating of inorganic material on geometrically transformed viruses . Gold-binding filamentous phage were converted to spheroid form with a short chloroform treatment, and the resulting morphology was investigated with electron microscopy. Binding studies revealed that spheroid-shaped gold-binding phage preserved its affinity for gold. Spheroids adhered to a planar substrate assembled clusters or rings of gold nanoparticles. This gold-binding phage served as a demonstration of a highly shape-modifiable viral-template for inorganic materials.

Phage display of engineered binding proteins.

PubMed

Levisson, Mark; Spruijt, Ruud B; Winkel, Ingrid Nolla; Kengen, Servé W M; van der Oost, John

2014-01-01

In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity.

PubMed

Ren, Huimiao; Bandyopadhyay, Sanjay; Allison, William S

2006-05-16

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.

An additional role for the Brønsted acid-base catalysts of mandelate racemase in transition state stabilization.

PubMed

Nagar, Mitesh; Bearne, Stephen L

2015-11-10

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate and serves as a paradigm for understanding the enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high pKa. The enzyme utilizes a two-base mechanism with Lys 166 and His 297 acting as Brønsted acid and base catalysts, respectively, in the R → S reaction direction. In the S → R reaction direction, their roles are reversed. Using isothermal titration calorimetry (ITC), MR is shown to bind the intermediate/transition state (TS) analogue inhibitor benzohydroxamate (BzH) in an entropy-driven process with a value of ΔCp equal to -358 ± 3 cal mol(-1) K(-1), consistent with an increased number of hydrophobic interactions. However, MR binds BzH with an affinity that is ∼2 orders of magnitude greater than that predicted solely on the basis of hydrophobic interactions [St. Maurice, M., and Bearne, S. L. (2004) Biochemistry 43, 2524], suggesting that additional specific interactions contribute to binding. To test the hypothesis that cation-π/NH-π interactions between the side chains of Lys 166 and His 297 and the aromatic ring and/or the hydroxamate/hydroximate moiety of BzH contribute to the binding of BzH, site-directed mutagenesis was used to generate the MR variants K166M, K166C, H297N, and K166M/H297N and their binding affinity for various ligands determined using ITC. Comparison of the binding affinities of these MR variants with the intermediate/TS analogues BzH and cyclohexanecarbohydroxamate revealed that cation-π/NH-π interactions between His 297 and the hydroxamate/hydroximate moiety and the phenyl ring of BzH contribute approximately 0.26 and 0.91 kcal/mol to binding, respectively, while interactions with Lys 166 contribute approximately 1.74 and 1.74 kcal/mol, respectively. Similarly, comparison of the binding affinities of these mutants with substrate analogues revealed that Lys 166 contributes >2.93 kcal/mol to the binding of (R)-atrolactate, and His 297 contributes 2.46 kcal/mol to the binding of (S)-atrolactate. These results are consistent with Lys 166 and His 297 playing dual roles in catalysis: they act as Brønsted acid-base catalysts, and they stabilize both the enolate moiety and phenyl ring of the altered substrate in the TS.

Use of an uncertainty analysis for genome-scale models as a prediction tool for microbial growth processes in subsurface environments.

PubMed

Klier, Christine

2012-03-06

The integration of genome-scale, constraint-based models of microbial cell function into simulations of contaminant transport and fate in complex groundwater systems is a promising approach to help characterize the metabolic activities of microorganisms in natural environments. In constraint-based modeling, the specific uptake flux rates of external metabolites are usually determined by Michaelis-Menten kinetic theory. However, extensive data sets based on experimentally measured values are not always available. In this study, a genome-scale model of Pseudomonas putida was used to study the key issue of uncertainty arising from the parametrization of the influx of two growth-limiting substrates: oxygen and toluene. The results showed that simulated growth rates are highly sensitive to substrate affinity constants and that uncertainties in specific substrate uptake rates have a significant influence on the variability of simulated microbial growth. Michaelis-Menten kinetic theory does not, therefore, seem to be appropriate for descriptions of substrate uptake processes in the genome-scale model of P. putida. Microbial growth rates of P. putida in subsurface environments can only be accurately predicted if the processes of complex substrate transport and microbial uptake regulation are sufficiently understood in natural environments and if data-driven uptake flux constraints can be applied.

Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate.

PubMed

Maloney, Finn P; Gerwick, Lena; Gerwick, William H; Sherman, David H; Smith, Janet L

2016-09-13

Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (Kd = 0.5 μM) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre- and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA.

Apolar Distal Pocket Mutants of Yeast Cytochrome c Peroxidase: Hydrogen Peroxide Reactivity and Cyanide Binding of the TriAla, TriVal, and TriLeu Variants

PubMed Central

Bidwai, Anil K.; Meyen, Cassandra; Kilheeney, Heather; Wroblewski, Damian; Vitello, Lidia B.; Erman, James E.

2012-01-01

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H2O2 compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H2O2 and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H2O2 and HCN while features that inhibit substrate ionization slow the reactions. PMID:23022490

The oligomeric architecture of the archaeal exosome is important for processive and efficient RNA degradation.

PubMed

Audin, Maxime J C; Wurm, Jan Philip; Cvetkovic, Milos A; Sprangers, Remco

2016-04-07

The exosome plays an important role in RNA degradation and processing. In archaea, three Rrp41:Rrp42 heterodimers assemble into a barrel like structure that contains a narrow RNA entrance pore and a lumen that contains three active sites. Here, we demonstrate that this quaternary structure of the exosome is important for efficient RNA degradation. We find that the entrance pore of the barrel is required for nM substrate affinity. This strong interaction is crucial for processive substrate degradation and prevents premature release of the RNA from the enzyme. Using methyl TROSY NMR techniques, we establish that the 3' end of the substrate remains highly flexible inside the lumen. As a result, the RNA jumps between the three active sites that all equally participate in substrate degradation. The RNA jumping rate is, however, much faster than the cleavage rate, indicating that not all active site:substrate encounters result in catalysis. Enzymatic turnover therefore benefits from the confinement of the active sites and substrate in the lumen, which ensures that the RNA is at all times bound to one of the active sites. The evolution of the exosome into a hexameric complex and the optimization of its catalytic efficiency were thus likely co-occurring events. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

MacA, a periplasmic membrane fusion protein of the macrolide transporter MacAB-TolC, binds lipopolysaccharide core specifically and with high affinity.

PubMed

Lu, Shuo; Zgurskaya, Helen I

2013-11-01

The Escherichia coli MacAB-TolC transporter has been implicated in efflux of macrolide antibiotics and secretion of enterotoxin STII. In this study, we found that purified MacA, a periplasmic membrane fusion protein, contains one tightly bound rough core lipopolysaccharide (R-LPS) molecule per MacA molecule. R-LPS was bound specifically to MacA protein with affinity exceeding that of polymyxin B. Sequence analyses showed that MacA contains two high-density clusters of positively charged amino acid residues located in the cytoplasmic N-terminal domain and the periplasmic C-terminal domain. Substitutions in the C-terminal cluster reducing the positive-charge density completely abolished binding of R-LPS. At the same time, these substitutions significantly reduced the functionality of MacA in the protection of E. coli against macrolides in vivo and in the in vitro MacB ATPase stimulation assays. Taken together, our results suggest that R-LPS or a similar glycolipid is a physiological substrate of MacAB-TolC.

MacA, a Periplasmic Membrane Fusion Protein of the Macrolide Transporter MacAB-TolC, Binds Lipopolysaccharide Core Specifically and with High Affinity

PubMed Central

Lu, Shuo

2013-01-01

The Escherichia coli MacAB-TolC transporter has been implicated in efflux of macrolide antibiotics and secretion of enterotoxin STII. In this study, we found that purified MacA, a periplasmic membrane fusion protein, contains one tightly bound rough core lipopolysaccharide (R-LPS) molecule per MacA molecule. R-LPS was bound specifically to MacA protein with affinity exceeding that of polymyxin B. Sequence analyses showed that MacA contains two high-density clusters of positively charged amino acid residues located in the cytoplasmic N-terminal domain and the periplasmic C-terminal domain. Substitutions in the C-terminal cluster reducing the positive-charge density completely abolished binding of R-LPS. At the same time, these substitutions significantly reduced the functionality of MacA in the protection of E. coli against macrolides in vivo and in the in vitro MacB ATPase stimulation assays. Taken together, our results suggest that R-LPS or a similar glycolipid is a physiological substrate of MacAB-TolC. PMID:23974027

Characteristics of a ugp-encoded and phoB-dependent glycerophosphoryl diester phosphodiesterase which is physically dependent on the ugp transport system of Escherichia coli.

PubMed

Brzoska, P; Boos, W

1988-09-01

The ugp-encoded transport system of Escherichia coli accumulates sn-glycerol-3-phosphate with high affinity; it is binding protein mediated and part of the pho regulon. Here, we report that glycerophosphoryl diesters (deacylated phospholipids) are also high-affinity substrates for the ugp-encoded system. The diesters are not taken up in an unaltered form but are hydrolyzed during transport to sn-glycerol-3-phosphate plus the corresponding alcohols. The enzyme responsible for this reaction is not essential for the translocation of sn-glycerol-3-phosphate or for the glycerophosphoryl diesters but can only hydrolyze diesters that are in the process of being transported. Diesters in the periplasm or in the cytoplasm were not recognized, and no enzymatic activity could be detected in cellular extracts. The enzyme is encoded by the last gene in the ugp operon, termed ugpQ. The product of the ugpQ gene, expressed in minicells, has an apparent molecular weight of 17,500. We present evidence that only one major phoB-dependent promoter controls all ugp genes.

Substrate-Independent Robust and Heparin-Mimetic Hydrogel Thin Film Coating via Combined LbL Self-Assembly and Mussel-Inspired Post-Cross-linking.

PubMed

Ma, Lang; Cheng, Chong; He, Chao; Nie, Chuanxiong; Deng, Jie; Sun, Shudong; Zhao, Changsheng

2015-12-02

In this work, we designed a robust and heparin-mimetic hydrogel thin film coating via combined layer-by-layer (LbL) self-assembly and mussel-inspired post-cross-linking. Dopamine-grafted heparin-like/-mimetic polymers (DA-g-HepLP) with abundant carboxylic and sulfonic groups were synthesized by the conjugation of adhesive molecule, DA, which exhibited substrate-independent adhesive affinity to various solid surfaces because of the formation of irreversible covalent bonds. The hydrogel thin film coated substrates were prepared by a three-step reaction: First, the substrates were coated with DA-g-HepLP to generate negatively charged surfaces. Then, multilayers were obtained via LbL coating of chitosan and the DA-g-HepLP. Finally, the noncovalent multilayers were oxidatively cross-linked by NaIO4. Surface ATR-FTIR and XPS spectra confirmed the successful fabrication of the hydrogel thin film coatings onto membrane substrates; SEM images revealed that the substrate-independent coatings owned 3D porous morphology. The soaking tests in highly alkaline, acid, and concentrated salt solutions indicated that the cross-linked hydrogel thin film coatings owned high chemical resistance. In comparison, the soaking tests in physiological solution indicated that the cross-linked hydrogel coatings owned excellent long-term stability. The live/dead cell staining and morphology observations of the adhered cells revealed that the heparin-mimetic hydrogel thin film coated substrates had low cell toxicity and high promotion ability for cell proliferation. Furthermore, systematic in vitro investigations of protein adsorption, platelet adhesion, blood clotting, and blood-related complement activation confirmed that the hydrogel film coated substrates showed excellent hemocompatibility. Both the results of inhibition zone and bactericidal activity indicated that the gentamycin sulfate loaded hydrogel thin films had significant inhibition capability toward both Escherichia coli and Staphylococcus aureus bacteria. Combined the above advantages, it is believed that the designed heparin-mimetic hydrogel thin films may show high potential for applications in various biological and clinical fields, such as long-term hemocompatible and drug-loading materials for implants.

Highly Efficient Photothermal Semiconductor Nanocomposites for Photothermal Imaging of Latent Fingerprints.

PubMed

Cui, Jiabin; Xu, Suying; Guo, Chang; Jiang, Rui; James, Tony D; Wang, Leyu

2015-11-17

Optical imaging of latent fingerprints (LFPs) has been widely used in forensic science and for antiterrorist applications, but it suffers from interference from autofluorescence and the substrates background color. Cu7S4 nanoparticles (NPs), with excellent photothermal properties, were synthesized using a new strategy and then fabricated into amphiphilic nanocomposites (NCs) via polymerization of allyl mercaptan coated on Cu7S4 NPs to offer good affinities toward LFPs. Here, we develop a facile and versatile photothermal LFP imaging method based on the high photothermal conversion efficiency (52.92%, 808 nm) of Cu7S4 NCs, indicating its effectiveness for imaging LFPs left on different substrates (with various background colors), which will be extremely useful for crime scene investigations. Furthermore, by fabricating Cu7S4-CdSe@ZnS NCs, a fluorescent-photothermal dual-mode imaging strategy was used to detect trinitrotoluene (TNT) in LFPs while still maintaining a complete photothermal image of LFP.

Tripartite ATP-independent periplasmic (TRAP) transporters in bacteria and archaea.

PubMed

Mulligan, Christopher; Fischer, Marcus; Thomas, Gavin H

2011-01-01

The tripartite ATP-independent periplasmic (TRAP) transporters are the best-studied family of substrate-binding protein (SBP)-dependent secondary transporters and are ubiquitous in prokaryotes, but absent from eukaryotes. They are comprised of an SBP of the DctP or TAXI families and two integral membrane proteins of unequal sizes that form the DctQ and DctM protein families, respectively. The SBP component has a structure comprised of two domains connected by a hinge that closes upon substrate binding. In DctP-TRAP transporters, substrate binding is mediated through a conserved and specific arginine/carboxylate interaction in the SBP. While the SBP component has now been relatively well characterized, the membrane components of TRAP transporters are still poorly understood both in terms of their structure and function. We review the expanding repertoire of substrates and physiological roles for experimentally characterized TRAP transporters in bacteria and discuss mechanistic aspects of these transporters using data primarily from the sialic acid-specific TRAP transporter SiaPQM from Haemophilus influenzae, which suggest that TRAP transporters are high-affinity, Na(+)-dependent unidirectional secondary transporters. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Bacteria-Affinity 3D Macroporous Graphene/MWCNTs/Fe3O4 Foams for High-Performance Microbial Fuel Cells.

PubMed

Song, Rong-Bin; Zhao, Cui-E; Jiang, Li-Ping; Abdel-Halim, Essam Sayed; Zhang, Jian-Rong; Zhu, Jun-Jie

2016-06-29

Promoting the performance of microbial fuel cells (MFCs) relies heavily on the structure design and composition tailoring of electrode materials. In this work, three-dimensional (3D) macroporous graphene foams incorporated with intercalated spacer of multiwalled carbon nanotubes (MWCNTs) and bacterial anchor of Fe3O4 nanospheres (named as G/MWCNTs/Fe3O4 foams) were first synthesized and used as anodes for Shewanella-inoculated microbial fuel cells (MFCs). Thanks to the macroporous structure of 3D graphene foams, the expanded electrode surface by MWCNTs spacing, as well as the high affinity of Fe3O4 nanospheres toward Shewanella oneidensis MR-1, the anode exhibited high bacterial loading capability. In addition to spacing graphene nanosheets for accommodating bacterial cells, MWCNTs paved a smoother way for electron transport in the electrode substrate of MFCs. Meanwhile, the embedded bioaffinity Fe3O4 nanospheres capable of preserving the bacterial metabolic activity provided guarantee for the long-term durability of the MFCs. With these merits, the constructed MFC possessed significantly higher power output and stronger stability than that with conventional graphite rod anode.

Multiple allosteric sites are involved in the modulation of insulin-degrading-enzyme activity by somatostatin.

PubMed

Tundo, Grazia R; Di Muzio, Elena; Ciaccio, Chiara; Sbardella, Diego; Di Pierro, Donato; Polticelli, Fabio; Coletta, Massimo; Marini, Stefano

2016-10-01

Somatostatin is a cyclic peptide, released in the gastrointestinal system and the central nervous system, where it is involved in the regulation of cognitive and sensory functions, motor activity and sleep. It is a substrate of insulin-degrading enzyme (IDE), as well as a modulator of its activity and expression. In the present study, we have investigated the modulatory role of somatostatin on IDE activity at 37 °C and pH 7.3 for various substrates [i.e. insulin, β-amyloid (Aβ) 1-40 and bradykinin], aiming to quantitatively characterize the correlation between the specific features of the substrates and the regulatory mechanism. Functional data indicate that somatostatin, in addition to the catalytic site of IDE (being a substrate), is also able to bind to two additional exosites, which play different roles according to the size of the substrate and its binding mode to the IDE catalytic cleft. In particular, one exosite, which displays high affinity for somatostatin, regulates only the interaction of IDE with larger substrates (such as insulin and Aβ 1-40 ) in a differing fashion according to their various modes of binding to the enzyme. A second exosite, which is involved in the regulation of enzymatic processing by IDE of all substrates investigated (including a 10-25 amino acid long amyloid-like peptide, bradykinin and somatostatin itself, which had been studied previously), probably acts through the alteration of an 'open-closed' equilibrium. © 2016 Federation of European Biochemical Societies.

Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcella, Aaron M.; Culbertson, Sannie J.; Shogren-Knaak, Michael A.

The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05 and 4.10 Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determiningmore » the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD = 62 ± 13 nM, followed by the binding of two more equivalents of holo-ACPP with KD = 1.2 ± 0.2 μM. Cooperativity was not observed for apo-ACPP which bound with KD = 2.4 ± 0.1 μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.« less

Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

NASA Astrophysics Data System (ADS)

Liu, Feng; Roth, Richard A.

1995-10-01

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

BiPPred: Combined sequence- and structure-based prediction of peptide binding to the Hsp70 chaperone BiP.

PubMed

Schneider, Markus; Rosam, Mathias; Glaser, Manuel; Patronov, Atanas; Shah, Harpreet; Back, Katrin Christiane; Daake, Marina Angelika; Buchner, Johannes; Antes, Iris

2016-10-01

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

NASA Astrophysics Data System (ADS)

Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

2016-06-01

Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh,S.; Yamashita, A.; Gouaux, E.

Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibitionmore » exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.« less

Kinetics and thiol requirements of iodothyronine 5'-deiodination are tissue-specific in common carp (Cyprinus carpio L.).

PubMed

Klaren, Peter H M; Geven, Edwin J W; Nagelkerke, Anika; Flik, Gert

2012-03-01

Iodothyronine deiodinases determine the biological activity of thyroid hormones. Despite the homology of the catalytic sites of mammalian and teleostean deiodinases, in-vitro requirements for the putative thiol co-substrate dithiothreitol (DTT) vary considerably between vertebrate species. To further our insights in the interactions between the deiodinase protein and its substrates: thyroid hormone and DTT, we measured enzymatic iodothyronine 5'-deiodination, Dio1 and Dio2 mRNA expression, and Dio1 affinity probe binding in liver and kidney preparations from a freshwater teleost, the common carp (Cyprinus carpio L.). Deiodination rates, using reverse T3 (rT3, 3,3',5'-triiodothyronine) as the substrate, were analysed as a function of the iodothyronine and DTT concentrations. In kidney rT3 5'-deiodinase activity measured at rT3 concentrations up to 10 μM and in the absence of DTT does not saturate appreciably. In the presence of 1mM DTT, renal rT3 deiodination rates are 20-fold lower. In contrast, rT3 5'-deiodination in liver is potently stimulated by 1mM DTT. The marked biochemical differences between 5'-deiodination in liver and kidney are not associated with the expression of either Dio1 or Dio2 mRNA since both organs express both deiodinase types. In liver and kidney, DTT stimulates the incorporation of N-bromoacetylated affinity labels in proteins with estimated molecular masses of 57 and 55, and 31 and 28 kDa, respectively. Although primary structures are highly homologous, the biochemistry of carp deiodinases differs markedly from their mammalian counterparts. Copyright © 2011 Elsevier Inc. All rights reserved.

Enzymatic characterization of a novel bovine liver dihydrodiol dehydrogenase--reaction mechanism and bile acid dehydrogenase activity.

PubMed

Nanjo, H; Adachi, H; Morihana, S; Mizoguchi, T; Nishihara, T; Terada, T

1995-05-11

Bovine liver cytosolic dihydrodiol dehydrogenase (DD3) has been characterized by its unique dihydrodiol dehydrogenase activity for trans-benzenedihydrodiol (trans-1,2-dihydrobenzene-1,2-diol) with the highest affinity and the greatest velocity among three multiple forms of dihydrodiol dehydrogenases (DD1-DD3). It is the first time that DD3 has shown a significant dehydrogenase activity for (S)-(+)-1-indanol with low Km value (0.33 +/- 0.022 mM) and high K(cat) value (25 +/- 0.79 min-1). The investigation of the product inhibition of (S)-(+)-1-indanol with NADP+ versus 1-indanone and NADPH clearly showed that the enzymatic reaction of DD3 may follow a typical ordered Bi Bi mechanism similar to many aldo/keto reductases. Additionally, DD3 was shown to catalyze the dehydrogenation of bile acids (lithocholic acid, taurolithocholic acid and taurochenodeoxycholic acid) having no 12-hydroxy groups with low Km values (17 +/- 0.65, 33 +/- 1.9 and 890 +/- 73 microM, respectively). In contrast, DD1, 3 alpha-hydroxysteroid dehydrogenase, shows a broad substrate specificity for many bile acids with higher affinity than those of DD3. Competitive inhibition of DD3 with androsterone against dehydrogenase activity for (S)-(+)-1-indanol, trans-benzenedihydrodiol or lithocholic acid suggests that these three substrates bind to the same substrate binding site of DD3, different from the case of human liver bile acid binder/dihydrodiol dehydrogenase (Takikawa, H., Stolz, A., Sugiyama, Y., Yoshida, H., Yamamoto, M. and Kaplowitz, N. (1990) J. Biol. Chem. 265, 2132-2136). Considering the reaction mechanism, DD3 may also play an important role in bile acids metabolism as well as the detoxication of aromatic hydrocarbons.

Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies.

PubMed

Fong, Monica; Berrin, Jean-Guy; Paës, Gabriel

2016-01-01

Enzymes degrading plant biomass polymers are widely used in biotechnological applications. Their efficiency can be limited by non-specific interactions occurring with some chemical motifs. In particular, the lignin component is known to bind enzymes irreversibly. In order to determine interactions of enzymes with their substrates, experiments are usually performed on isolated simple polymers which are not representative of plant cell wall complexity. But when using natural plant substrates, the role of individual chemical and structural features affecting enzyme-binding properties is also difficult to decipher. We have designed and used lignified model assemblies of plant cell walls as templates to characterize binding properties of multi-modular cellulases. These three-dimensional assemblies are modulated in their composition using the three principal polymers found in secondary plant cell walls (cellulose, hemicellulose, and lignin). Binding properties of enzymes are obtained from the measurement of their mobility that depends on their interactions with the polymers and chemical motifs of the assemblies. The affinity of the multi-modular GH45 cellulase was characterized using a statistical analysis to determine the role played by each assembly polymer. Presence of hemicellulose had much less impact on affinity than cellulose and model lignin. Depending on the number of CBMs appended to the cellulase catalytic core, binding properties toward cellulose and lignin were highly contrasted. Model assemblies bring new insights into the molecular determinants that are responsible for interactions between enzymes and substrate without the need of complex analysis. Consequently, we believe that model bioinspired assemblies will provide relevant information for the design and optimization of enzyme cocktails in the context of biorefineries.

Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates.

PubMed

Addington, Trevor; Calisto, Barbara; Alfonso-Prieto, Mercedes; Rovira, Carme; Fita, Ignasi; Planas, Antoni

2011-02-01

Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity. © 2010 Wiley-Liss, Inc.

Effects of metal ions on the catalytic degradation of dicofol by cellulase.

PubMed

Zhai, Zihan; Yang, Ting; Zhang, Boya; Zhang, Jianbo

2015-07-01

A new technique whereby cellulase immobilized on aminated silica was applied to catalyze the degradation of dicofol, an organochlorine pesticide. In order to evaluate the performance of free and immobilized cellulase, experiments were carried out to measure the degradation efficiency. The Michaelis constant, Km, of the reaction catalyzed by immobilized cellulase was 9.16 mg/L, and the maximum reaction rate, Vmax, was 0.40 mg/L/min, while that of free cellulase was Km=8.18 mg/L, and Vmax=0.79 mg/L/min, respectively. The kinetic constants of catalytic degradation were calculated to estimate substrate affinity. Considering that metal ions may affect enzyme activity, the effects of different metal ions on the catalytic degradation efficiency were explored. The results showed that the substrate affinity decreased after immobilization. Monovalent metal ions had no effect on the reaction, while divalent metal ions had either positive or inhibitory effects, including activation by Mn2+, reversible competition with Cd2+, and irreversible inhibition by Pb2+. Ca2+ promoted the catalytic degradation of dicofol at low concentrations, but inhibited it at high concentrations. Compared with free cellulase, immobilized cellulase was affected less by metal ions. This work provided a basis for further studies on the co-occurrence of endocrine-disrupting chemicals and heavy metal ions in the environment. Copyright © 2015. Published by Elsevier B.V.

Determination of trace alkaline phosphatase by solid-substrate room-temperature phosphorimetry based on Triticum vulgare lectin labeled with fullerenol.

PubMed

Liu, Jia-Ming; Gao, Fei; Huang, Hong-Hua; Zeng, Li-Qing; Huang, Xiao-Mei; Zhu, Guo-Hui; Li, Zhi-Ming

2008-04-01

Fullerenol (F) shows a strong and stable room-temperature phosphorescence (RTP) signal on the surface of nitrocellulose membrane (NCM) at lambda ex max/ lambda em max =542.0/709.4 nm. When modified by dodecylbenzenesulfonic acid sodium salt (DBS), fullerenol emits a stronger signal. It was also found that quantitative specific affinity-adsorption reaction can be carried out between Triticum vulgare lectin (WGA) labeled with DBS-F and alkaline phosphatase (ALP) on the surface of NCM, and the product obtained (WGA-ALP-WGA-F-DBS) emits a strong and stable RTP signal. Furthermore, the content of ALP was proportional to the DeltaI(p) value. Based on the facts above, a new method for the determination of trace amounts of ALP by affinity-adsorption solid-substrate room-temperature phosphorimetry (AA-SS-RTP) was established, using fullerenol modified with DBS to label WGA. The detection limit was 0.011 fg spot(-1) (corresponding concentration: 2.8x10(-14) g ml(-1), namely 2.8x10(-16) mol l(-1)). This method with high sensitivity, accuracy, and precision has been successfully applied to the determination of the content of ALP in human serum survey and forecast human disease, and the results are tallied with those using alkaline phosphatase kits. The mechanism for the determination of ALP using AA-SS-RTP was also discussed.

Crossing borders to bind proteins--a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set.

PubMed

Baltzer, Lars

2011-06-01

A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.

Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

PubMed

Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

2003-05-13

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate constants in the initial intermediate formed during acylation (EAP, where EA is the acyl enzyme and P is the alcohol leaving group cleaved from the ester substrate) may be constrained such that the leaving group P must dissociate before hydrolytic deacylation can occur.

Using the lentiviral vector system to stably express chicken P-gp and BCRP in MDCK cells for screening the substrates and studying the interplay of both transporters.

PubMed

Zhang, Yujuan; Huang, Jinhu; Liu, Yang; Guo, Tingting; Wang, Liping

2018-06-01

Transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are known to influence the pharmacokinetics and toxicity of substrate drugs. However, no detailed information is as yet available about functional activity and substrate spectra of chicken P-gp and BCRP. In this study, BCRP single and BCRP/P-gp double-transfected MDCK cell lines (named MDCK-chAbcg2 and MDCK-chAbcg2/Abcb1, respectively) were generated using lentiviral vector system to develop reliable systems for screening the substrates for these two transporters and study the interplay between them. The constructed cell lines significantly expressed functional exogenous proteins and expression persisted for at least 50 generations with no decrease. Enrofloxacin, ciprofloxacin, tilmicosin, sulfadiazine, ampicillin and clindamycin were classified as the substrates of chicken P-gp according to the rules suggested by FDA, as their net efflux ratios were greater than two. Similarly, enrofloxacin, ciprofloxacin, tilmicosin, florfenicol, ampicillin and clindamycin were classified as the substrates of BCRP. Among these drugs, enrofloxacin, ciprofloxacin, tilmicosin, ampicillin, and clindamycin were the cosubstrates of P-gp and BCRP, however, chicken BCRP and P-gp exhibit different affinities to the shared substrates at different concentrations by blocking either one or both transport with specific inhibitors in the coexpression system. It was also found that ceftiofur, amoxicillin and doxycycline were not substrates of either chicken BCRP or the substrates of chicken P-gp. These constructed cell models provide useful systems for high-throughput screening of the potential substrates of chicken BCRP and P-gp as well as the drug-drug interaction mediated via chicken BCRP and P-gp.

Kinetic Measurements of Di- and Tripeptide and Peptidomimetic Drug Transport in Different Kidney Regions Using the Fluorescent Membrane Potential-Sensitive Dye, DiS-C3-(3).

PubMed

Alghamdi, Othman A; King, Nicola; Jones, Graham L; Moens, Pierre D J

2017-12-01

Tri- and dipeptides are transported in the kidney by PEPT1 and PEPT2 isoforms. The aim of this study was to investigate differences in transport kinetics between renal brush border (BBMV) and outer medulla (OMMV) membrane vesicles (where PEPT1 and PEPT2 are sequentially available) for a range of di- and tripeptides and peptidomimetic drugs. This was accomplished through the use of the potential-sensitive fluorescent dye 3,3'-dipropylthiacarbocyanine iodide [DiS-C 3 -(3)]. BBMV and OMMV were prepared from the rat kidney using standard techniques. The presence of PEPT1 in BBMV and PEPT2 in OMMV was confirmed using Western blotting. Fluorescence changes were measured when extravesicular medium at pH 6.6 containing 0-1 mM substrates was added to a cuvette containing vesicles pre-equilibrated at pH 7.4 and 2.71 μM DiS-C 3 -(3). An increase in fluorescence intensity occurred upon substrate addition reflecting the expected positive change in membrane potential difference. Of the range of substrates studied, OMMV manifested the highest affinity to cefadroxil and valacyclovir (K m 4.3 ± 1.2 and 11.7 ± 3.2 µM, respectively) compared to other substrates, whilst the BBMV showed a higher affinity to Gly-His (K m 15.4 ± 3.1 µM) compared to other substrates. In addition, OMMV showed higher affinity and capacity to Gly-Gln (K m 47.1 ± 9.8 µM, 55.5 ± 2.8 ΔF/s/mg protein) than BBMV (K m 78.1 ± 13.3 µM and 35.5 ± 1.7 ΔF/s/mg protein, respectively). In conclusion, this study successfully separated the expression of PEPT1 and PEPT2 into different vesicle preparations inferring their activity in different regions of the renal proximal tubule.

Temperature Dependence of Inorganic Nitrogen Uptake: Reduced Affinity for Nitrate at Suboptimal Temperatures in Both Algae and Bacteria

PubMed Central

Reay, David S.; Nedwell, David B.; Priddle, Julian; Ellis-Evans, J. Cynan

1999-01-01

Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 ≈ 3, where Q10 is the proportional change with a 10°C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures. PMID:10347046

The Effects of Protein-Ligand Associations on the Subunit Interactions of Phosphofructokinase from B. stearothermophilus†

PubMed Central

Quinlan, R. Jason; Reinhart, Gregory D.

2008-01-01

Differences between the crystal structures of inhibitor-bound and uninihibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits containing two different extrinsic fluorophores simultaneously in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel Fluorescence Correlation Spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor-binding, as binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated. PMID:16981693

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan

2010-11-22

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues thatmore » flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.« less

Identification of Breast Cancer Specific Proteolytic Activities for Targeted Prodrug Activation

DTIC Science & Technology

2006-05-01

volume of fluid that can be obtained from ECF of human breast cancers is to use a phage display approach. To accomplish this, we have designed a...affinity support, followed by a randomized protease substrate sequence and the carboxyl-terminal domain of M13 gene III. Each fusion protein was displayed ...PSMA) (35). Substrate phage can be created either as a monovalent or as pentavalent display (34). Both approaches have their own advantages and

Functional Role of Tyr12 in the Catalytic Activity of Novel Zeta-like Glutathione S-transferase from Acidovorax sp. KKS102.

PubMed

Shehu, Dayyabu; Alias, Zazali

2018-05-19

Glutathione S-transferases (GSTs) are a family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide. The enzyme also displayed dehalogenation function against dichloroacetate, permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.

Three cell recognition changes accompany the ingression of sea urchin primary mesenchyme cells.

PubMed

Fink, R D; McClay, D R

1985-01-01

At gastrulation the primary mesenchyme cells of sea urchin embryos lose contact with the extracellular hyaline layer and with neighboring blastomeres as they pass through the basal lamina and enter the blastocoel. This delamination process was examined using a cell-binding assay to follow changes in affinities between mesenchyme cells and their three substrates: hyalin, early gastrula cells, and basal lamina. Sixteen-cell-stage micromeres (the precursors of primary mesenchyme cells), and mesenchyme cells obtained from mesenchyme-blastula-stage embryos were used in conjunction with micromeres raised in culture to intermediate ages. The micromeres exhibited an affinity for hyalin, but the affinity was lost at the time of mesenchyme ingression in vivo. Similarly, micromeres had an affinity for monolayers of gastrula cells but the older mesenchyme cells lost much of their cell-to-cell affinity. Presumptive ectoderm and endoderm cells tested against the gastrula monolayers showed no decrease in binding over the same time interval. When micromeres and primary mesenchyme cells were tested against basal lamina preparations, there was an increase in affinity that was associated with developmental time. Presumptive ectoderm and endoderm cells showed no change in affinity over the same interval. Binding measurements using isolated basal laminar components identified fibronectin as one molecule for which the wandering primary mesenchyme cells acquired a specific affinity. The data indicate that as the presumptive mesenchyme cells leave the vegetal plate of the embryo they lose affinities for hyalin and for neighboring cells, and gain an affinity for fibronectin associated with the basal lamina and extracellular matrix that lines the blastocoel.

Modular, Antibody-free Time-Resolved LRET Kinase Assay Enabled by Quantum Dots and Tb3+-sensitizing Peptides

NASA Astrophysics Data System (ADS)

Cui, Wei; Parker, Laurie L.

2016-07-01

Fluorescent drug screening assays are essential for tyrosine kinase inhibitor discovery. Here we demonstrate a flexible, antibody-free TR-LRET kinase assay strategy that is enabled by the combination of streptavidin-coated quantum dot (QD) acceptors and biotinylated, Tb3+ sensitizing peptide donors. By exploiting the spectral features of Tb3+ and QD, and the high binding affinity of the streptavidin-biotin interaction, we achieved multiplexed detection of kinase activity in a modular fashion without requiring additional covalent labeling of each peptide substrate. This strategy is compatible with high-throughput screening, and should be adaptable to the rapidly changing workflows and targets involved in kinase inhibitor discovery.

Utilization of Nitrophenylphosphates and Oxime-Based Ligation for the Development of Nanomolar Affinity Inhibitors of the Yersinia Pestis Outer Protein H (YopH) Phosphatase†, §, ¶

PubMed Central

Bahta, Medhanit; Lountos, George T.; Dyas, Beverly; Kim, Sung-Eun; Ulrich, Robert G.; Waugh, David S.; Burke, Terrence R.

2011-01-01

Our current study reports the first KM optimization of a library of nitrophenylphosphate-containing substrates for generating an inhibitor lead against the Yersinia pestis outer protein phosphatase (YopH). A high activity substrate identified by this method (KM = 80 μM was converted from a substrate into an inhibitor by replacement of its phosphate group with difluoromethylphosphonic acid and by attachment of an aminooxy handle for further structural optimization by oxime-ligation. A co-crystal structure of this aminooxy-containing platform in complex with YopH allowed the identification of a conserved water molecule proximal to the aminooxy group that was subsequently employed for the design of furanyl-based oxime derivatives. By this process, a potent (IC50 = 190 nM) and non-promiscuous inhibitor was developed with good YopH selectivity relative to a panel of phosphatases. The inhibitor showed significant inhibition of intracellular Y. pestis replication at a non-cytotoxic concentration. The current work presents general approaches to PTP inhibitor development that may be useful beyond YopH. PMID:21443195

Cu-rGO subsurface layer creation on copper substrate and its resistance to oxidation

NASA Astrophysics Data System (ADS)

Pietrzak, Katarzyna; Strojny-Nędza, Agata; Olesińska, Wiesława; Bańkowska, Anna; Gładki, Andrzej

2017-11-01

On the basis of a specially designed experiment, this paper presents a model, which is an attempt to explain the mechanism of formatting and creating oxidation resistance of Cu-rGO subsurface layers. Practically zero chemical affinity of copper to carbon is a fundamental difficulty in creating composite structures of Cu-C, properties which are theoretically possible to estimate. In order to bind the thermally reduced graphene oxide with copper surface, the effect of structural rebuilding of the copper oxide, in the process of annealing in a nitrogen atmosphere, have been used. On intentionally oxidized and anoxic copper substrates the dispersed graphene oxide (GO) and thermally reduced graphene oxide (rGO) were loaded. Annealing processes after the binding effects of both graphene oxide forms to Cu substrates were tested. The methods for high-resolution electron microscopy were found subsurface rGO-Cu layer having a substantially greater resistance to oxidation than pure copper. The mechanism for the effective resistance to oxidation of the Cu-rGO has been presented in a hypothetical form.

Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease.

PubMed

Louis, John M; Tözsér, József; Roche, Julien; Matúz, Krisztina; Aniana, Annie; Sayer, Jane M

2013-10-29

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, M.H.; Neubig, R.R.

1986-03-05

High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonistmore » (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.« less

The 30 kDa protein co-purified with chick liver glutathione S-transferases is a carbonyl reductase.

PubMed

Tsai, S P; Wang, L Y; Yeh, H I; Tam, M F

1996-02-08

An unidentified 30 kDa protein was co-purified with chick liver glutathione S-transferases from S-hexylglutathione affinity column. The protein was isolated to apparent homogeneity with chromatofocusing. The molecular mass of the protein was determined to be 30 277 +/- 3 dalton by mass spectrometry. The protein was digested with Achromobacter proteinase I. Amino-acid sequence analyses of the resulting peptides show a high degree of identity with those of human carbonyl reductase. The protein is active with menadione as substrate. Thus, it is identified as chick liver carbonyl reductase.

Complex phylogeny and gene expression patterns of members of the NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) in wheat

PubMed Central

Buchner, Peter; Hawkesford, Malcolm J.

2014-01-01

NPF (formerly referred to as low-affinity NRT1) and ‘high-affinity’ NRT2 nitrate transporter genes are involved in nitrate uptake by the root, and transport and distribution of nitrate within the plant. The NPF gene family consists of 53 members in Arabidopsis thaliana, however only 11 of these have been functionally characterized. Although homologous genes have been identified in genomes of different plant species including some cereals, there is little information available for wheat (Triticum aestivum). Sixteen genes were identified in wheat homologous to characterized Arabidopsis low-affinity nitrate transporter NPF genes, suggesting a complex wheat NPF gene family. The regulation of wheat NFP genes by plant N-status indicated involvement of these transporters in substrate transport in relation to N-metabolism. The complex expression pattern in relation to tissue specificity, nitrate availability and senescence may be associated with the complex growth patterns of wheat depending on sink/source demands, as well as remobilization during grain filling. PMID:24913625

Molecular modeling and dynamics simulations of PNP from Streptococcus agalactiae.

PubMed

Caceres, Rafael Andrade; Saraiva Timmers, Luis Fernando; Dias, Raquel; Basso, Luiz Augusto; Santos, Diogenes Santiago; de Azevedo, Walter Filgueira

2008-05-01

This work describes for the first time a structural model of purine nucleoside phosphorylase from Streptococcus agalactiae (SaPNP). PNP catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is a potential target for the development of antibacterial drugs. We modeled the complexes of SaPNP with 15 different ligands in order to determine the structural basis for the specificity of these ligands against SaPNP. The application of a novel empirical scoring function to estimate the affinity of a ligand for a protein was able to identify the ligands with high affinity for PNPs. The analysis of molecular dynamics trajectory for SaPNP indicates that the functionally important motifs have a very stable structure. This new structural model together with a novel empirical scoring function opens the possibility to explorer larger library of compounds in order to identify the new inhibitors for PNPs in virtual screening projects.

Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY†

PubMed Central

Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Choe, Jun-Yong; Kaback, H. Ronald

2009-01-01

A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (Kdapp) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low Kdapp) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates koff as a major factor for the affinity change at alkaline pH and confirms the effects of pH on Kdapp inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding. PMID:19689129

Determination of trace alkaline phosphatase by affinity adsorption solid substrate room temperature phosphorimetry based on wheat germ agglutinin labeled with 8-quinolineboronic acid phosphorescent molecular switch and prediction of diseases

NASA Astrophysics Data System (ADS)

Liu, Jia-Ming; Gao, Hui; Li, Fei-Ming; Shi, Xiu-Mei; Lin, Chang-Qing; Lin, Li-Ping; Wang, Xin-Xing; Li, Zhi-Ming

2010-09-01

The 8-quinolineboronic acid phosphorescent molecular switch (abbreviated as PMS-8-QBA. Thereinto, 8-QBA is 8-quinolineboronic acid, and PMS is phosphorescent molecular switch) was found for the first time. PMS-8-QBA, which was in the "off" state, could only emit weak room temperature phosphorescence (RTP) on the acetyl cellulose membrane (ACM). However, PMS-8-QBA turned "on" automatically for its changed structure, causing that the RTP of 8-QBA in the system increased, after PMS-8-QBA-WGA (WGA is wheat germ agglutinin) was formed by reaction between -OH of PMS-8-QBA and -COOH of WGA. More interesting is that the -NH 2 of PMS-8-QBA-WGA could react with the -COOH of alkaline phosphatase (AP) to form the affinity adsorption (AA) product WGA-AP-WGA-8-QBA-PMS (containing -NH-CO- bond), which caused RTP of the system to greatly increase. Thus, affinity adsorption solid substrate room temperature phosphorimetry using PMS-8-QBA as labelling reagent (PMS-8-QBA-AA-SSRTP) for the determination of trace AP was established. The method had many advantages, such as high sensitivity (the detection limit (LD) was 2.5 zg spot -1. For sample volume of 0.40 μl spot -1, corresponding concentration was 6.2 × 10 -18 g ml -1), good selectivity (the allowed concentration of coexisting material was higher, when the relative error was ±5%), high accuracy (applied to detection of AP content in serum samples, the result was coincided with those obtained by enzyme-linked immunoassay), which was suitable for the detection of trace AP content in serum samples and the forecast of human diseases. Meanwhile, the mechanism of PMS-8-QBA-AASSRTP was discussed. The new field of analytical application and clinic diagnosis technique of molecule switch are exploited, based on the phosphorescence characteristic of PMS-8-QBA, the AA reaction between WGA and AP, as well as the relation between AP content and human diseases. The research results promote the development and interpenetrate among molecule switch technique, lectin science and SSRTP.

Determination of trace alkaline phosphatase by affinity adsorption solid substrate room temperature phosphorimetry based on wheat germ agglutinin labeled with 8-quinolineboronic acid phosphorescent molecular switch and prediction of diseases.

PubMed

Liu, Jia-Ming; Gao, Hui; Li, Fei-Ming; Shi, Xiu-Mei; Lin, Chang-Qing; Lin, Li-Ping; Wang, Xin-Xing; Li, Zhi-Ming

2010-09-01

The 8-quinolineboronic acid phosphorescent molecular switch (abbreviated as PMS-8-QBA. Thereinto, 8-QBA is 8-quinolineboronic acid, and PMS is phosphorescent molecular switch) was found for the first time. PMS-8-QBA, which was in the "off" state, could only emit weak room temperature phosphorescence (RTP) on the acetyl cellulose membrane (ACM). However, PMS-8-QBA turned "on" automatically for its changed structure, causing that the RTP of 8-QBA in the system increased, after PMS-8-QBA-WGA (WGA is wheat germ agglutinin) was formed by reaction between -OH of PMS-8-QBA and -COOH of WGA. More interesting is that the -NH(2) of PMS-8-QBA-WGA could react with the -COOH of alkaline phosphatase (AP) to form the affinity adsorption (AA) product WGA-AP-WGA-8-QBA-PMS (containing -NH-CO- bond), which caused RTP of the system to greatly increase. Thus, affinity adsorption solid substrate room temperature phosphorimetry using PMS-8-QBA as labelling reagent (PMS-8-QBA-AA-SSRTP) for the determination of trace AP was established. The method had many advantages, such as high sensitivity (the detection limit (LD) was 2.5zgspot(-1). For sample volume of 0.40mulspot(-1), corresponding concentration was 6.2x10(-18)gml(-1)), good selectivity (the allowed concentration of coexisting material was higher, when the relative error was +/-5%), high accuracy (applied to detection of AP content in serum samples, the result was coincided with those obtained by enzyme-linked immunoassay), which was suitable for the detection of trace AP content in serum samples and the forecast of human diseases. Meanwhile, the mechanism of PMS-8-QBA-AASSRTP was discussed. The new field of analytical application and clinic diagnosis technique of molecule switch are exploited, based on the phosphorescence characteristic of PMS-8-QBA, the AA reaction between WGA and AP, as well as the relation between AP content and human diseases. The research results promote the development and interpenetrate among molecule switch technique, lectin science and SSRTP. Copyright 2009 Elsevier B.V. All rights reserved.

Purification and characterization of peroxidase from avocado (Persea americana Mill, cv. Hass).

PubMed

Rojas-Reyes, José O; Robles-Olvera, Victor; Carvajal-Zarrabal, Octavio; Castro Matinez, Claudia; Waliszewski, Krzysztof N; Aguilar-Uscanga, María Guadalupe

2014-07-01

Avocado (Persea americana Mill, cv. Hass) fruit ranks tenth in terms of the most important products for Mexico. Avocado products are quite unstable due to the presence of oxidative enzymes such as polyphenol oxidase and peroxidase. The present study is to characterize the activity of purified avocado peroxidase from avocado in order to ascertain the biochemical and kinetic properties and their inhibition conditions. Purification was performed by Sephacryl S 200 HR gel filtration chromatography and its estimated molecular weight was 40 kDa. The zymogram showed an isoelectric point of 4.7. Six substrates were tested in order to ascertain the affinity of the enzyme for these substrates. The purified peroxidase was found to have low Km (0.296 mM) and high catalytic efficiency (2688 mM(-1) s(-1)) using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), optimum activity being reached at 51°C, pH 3.8. The addition of dithiothreitol, β-mercaptoethanol, ascorbic acid, sodium azide, L-cysteine and Tween-20 had high inhibitory effects, while metals ions such as Cu(+), Fe(2+) and Mn(2+) had weak inhibitory activity on purified avocado peroxidase. The purified avocado peroxidase exhibits high inhibition (Ki = 0.37 µM) with 1.97 µM n-propyl gallate using ABTS as substrate at 51°C, pH 3.8 for 10 min. © 2013 Society of Chemical Industry.

Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

NASA Astrophysics Data System (ADS)

Keefe, Andrew J.; Jiang, Shaoyi

2012-01-01

Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein-substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.

Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

PubMed

Kandeel, Mahmoud; Kitade, Yukio

2018-02-01

RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, K dPolyU and K dPolyC were 15.8 and 9.3μM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and β5 in the β-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding. Copyright © 2017 Elsevier B.V. All rights reserved.

A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding

PubMed Central

Kostrhon, Sebastian; Kontaxis, Georg; Kaufmann, Tanja; Schirghuber, Erika; Kubicek, Stefan; Konrat, Robert

2017-01-01

N-terminal histone tails are subject to many posttranslational modifications that are recognized by and interact with designated reader domains in histone-binding proteins. BROMO domain adjacent to zinc finger 2B (BAZ2B) is a multidomain histone-binding protein that contains two histone reader modules, a plant homeodomain (PHD) and a bromodomain (BRD), linked by a largely disordered linker. Although previous studies have reported specificity of the PHD domain for the unmodified N terminus of histone H3 and of the BRD domain for H3 acetylated at Lys14 (H3K14ac), the exact mode of H3 binding by BAZ2B and its regulation are underexplored. Here, using isothermal titration calorimetry and NMR spectroscopy, we report that acidic residues in the BAZ2B PHD domain are essential for H3 binding and that BAZ2B PHD–BRD establishes a polyvalent interaction with H3K14ac. Furthermore, we provide evidence that the disordered interdomain linker modulates the histone-binding affinity by interacting with the PHD domain. In particular, lysine-rich stretches in the linker, which resemble the positively charged N terminus of histone H3, reduce the binding affinity of the PHD finger toward the histone substrate. Phosphorylation, acetylation, or poly(ADP-ribosyl)ation of the linker residues may therefore act as a cellular mechanism to transiently tune BAZ2B histone-binding affinity. Our findings further support the concept of interdomain linkers serving a dual role in substrate binding by appropriately positioning the adjacent domains and by electrostatically modulating substrate binding. Moreover, inhibition of histone binding by a histone-mimicking interdomain linker represents another example of regulation of protein–protein interactions by intramolecular mimicry. PMID:28864776

Betaxanthins as Substrates for Tyrosinase. An Approach to the Role of Tyrosinase in the Biosynthetic Pathway of Betalains1

PubMed Central

Gandía-Herrero, Fernando; Escribano, Josefa; García-Carmona, Francisco

2005-01-01

Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is the key enzyme in melanin biosynthesis and in the enzymatic browning of fruits and vegetables. The role of tyrosinase in the secondary metabolism of plants still remains unclear, but its implication in betalain biosynthesis has been proposed. Betalains are an important class of water-soluble pigments, characteristic of plants belonging to the order Caryophyllales. In this article, the betaxanthins, tyrosine-betaxanthin (portulacaxanthin II) and dopaxanthin, are reported to be physiological substrates for tyrosinase. The direct activity of tyrosinase on selected betaxanthins is characterized in depth, and conversion of tyrosine-betaxanthin to dopaxanthin and its further oxidation to a series of compounds are described. Identity of the reaction products was studied by high-performance liquid chromatography and electrospray ionization-mass spectrometry. Masses determined for the reaction products were the same in all cases, 389 m/z ([M + H]+) and equal to that determined for betanidin. Data indicate that dopaxanthin-quinone is obtained and evolves to more stable species by intramolecular cyclization. Kinetic parameters for tyrosinase acting on dopaxanthin were evaluated, showing a high affinity for this substrate (Km = 84.3 μm). The biosynthetic scheme of betalains is reviewed and a branch is proposed based on the description of physiological substrates for tyrosinase. Lampranthus productus, Glottiphylum oligocarpum, and Glottiphylum pigmaeum are described as sources of stereopure (2S/S)-dopaxanthin. PMID:15805475

Characterisation of the two malate dehydrogenases from Phytomonas sp. Purification of the glycosomal isoenzyme.

PubMed

Uttaro, A D; Opperdoes, F R

1997-10-01

Two NAD(H)-dependent malate dehydrogenase (MDH) isoenzymes were detected in Phytomonas isolated from the lactiferous tubes of Euphorbia characias. The total specific activity in crude extracts using oxaloacetate as substrate was 3.3 U mg-1 of protein. The two isoenzymes had isoelectric points of 6.0 and 7.2, respectively. The acidic isoform represented 80% of the total activity in the cell and was present in the glycosome. It was purified to homogeneity by a method involving hydrophobic interaction chromatography on Phenyl-Sepharose followed by ionic exchange on CM-Sepharose and affinity chromatography on Blue-Sepharose. The purified glycosomal MDH is a homodimeric protein with a subunit molecular mass of 37 kDa and it has a low substrate specificity, since it was able to reduce both aromatic and aliphatic alpha-ketoacids as substrate including oxaloacetate, phenyl pyruvate, alpha-keto iso-caproate and pyruvate. The apparent K(m)s for oxaloacetate and NADH were 166 and 270 microM, respectively and for L-malate and NAD+, 3000 and 246 microM, respectively. The basic isoform was present in the mitochondrion. It has a high substrate specificity and an apparent K(m) of 132 and 63 microM for oxaloacetate and NADH, respectively, and of 450 and 91 microM, respectively, with L-malate and NAD+.

Two Tropinone Reductases with Distinct Stereospecificities from Cultured Roots of Hyoscyamus niger1

PubMed Central

Hashimoto, Takashi; Nakajima, Keiji; Ongena, Godelieve; Yamada, Yasuyuki

1992-01-01

Tropinone is an alkamine intermediate at the branch point of biosynthetic pathways leading to various tropane alkaloids. Two stereospecifically distinct NADPH-dependent oxidoreductases, TR-I and TR-II, which, respectively, reduce tropinone to 3α-hydroxytropane (tropine) and 3β-hydroxytropane (ψ-tropine), were detected mainly in the root of tropane alkaloid-producing plants but not in nonproducing cultured root. Both reductases were purified to near homogeneity from cultured root of Hyoscyamus niger and characterized. The TR-I reaction was reversible, whereas the TR-II reaction was essentially irreversible, reduction of the ketone being highly favored over oxidation of the alcohol ψ-tropine. Marked differences were found between the two reductase in their affinities for tropinone substrate and in the effects of amino acid modification reagents. Some differences in substrate specificity were apparent. For example, N-propyl-4-piperidone was reduced by TR-II but not by TR-I. Conversely, 3-quinuclidinone and 8-thiabicyclo[3,2,1]octane-3-one were accepted as substrates by TR-I but hardly at all by TR-II. Both enzymes were shown to be class B oxidoreductases, which transfer the pro-S hydrogen of NAD(P)H to their substrates. Possible roles of these tropinone reductases in alkaloid biosynthesis are discussed. Images Figure 6 PMID:16653065

Recent advances in the in silico modelling of UDP glucuronosyltransferase substrates.

PubMed

Sorich, Michael J; Smith, Paul A; Miners, John O; Mackenzie, Peter I; McKinnon, Ross A

2008-01-01

UDP glucurononosyltransferases (UGT) are a superfamily of enzymes that catalyse the conjugation of a range of structurally diverse drugs, environmental and endogenous chemicals with glucuronic acid. This process plays a significant role in the clearance and detoxification of many chemicals. Over the last decade the regulation and substrate profiles of UGT isoforms have been increasingly characterised. The resulting data has facilitated the prototyping of ligand based in silico models capable of predicting, and gaining insights into, binding affinity and the substrate- and regio- selectivity of glucuronidation by UGT isoforms. Pharmacophore modelling has produced particularly insightful models and quantitative structure-activity relationships based on machine learning algorithms result in accurate predictions. Simple structural chemical descriptors were found to capture much of the chemical information relevant to UGT metabolism. However, quantum chemical properties of molecules and the nucleophilic atoms in the molecule can enhance both the predictivity and chemical intuitiveness of structure-activity models. Chemical diversity analysis of known substrates has shown some bias towards chemicals with aromatic and aliphatic hydroxyl groups. Future progress in in silico development will depend on larger and more diverse high quality metabolic datasets. Furthermore, improved protein structure data on UGTs will enable the application of structural modelling techniques likely leading to greater insight into the binding and reactive processes of UGT catalysed glucuronidation.

Remote Exosites of the Catalytic Domain of Matrix Metalloproteinase-12 Enhance Elastin Degradation┼

PubMed Central

Fulcher, Yan G.; Van Doren, Steven R.

2011-01-01

How does matrix metalloproteinase-12 (MMP-12 or metalloelastase) degrade elastin with high specific activity? NMR suggested soluble elastin to cover surfaces of MMP-12 far from its active site. Two of these surfaces have been found, by mutagenesis guided by the BINDSIght approach, to affect degradation and affinity for elastin substrates but not a small peptide substrate. Main exosite 1 has been extended out to Asp124 that binds calcium. Novel exosite 2 comprises residues from the II–III loop and β-strand I near the back of the catalytic domain. The high exposure of these distal exosites may make them accessible to elastin made more flexible by partial hydrolysis. Importantly, combination of a lesion at each of exosites 1 and 2 and active site decreased catalytic competence towards soluble elastin by 13- to 18-fold to the level of MMP-3, homologue and poor elastase. Double mutant cycle analysis of conservative mutations of Met156 (exosite 2) and either Asp124 (exosite 1) or Ile180 (active site) had additive effects. Compared to polar substitutions observed in other MMPs, Met156 enhanced affinity and Ile180 kcat for soluble elastin. Both residues detracted from the higher folding stability with polar mutations. This resembles the trend in enzymes of an inverse relationship between folding stability and activity. Restoring Asp124 from combination mutants enhanced kcat for soluble elastin. In elastin degradation, exosites 1 and 2 contributed independently of each other and Ile180 at the active site, but with partial coupling to Ala182 near the active site. The concept of weak, separated interactions coalescing somewhat independently can be extended to this proteolytic digestion of a protein from fibrils. PMID:21967233

Identification of Ftr1 and Zrt1 as iron and zinc micronutrient transceptors for activation of the PKA pathway in Saccharomyces cerevisiae

PubMed Central

Schothorst, Joep; Zeebroeck, Griet V.; Thevelein, Johan M.

2017-01-01

Multiple types of nutrient transceptors, membrane proteins that combine a transporter and receptor function, have now been established in a variety of organisms. However, so far all established transceptors utilize one of the macronutrients, glucose, amino acids, ammonium, nitrate, phosphate or sulfate, as substrate. This is also true for the Saccharomyces cerevisiae transceptors mediating activation of the PKA pathway upon re-addition of a macronutrient to glucose-repressed cells starved for that nutrient, re-establishing a fermentable growth medium. We now show that the yeast high-affinity iron transporter Ftr1 and high-affinity zinc transporter Zrt1 function as transceptors for the micronutrients iron and zinc. We show that replenishment of iron to iron-starved cells or zinc to zinc-starved cells triggers within 1-2 minutes a rapid surge in trehalase activity, a well-established PKA target. The activation with iron is dependent on Ftr1 and with zinc on Zrt1, and we show that it is independent of intracellular iron and zinc levels. Similar to the transceptors for macronutrients, Ftr1 and Zrt1 are strongly induced upon iron and zinc starvation, respectively, and they are rapidly downregulated by substrate-induced endocytosis. Our results suggest that transceptor-mediated signaling to the PKA pathway may occur in all cases where glucose-repressed yeast cells have been starved first for an essential nutrient, causing arrest of growth and low activity of the PKA pathway, and subsequently replenished with the lacking nutrient to re-establish a fermentable growth medium. The broadness of the phenomenon also makes it likely that nutrient transceptors use a common mechanism for signaling to the PKA pathway. PMID:28357393

Mechanistic insights into PEPT1-mediated transport of a novel antiepileptic, NP-647.

PubMed

Khomane, Kailas S; Nandekar, Prajwal P; Wahlang, Banrida; Bagul, Pravin; Shaikh, Naeem; Pawar, Yogesh B; Meena, Chhuttan Lal; Sangamwar, Abhay T; Jain, Rahul; Tikoo, K; Bansal, Arvind K

2012-09-04

The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 μM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.

Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex.

PubMed

Marcella, Aaron M; Culbertson, Sannie J; Shogren-Knaak, Michael A; Barb, Adam W

2017-11-24

The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a K D =62±13nM, followed by the binding of two more equivalents of holo-ACPP with K D =1.2±0.2μM. Cooperativity was not observed for apo-ACPP which bound with K D =2.4±0.1μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dye-ligand affinity systems.

PubMed

Denizli, A; Pişkin, E

2001-10-30

Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (often a mono- or dichlorotriazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consideration in dye-affinity systems. There are several methods for immobilization of dye molecules onto the support matrix, in which usually several intermediate steps are followed. Both the adsorption and elution steps should carefully be optimized/designed for a successful separation. Dye-affinity systems in the form of spherical sorbents or as affinity membranes have been used in protein separation.

Real-time Full-spectral Imaging and Affinity Measurements from 50 Microfluidic Channels using Nanohole Surface Plasmon Resonance†

PubMed Central

Lee, Si Hoon; Lindquist, Nathan C.; Wittenberg, Nathan J.; Jordan, Luke R.; Oh, Sang-Hyun

2012-01-01

With recent advances in high-throughput proteomics and systems biology, there is a growing demand for new instruments that can precisely quantify a wide range of receptor-ligand binding kinetics in a high-throughput fashion. Here we demonstrate a surface plasmon resonance (SPR) imaging spectroscopy instrument capable of extracting binding kinetics and affinities from 50 parallel microfluidic channels simultaneously. The instrument utilizes large-area (~cm2) metallic nanohole arrays as SPR sensing substrates and combines a broadband light source, a high-resolution imaging spectrometer and a low-noise CCD camera to extract spectral information from every channel in real time with a refractive index resolution of 7.7 × 10−6. To demonstrate the utility of our instrument for quantifying a wide range of biomolecular interactions, each parallel microfluidic channel is coated with a biomimetic supported lipid membrane containing ganglioside (GM1) receptors. The binding kinetics of cholera toxin b (CTX-b) to GM1 are then measured in a single experiment from 50 channels. By combining the highly parallel microfluidic device with large-area periodic nanohole array chips, our SPR imaging spectrometer system enables high-throughput, label-free, real-time SPR biosensing, and its full-spectral imaging capability combined with nanohole arrays could enable integration of SPR imaging with concurrent surface-enhanced Raman spectroscopy. PMID:22895607

A Peptide/MHCII conformer generated in the presence of exchange peptide is substrate for HLA-DM editing

PubMed Central

Ferrante, Andrea; Gorski, Jack

2012-01-01

The mechanism of HLA-DM (DM) activity is still unclear. We have shown that DM-mediated peptide release from HLA-DR (DR) is dependent on the presence of exchange peptide. However, DM also promotes a small amount of peptide release in the absence of exchange peptide. Here we show that SDS-PAGE separates purified peptide/DR1 complexes (pDR1) into two conformers whose ratio is peptide Kd-dependent. In the absence of exchange peptide, DM only releases peptide from the slower migrating conformer. Addition of exchange peptide converts the DM-resistant conformer to the slower migrating conformer, which is DM labile. Thus, exchange peptide generates a conformer of pDR1 which constitutes the intermediate for peptide exchange and the substrate for DM activity. The resolution of the intermediate favors the highest affinity peptide. However, once folded into the DM-resistant conformer, even low affinity peptides can be presented in the absence of free peptide, broadening the repertoire available for presentation. PMID:22545194

Structural and Molecular Mechanism for Autoprocessing of MARTX Toxin of Vibrio cholerae at Multiple Sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prochazkova, Katerina; Shuvalova, Ludmilla A.; Minasov, George

2009-10-05

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP{sub 6}). In this study, we demonstrated that InsP{sub 6} is not simply an allosteric cofactor, but rather binding of InsP{sub 6} stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-{angstrom} crystal structure of thismore » InsP{sub 6}-bound unprocessed form of CPD was determined and revealed the scissile bond Leu{sup 3428}-Ala{sup 3429} captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP{sub 6}, but was reactivated for high affinity binding of InsP{sub 6} by cooperative binding of both a new substrate and InsP{sub 6}. Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.« less

Suicide inactivation of hydroxylamine oxidoreductase of Nitrosomonas europaea by organohydrazines.

PubMed

Logan, M S; Hooper, A B

1995-07-18

In the presence of a suitable electron acceptor such as mammalian cytochrome c, hydroxylamine oxidoreductase (HAO) from the chemolithotrophic bacterium Nitrosomonas europaea catalyzes the oxidation of hydroxylamine or hydrazine to nitrite or dinitrogen, respectively. Each subunit of HAO contains 7 c-hemes and a chromophore of the active site called heme P460, a c-heme bridged from a methylene carbon to a ring carbon of a tyrosine of the peptide chain. Reaction with either substrate results in reduction of several c-hemes of HAO. The reaction of organohydrazines with HAO was investigated in this work. HAO was inactivated by (phenyl-, (methyl-, or (hydroxyethyl)hydrazine. The process followed first order kinetics and was inhibited by the substrates, hydroxylamine or hydrazine. Complete loss of enzyme activity and absorbancy characteristic of native heme P460 of HAO occurred at a 1:1 ratio of phenylhydrazine and HAO. HAO was covalently derivatized by two molecules of [14C]-phenylhydrazine per subunit. Heme P460 was derivatized with high affinity, and an amino acid residue was derivatized with lower affinity. c-Hemes were not derivatized except for the partial reaction of (hydroxyethyl)hydrazine with one heme. As with hydroxylamine and hydrazine, incubation with organohydrazines resulted in reduction of c-heme of HAO. Derivatized minus native optical difference spectra of ferric or ferrous HAO revealed changes in the optical properties of heme P460 which were generally similar to shifts seen in the reaction of the heme of other hemoproteins with organohydrazines.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylosuccinate synthetase: recent developments.

PubMed

Honzatko, R B; Stayton, M M; Fromm, H J

1999-01-01

By exerting strategic control on purine nucleotide biosynthesis, and by engaging GTP-dependent transphosphorylation of IMP to activate loss of an oxygen atom during catalysis, adenylosuccinate synthetase remains as enzyme that justifiably fascinates students of enzyme catalysis. This review describes how the balanced application of X-ray crystallography and enzyme kinetics has advanced the comprehension of the catalytic and regulatory properties of adenylosuccinate synthetase. Detailed analysis has demonstrated the formation of 6-phosphoryl-IMP, an intermediate originally postulated over 40 years ago on the basis of oxygen-18 exchange experiments showing that position-6 oxygen of IMP becomes incorporated into phosphate. Inferences about the participation of amino acid side-chains that stabilize 6-P-IMP during catalysis have also been confirmed by site-directed mutagenesis and examination of such mutations on various kinetic parameters. Moreover, the action of certain regulatory ligands have also been viewed at atomic level resolution. For example, magnesium ion and GDP can induce conformational changes linked to the stabilization of one of two known conformations of the so-called 40s loop. Another significant finding is that two magnesium ions play fundamental roles: one binding with high affinity to the substrate GTP, and a second binding with lower affinity to the co-substrate aspartate. These structural and kinetic studies have also formed the basis for clarifying the action of various inhibitors and potentially important pharmacologic agents with this key regulatory enzyme. Finally, this review explores the current status of investigations on gene structure and gene expression in a number of organisms.

Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerol-specific enzyme for citral biosynthesis.

PubMed

Tan, Cheng Seng; Hassan, Maizom; Mohamed Hussein, Zeti Azura; Ismail, Ismanizan; Ho, Kok Lian; Ng, Chyan Leong; Zainal, Zamri

2018-02-01

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp 280 , Leu 294 and Ala 303 ). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces.

PubMed

Hakami, Abdulrahim R; Ball, Jonathan K; Tarr, Alexander W

2015-09-01

Phage-displayed random peptide libraries are widely used for identifying peptide interactions with proteins and other substrates. Selection of peptide ligands involves iterative rounds of affinity enrichment. The binding properties of the selected phage clones are routinely tested using immunoassay after propagation to high titre in a bacterial host and precipitation using polyethylene glycol (PEG) and high salt concentration. These immunoassays can suffer from low sensitivity and high background signals. Polysorbate 20 (Tween(®) 20) is a non-ionic detergent commonly used in immunoassay washing buffers to reduce non-specific binding, and is also used as a blocking reagent. We have observed that Tween 20 enhances non-specific M13 library phage binding in a peptide-independent manner. Other non-ionic detergents were also found to promote significant, dose-dependent non-specific phage binding in ELISA. This effect was not observed for assays using phage concentrated by ultracentrifugation, suggesting that interactions occur between detergents and the PEG-precipitated phage, irrespective of the displayed peptide motif. This artefact may impact on successful affinity selection of peptides from phage-display libraries. We propose alternative methods for screening phage libraries for identifying binding interactions with target ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification and characterization of a gene encoding for a nucleotidase from Phaseolus vulgaris.

PubMed

Cabello-Díaz, Juan Miguel; Gálvez-Valdivieso, Gregorio; Caballo, Cristina; Lambert, Rocío; Quiles, Francisco Antonio; Pineda, Manuel; Piedras, Pedro

2015-08-01

Nucleotidases are phosphatases that catalyze the removal of phosphate from nucleotides, compounds with an important role in plant metabolism. A phosphatase enzyme, with high affinity for nucleotides monophosphate previously identified and purified in embryonic axes from French bean, has been analyzed by MALDI TOF/TOF and two internal peptides have been obtained. The information of these peptide sequences has been used to search in the genome database and only a candidate gene that encodes for the phosphatase was identified (PvNTD1). The putative protein contains the conserved domains (motif I-IV) for haloacid dehalogenase-like hydrolases superfamily. The residues involved in the catalytic activity are also conserved. A recombinant protein overexpressed in Escherichia coli has shown molybdate resistant phosphatase activity with nucleosides monophosphate as substrate, confirming that the identified gene encodes for the phosphatase with high affinity for nucleotides purified in French bean embryonic axes. The activity of the purified protein was inhibited by adenosine. The expression of PvNTD1 gene was induced at the specific moment of radicle protrusion in embryonic axes. The gene was also highly expressed in young leaves whereas the level of expression in mature tissues was minimal. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

Characterization of the three different states of the cholecystokinin (CCK) receptor in pancreatic acini.

PubMed

Talkad, V D; Patto, R J; Metz, D C; Turner, R J; Fortune, K P; Bhat, S T; Gardner, J D

1994-10-20

By measuring binding of [125I]CCK-8 and [3H]L-364,718 to rat pancreatic acini we demonstrated directly that the pancreatic CCK receptor can exist in three different affinity states with respect to CCK--high affinity, low affinity and very low affinity. Binding of [125I]CCK-8 reflects interaction of the tracer with the high and low affinity states, whereas binding of [3H]L-364,718 reflects interaction of the tracer with the low and very low affinity states. Treating acini with carbachol abolished the high affinity state of the CCK receptor and converted approximately 25% of the low affinity receptors to the very low affinity state. Carbachol treatment was particularly useful in establishing the values of Kd for the high and low affinity states for different CCK receptor agonists and antagonists. Of the various CCK receptor agonists tested, CCK-8 had the highest affinity for the high affinity state (Kd approximately 1 nM), whereas CCK-JMV-180 had the highest affinity for the low (Kd 7 nM) and very low affinity (Kd 200 nM) states. Gastrin and de(SO4)CCK-8 had affinities for the high and low affinity states of the receptor that were 100- to 400-fold less than those of CCK-8 but had affinities for the very low affinity state that were only 3- to 10-fold less than that of CCK-8. CCK receptor antagonists showed several patterns in interacting with the different states of the CCK receptor. L-364,718 had the same affinity for each state of the CCK receptor. CR1409 and Bt2cGMP each had similar affinities for the high and low affinity states and lower affinity for the very low affinity state. L-365,260 and CCK-JMV-179 had the highest affinity for the low affinity state and lower affinities for the high and very low affinity states. Different CCK receptor agonists caused the same maximal stimulation of amylase secretion but showed different degrees of amplification in terms of the relationship between their abilities to stimulate amylase secretion and their abilities to occupy the low affinity state of the CCK receptor. When amplification was expressed quantitatively as the value of Kd for the low affinity state divided by the corresponding EC50 for stimulating amylase secretion the values were CCK-8 (1000), de(SO)CCK-8 (1500), gastrin (100) and CCK-JMV-180 (Menozzi, D., Vinayek, R., Jensen, R.T. and Gardner, J.D. (1991) J. Biol. Chem. 266, 10385-1091).(ABSTRACT TRUNCATED AT 400 WORDS)

Wetting and adhesion evaluation of cosmetic ingredients and products: correlation of in vitro-in vivo contact angle measurements.

PubMed

Capra, P; Musitelli, G; Perugini, P

2017-08-01

The aim of this work was to use the contact angle measurement in order to predict the behaviour of ingredients and finished cosmetic products on skin to improve skin feel and product texture. Different classes of cosmetic ingredients and formulations were evaluated. The contact angle measurements were carried out by the sessile drop method using an apparatus, designed and set up in laboratory. Glass, Teflon and human skin were the reference substrates. In a preliminary phase, TEWL parameter, sebum content and hydration of human skin were measured to set up method. Data demonstrated that glass substrate may be used as replacement of the skin:critical surface tension of skin and glass were about of 27 and 31 dyne cm -1 , respectively. Non-ionic surfactant with increasing HLB was evaluated: a correlation between contact angle measured and HLB was not observed because of different and complex molecular structure. In detail, ethylhexyl hydroxystearate (θ glass = 17.1°) showed lower contact angle value with respect to Polysorbate 20 (θ glass = 28.1°). Sodium laureth sulphate and stearalkonium chloride were also evaluated: anionic molecule showed more affinity for glass with respect to Teflon (θ glass = 21.7° and θ Teflon = 52.3°). Lipids and silicones showed different affinity for substrate according to hydrophilic groups and hydrocarbon chain: contact angles of silicones remained unchanged independently from substrate. Finished cosmetic products (O/W, W/O emulsions, cleansing oil, dry skin oil) showed different profiles according to surfactant and its affinity for continuous phase of the formulation. Comparing the values of the contact angle on skin of non-ionic surfactants, as ethylhexyl hydroxystearate and Polysorbate 20, they showed values lower (near to zero) than ones of sodium laureth sulphate and Stearalkonium Chloride (21.7° and 66.8°, respectively). Finally, finished cosmetic products tested on human skin showed different profile: corresponded contact angle values were less than 20°. The product tended to be quickly adsorbed on human skin. Systematic study carried out by evaluating the wettability of single cosmetic ingredients on different substrates allowed to find correlations between the use of certain ingredients and the final performance of a cosmetic product. © 2017 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

The “gating” residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition

PubMed Central

Milczek, Erika M.; Binda, Claudia; Rovida, Stefano; Mattevi, Andrea; Edmondson, Dale E.

2011-01-01

Summary The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 Å3 volume and MAO B contains a dipartite cavity structure with volumes of ~290 Å3 (entrance cavity) and ~400 Å3 (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared to WT enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100–103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine KM but unaltered kcat values. The altered KM values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to WT enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provides insights into specific reversible inhibitor design for these membrane-bound enzymes. PMID:21978362

Structural and Affinity Determinants in the Interaction between Alcohol Acyltransferase from F. x ananassa and Several Alcohol Substrates: A Computational Study

PubMed Central

Herrera, Raúl; Caballero, Julio; Alzate-Morales, Jans H.

2016-01-01

Aroma and flavor are important factors of fruit quality and consumer preference. The specific pattern of aroma is generated during ripening by the accumulation of volatiles compounds, which are mainly esters. Alcohol acyltransferase (AAT) (EC 2.3.1.84) catalyzes the esterification reaction of aliphatic and aromatic alcohols and acyl-CoA into esters in fruits and flowers. In Fragaria x ananassa, there are different volatiles compounds that are obtained from different alcohol precursors, where octanol and hexanol are the most abundant during fruit ripening. At present, there is not structural evidence about the mechanism used by the AAT to synthesize esters. Experimental data attribute the kinetic role of this enzyme to 2 amino acidic residues in a highly conserved motif (HXXXD) that is located in the middle of the protein. With the aim to understand the molecular and energetic aspects of volatiles compound production from F. x ananassa, we first studied the binding modes of a series of alcohols, and also different acyl-CoA substrates, in a molecular model of alcohol acyltransferase from Fragaria x ananassa (SAAT) using molecular docking. Afterwards, the dynamical behavior of both substrates, docked within the SAAT binding site, was studied using routine molecular dynamics (MD) simulations. In addition, in order to correlate the experimental and theoretical data obtained in our laboratories, binding free energy calculations were performed; which previous results suggested that octanol, followed by hexanol, presented the best affinity for SAAT. Finally, and concerning the SAAT molecular reaction mechanism, it is suggested from molecular dynamics simulations that the reaction mechanism may proceed through the formation of a ternary complex, in where the Histidine residue at the HXXXD motif deprotonates the alcohol substrates. Then, a nucleophilic attack occurs from alcohol charged oxygen atom to the carbon atom at carbonyl group of the acyl CoA. This mechanism is in agreement with previous results, obtained in our group, in alcohol acyltransferase from Vasconcellea pubescens (VpAAT1). PMID:27078149

Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: cholesterol acyltransferase.

PubMed

Barter, P J; Hopkins, G J; Gorjatschko, L

1984-01-17

A recent observation that lecithin: cholesterol acyltransferase (EC 2.3.1.43) interacts with both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in human plasma is in apparent conflict with an earlier finding that the purified enzyme, while highly reactive with isolated HDL, was only minimally reactive with LDL. There is evidence, however, that lecithin: cholesterol acyltransferase may exist physiologically as a component of a complex with other proteins and that studies with the isolated enzyme may therefore provide misleading results. Consequently, interactions of the enzyme with isolated human lipoproteins have been re-examined in incubations containing lecithin: cholesterol acyltransferase as a component of human lipoprotein-free plasma in which a physiologically active complex of the enzyme with other proteins may have been preserved. In this system there was a ready esterification of the free cholesterol associated with both LDL and HDL-subfraction 3 (HDL3) in reactions that obeyed typical enzyme-saturation kinetics. For a given preparation of lipoprotein-free plasma the Vmax values with LDL and with HDL3 were virtually identical. The apparent Km for free cholesterol associated with HDL3 was 5.6 X 10(-5) M, while for that associated with LDL it was 4.1 X 10(-4) M. This implied that, in terms of free cholesterol concentration, the affinity of HDL3 for lecithin: cholesterol acyltransferase was about 7-times greater than that of LDL. When expressed in terms of lipoprotein particle concentration, however, it was apparent that the affinity of LDL for the enzyme was considerably greater than that of HDL3. When the lipoprotein fractions were equated in terms of lipoprotein surface area, the apparent affinities of the two fractions for the enzyme were found to be comparable.

Stoichiometry and pH dependence of the rabbit proton-dependent oligopeptide transporter PepT1.

PubMed

Steel, A; Nussberger, S; Romero, M F; Boron, W F; Boyd, C A; Hediger, M A

1997-02-01

1. The intestinal H(+)-coupled peptide transporter PepT1, displays a broad substrate specificity and accepts most charged and neutral di- and tripeptides. To study the proton-to-peptide stoichiometry and the dependence of the kinetic parameters on extracellular pH (pHo), rabbit PepT1 was expressed in Xenopus laevis oocytes and used for uptake studies of radiolabelled neutral and charged dipeptides, voltage-clamp analysis and intracellular pH measurements. 2. PepT1 did not display the substrate-gated anion conductances that have been found to be characteristic of members of the Na(+)- and H(+)-coupled high-affinity glutamate transporter family. In conjunction with previous data on the ion dependence of PepT1, it can therefore be concluded that peptide-evoked charge fluxes of PepT1 are entirely due to H+ movement. 3. Neutral, acidic and basic dipeptides induced intracellular acidification. The rate of acidification, the initial rates of the uptake of radiolabelled peptides and the associated charge fluxes gave proton-substrate coupling ratios of 1:1, 2:1 and 1:1 for neutral, acidic and basic dipeptides, respectively. 4. Maximal transport of the neutral and charged dipeptides Gly-Leu, Gly-Glu, Gly-Lys and Ala-Lys occurred at pHo 5.5, 5.2, 6.2 and 5.8, respectively. The Imax values were relatively pHo independent but the apparent affinity (Km(app) values for these peptides were shown to be highly pHo dependent. 5. Our data show that at physiological pH (pHo 5.5-6.0) PepT1 prefers neutral and acidic peptides. The shift in transport maximum for the acidic peptide Gly-Glu to a lower pH value suggests that acidic dipeptides are transported in the protonated form. The shift in the transport maxima of the basic dipeptides to higher pH values may involve titration of a side-chain on the transporter molecule (e.g. protonation of a histidine group). These considerations have led us to propose a model for coupled transport of neutral, acidic and basic dipeptides.

Characterization of new recombinant 3-ketosteroid-Δ1-dehydrogenases for the biotransformation of steroids.

PubMed

Wang, Xiaojun; Feng, Jinhui; Zhang, Dalong; Wu, Qiaqing; Zhu, Dunming; Ma, Yanhe

2017-08-01

3-Ketosteroid-Δ 1 -dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ 1 -dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, we cloned 12 putative KstD-encoding (kstd) genes from both fungal and Gram-positive microorganisms and attempted to overproduce the recombinant proteins in E. coli BL21(DE3). Five successful recombinant enzymes catalyzed the Δ 1 -desaturation of a variety of steroidal compounds such as 4-androstene-3,17-dione (AD), 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), hydrocortisone, cortisone, and cortexolone. However, the substrate specificity and catalytic efficiency of the enzymes differ depending on their sources. The purified KstD from Mycobacterium smegmatis mc 2 155 (MsKstD1) displayed high catalytic efficiency toward hydrocortisone, progesterone, and 9-OH-AD, where it had the highest affinity (K m 36.9 ± 4.6 μM) toward 9-OH-AD. On the other hand, the KstD from Rhodococcus erythropolis WY 1406 (ReKstD) exhibited high catalytic efficiency toward androst-4,9(11)-diene-3,17-dione (Diene), 21-acetoxy-pregna-4,9(11),16-triene-3,20-dione (Triene), and cortexolone, where in all three cases the K m values (12.3 to 17.8 μM) were 2.5-4-fold lower than that toward hydrocortisone (46.3 μM). For both enzymes, AD was a good substrate although ReKstD had a 3-fold higher affinity than MsKstD1. Reaction conditions were optimized for the biotransformation of AD or hydrocortisone in terms of pH, temperature, and effects of hydrogen peroxide, solvent, and electron acceptor. For the biotransformation of hydrocortisone with 20 g/L wet resting E. coli cells harboring MsKstD1 enzyme, the yield of prednisolone was about 90% within 3 h at the substrate concentration of 6 g/L, demonstrating the application potential of the newly cloned KstDs.

Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein lipase and hepatic lipase: Implications for the mechanisms of lipase action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deckelbaum, R.J.; Hamilton, J.A.; Butbul, E.

1990-02-06

To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, the authors compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of ({sup 13}C)carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T{sub 1}, were also shorter for trioctanoin, showing greater mobility formore » MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, they found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. These results suggest that despite lower enzyme affinity for MCT emulsions, shorter chain triacylglycerols are more readily hydrolyzed by lipoprotein and hepatic lipases than long-chain triacylglycerols because of greater MCT solubility and mobility at the emulsion-water interface.« less

Disruption of Chemoreceptor Signaling Arrays by High Levels of CheW, the Receptor-Kinase Coupling Protein

PubMed Central

Cardozo, Marcos J.; Massazza, Diego A.; Parkinson, John S.; Studdert, Claudia A.

2017-01-01

Summary During chemotactic signaling by Escherichia coli, the small cytoplasmic CheW protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signaling complexes, CheW in stoichiometric excess disrupts chemotactic behavior. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability, and elevated receptor methylation levels. The severity of these effects paralleled the receptor binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signaling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signaling. PMID:20487303

Integrity of N- and C-termini is important for E. coli Hsp31 chaperone activity

PubMed Central

Sastry, M S R; Zhou, Weibin; Baneyx, François

2009-01-01

Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging. PMID:19517531

Purification and high-resolution top-down mass spectrometric characterization of human salivary α-amylase.

PubMed

Peng, Ying; Chen, Xin; Sato, Takuya; Rankin, Scott A; Tsuji, Ryohei F; Ge, Ying

2012-04-03

Human salivary α-amylase (HSAMY) is a major component of salivary secretions, possessing multiple important biological functions. Here we have established three methods to purify HSAMY in human saliva for comprehensive characterization of HSAMY by high-resolution top-down mass spectrometry (MS). Among the three purification methods, the affinity method based on the enzyme-substrate specific interaction between amylase and glycogen is preferred, providing the highest purity HSAMY with high reproducibility. Subsequently, we employed Fourier transform ion cyclotron resonance MS to analyze the purified HSAMY. The predominant form of α-amylase purified from saliva of various races and genders is nonglycosylated with the same molecular weight of 55,881.2, which is 1885.8 lower than the calculated value based on the DNA-predicted sequence. High-resolution MS revealed the truncation of the first 15 N-terminal amino acids (-1858.96) and the subsequent formation of pyroglutamic acid at the new N-terminus Gln (-17.03). More importantly, five disulfide bonds in HSAMY were identified (-10.08) and effectively localized by tandem MS in conjunction with complete and partial reduction by tris (2-carboxyethyl) phosphine. Overall, this study demonstrates that top-down MS combined with affinity purification and partial reduction is a powerful method for rapid purification and complete characterization of large proteins with complex and overlapping disulfide bond patterns.

Carbon-containing cathodes for enhanced electron emission

DOEpatents

Cao, Renyu; Pan, Lawrence; Vergara, German; Fox, Ciaran

2000-01-01

A cathode has electropositive atoms directly bonded to a carbon-containing substrate. Preferably, the substrate comprises diamond or diamond-like (sp.sup.3) carbon, and the electropositive atoms are Cs. The cathode displays superior efficiency and durability. In one embodiment, the cathode has a negative electron affinity (NEA). The cathode can be used for field emission, thermionic emission, or photoemission. Upon exposure to air or oxygen, the cathode performance can be restored by annealing or other methods. Applications include detectors, electron multipliers, sensors, imaging systems, and displays, particularly flat panel displays.

Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases.

PubMed

Viigand, Katrin; Visnapuu, Triinu; Mardo, Karin; Aasamets, Anneli; Alamäe, Tiina

2016-08-01

Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α-methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α-glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an α-glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose-like substrates (α-1,4-glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate-binding pocket of MAL1 has three subsites (-1, +1 and +2) and that binding is strongest at the -1 subsite. The DSF assay results were in good accordance with affinity (Km ) and inhibition (Ki ) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α-glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd.

Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics.

PubMed

Bertrand, Thomas; Jolivalt, Claude; Briozzo, Pierre; Caminade, Eliane; Joly, Nathalie; Madzak, Catherine; Mougin, Christian

2002-06-11

Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.

Interaction of environmental contaminants with zebrafish organic anion transporting polypeptide, Oatp1d1 (Slco1d1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popovic, Marta; Zaja, Roko; Fent, Karl

Polyspecific transporters from the organic anion transporting polypeptide (OATP/Oatp) superfamily mediate the uptake of a wide range of compounds. In zebrafish, Oatp1d1 transports conjugated steroid hormones and cortisol. It is predominantly expressed in the liver, brain and testes. In this study we have characterized the transport of xenobiotics by the zebrafish Oatp1d1 transporter. We developed a novel assay for assessing Oatp1d1 interactors using the fluorescent probe Lucifer yellow and transient transfection in HEK293 cells. Our data showed that numerous environmental contaminants interact with zebrafish Oatp1d1. Oatp1d1 mediated the transport of diclofenac with very high affinity, followed by high affinity towardsmore » perfluorooctanesulfonic acid (PFOS), nonylphenol, gemfibrozil and 17α-ethinylestradiol; moderate affinity towards carbaryl, diazinon and caffeine; and low affinity towards metolachlor. Importantly, many environmental chemicals acted as strong inhibitors of Oatp1d1. A strong inhibition of Oatp1d1 transport activity was found by perfluorooctanoic acid (PFOA), chlorpyrifos-methyl, estrone (E1) and 17β-estradiol (E2), followed by moderate to low inhibition by diethyl phthalate, bisphenol A, 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4 tetrahydronapthalene and clofibrate. In this study we identified Oatp1d1 as a first Solute Carrier (SLC) transporter involved in the transport of a wide range of xenobiotics in fish. Considering that Oatps in zebrafish have not been characterized before, our work on zebrafish Oatp1d1 offers important new insights on the understanding of uptake processes of environmental contaminants, and contributes to the better characterization of zebrafish as a model species. - Highlights: • We optimized a novel assay for determination of Oatp1d1 interactors • Oatp1d1 is the first SLC characterized fish xenobiotic transporter • PFOS, nonylphenol, diclofenac, EE2, caffeine are high affinity Oatp1d1substrates • PFOA, chlorpyrifos-methyl, E1, E2 are strong inhibitors of Oatp1d1 • PFOA and diclofenac can block Oatp1d1 binding of DHEAS, E3S and E17ß-glucuronide.« less

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis *

PubMed Central

Schiebel, Johannes; Kapilashrami, Kanishk; Fekete, Agnes; Bommineni, Gopal R.; Schaefer, Christin M.; Mueller, Martin J.; Tonge, Peter J.; Kisker, Caroline

2013-01-01

The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60–90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C38–42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors. PMID:24108128

Utilization of Nitrophenylphosphates and Oxime-Based Ligation for the Development of Nanomolar Affinity Inhibitors of the Yersinia pestis Outer Protein H (YopH) Phosphatase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bahta, Medhanit; Lountos, George T.; Dyas, Beverly

Our current study reports the first K{sub M} optimization of a library of nitrophenylphosphate-containing substrates for generating an inhibitor lead against the Yersinia pestis outer protein phosphatase (YopH). A high activity substrate identified by this method (K{sub M} = 80 {micro}M) was converted from a substrate into an inhibitor by replacement of its phosphate group with difluoromethylphosphonic acid and by attachment of an aminooxy handle for further structural optimization by oxime ligation. A cocrystal structure of this aminooxy-containing platform in complex with YopH allowed the identification of a conserved water molecule proximal to the aminooxy group that was subsequently employedmore » for the design of furanyl-based oxime derivatives. By this process, a potent (IC{sub 50} = 190 nM) and nonpromiscuous inhibitor was developed with good YopH selectivity relative to a panel of phosphatases. The inhibitor showed significant inhibition of intracellular Y. pestis replication at a noncytotoxic concentration. The current work presents general approaches to PTP inhibitor development that may be useful beyond YopH.« less

Introduction of a specific binding domain on myoglobin surface by new chemical modification.

PubMed

Hayashi, T; Ando, T; Matsuda, T; Yonemura, H; Yamada, S; Hisaeda, Y

2000-11-01

A new myoglobin, reconstituted with a modified zinc protoporphyrin, having a total of four ammonium groups at the terminal of the two propionate side chains was constructed to introduce a substrate binding site. The protein with a positively charged patch on the surface formed a stable complex with negatively charged substrates, such as hexacyanoferrate(III) and anthraquinonesulfonate via an electrostatic interaction. The complexation was monitored by fluorescence quenching due to singlet electron transfer from the photoexcited reconstituted zinc myoglobin to the substrates. The binding properties were evaluated by Stern-Volmer plots from the fluorescence quenching of the zinc myoglobin by a quencher. Particularly, anthraquinone-2,7-disulfonic acid showed a high affinity with a binding constant of 1.5 x 10(5) M(-1) in 10 mM phosphate buffer, pH 7.0. In contrast, the plots upon the addition of anthraquinone-2-sulfonic acid at different ionic strengths indicated that the complex was formed not only by an electrostatic interaction but also by a hydrophobic contact. The findings from the fluorescence studies conclude that the present system is a useful model for discussion of electron transfer via non-covalently linked donor-acceptor pairing on the protein surface.

Hydrolase and fructosyltransferase activities implicated in the accumulation of different chain size fructans in three Asteraceae species.

PubMed

Itaya, Nair M; Asega, Amanda F; Carvalho, Maria Angela M; Figueiredo-Ribeiro, Rita de Cássia L

2007-09-01

Fructans are widely distributed in Asteraceae from floras with seasonal growth and are thought to be involved in drought and freezing tolerance, in addition to storage function. Reserve organs of Vernonia herbacea and Viguiera discolor, from the cerrado, and of the perennial herb Smallanthus sonchifolius, endemic to Andean region, store over 80% inulin, with different DP (35, 150, and 15, respectively). The fructan pattern in Asteraceae species could be explained by characteristics of their respective 1-FFTs. Hydrolases and fructosyltransferases from S. sonchifolius, V. herbacea and V. discolor were analyzed in plants at the same environmental conditions. The higher 1-FEH activities found in the species with lower DP, S. sonchifolius and V. herbacea reinforce the hypothesis of the involvement of 1-FEH in fructan profile and suggest that the high DP fructan of V. discolor is a consequence of the low affinity of its 1-FEH to the native long chain inulin. Long term incubation with sucrose suggested that the affinity of 1-FFT of V. discolor for 1-kestose is low when compared to that of V. herbacea. Indeed 1-FFT from V. discolor was shown to be an hDP 1-FFT, preferring longer inulins as acceptors. Conversely, 1-FFT from V. herbacea seems to have a higher affinity for short fructo-oligosaccharides, including 1-kestose, as acceptor substrates. Differences in fructan enzymes of the three Asteraceae provide new information towards the understanding of fructan metabolism and control of carbon flow between low and high DP fructans.

Adhesion between a rutile surface and a polyimide: a coarse grained molecular dynamics study

NASA Astrophysics Data System (ADS)

Kumar, Arun; Sudarkodi, V.; Parandekar, Priya V.; Sinha, Nishant K.; Prakash, Om; Nair, Nisanth N.; Basu, Sumit

2018-04-01

Titanium, due to its high strength to weight ratio and polyimides, due to their excellent thermal stability are being increasingly used in aerospace applications. We investigate the bonding between a (110) rutile substrate and a popular commercial polyimide, PMR-15, starting from the known atomic structure of the rutile substrate and the architecture of the polymer. First, the long PMR-15 molecule is divided into four fragments and an all-atom non-bonded forcefield governing the interaction between PMR-15 and a rutile substrate is developed. To this end, parameters of Buckingham potential for interaction between each atom in the fragments and the rutile surface are fitted, so as to ensure that the sum of non-bonded and electrostatic interaction energy between the substrate and a large number of configurations of each fragment, calculated by the quantum mechanical route and obtained from the fitted potential, is closely matched. Further, two coarse grained models of PMR-15 are developed—one for interaction between two coarse grained PMR-15 molecules and another for that between a coarse grained PMR-15 and the rutile substrate. Molecular dynamics simulations with the coarse grained models yields a traction separation law—a very useful tool for conducting continuum level finite element simulations of rutile-PMR-15 joints—governing the normal separation of a PMR-15 block from a flat rutile substrate. Moreover, detailed information about the affinity of various fragments to the substrate are also obtained. In fact, though the separation energy between rutile and PMR-15 turns out to be rather low, our analysis—with merely the molecular architecture of the polyimide as the starting point—provides a scheme for in-silico prediction of adhesion energies for new polyimide formulations.

Direct and selective immobilization of proteins by means of an inorganic material-binding peptide: discussion on functionalization in the elongation to material-binding peptide.

PubMed

Yokoo, Nozomi; Togashi, Takanari; Umetsu, Mitsuo; Tsumoto, Kouhei; Hattori, Takamitsu; Nakanishi, Takeshi; Ohara, Satoshi; Takami, Seiichi; Naka, Takashi; Abe, Hiroya; Kumagai, Izumi; Adschiri, Tadafumi

2010-01-14

Using an artificial peptide library, we have identified a peptide with affinity for ZnO materials that could be used to selectively accumulate ZnO particles on polypropylene-gold plates. In this study, we fused recombinant green fluorescent protein (GFP) with this ZnO-binding peptide (ZnOBP) and then selectively immobilized the fused protein on ZnO particles. We determined an appropriate condition for selective immobilization of recombinant GFP, and the ZnO-binding function of ZnOBP-fused GFP was examined by elongating the ZnOBP tag from a single amino acid to the intact sequence. The fusion of ZnOBP with GFP enabled specific adsorption of GFP on ZnO substrates in an appropriate solution, and thermodynamic studies showed a predominantly enthalpy-dependent electrostatic interaction between ZnOBP and the ZnO surface. The ZnOBP's binding affinity for the ZnO surface increased first in terms of material selectivity and then in terms of high affinity as the GFP-fused peptide was elongated from a single amino acid to intact ZnOBP. We concluded that the enthalpy-dependent interaction between ZnOBP and ZnO was influenced by the presence of not only charged amino acids but also their surrounding residues in the ZnOBP sequence.

The actions of some esters of 4-hydroxyquinuclidine on guinea-pig ileum, atria and rat fundus strip.

PubMed

Barlow, R B; Kitchen, R

1982-11-01

1 The acetyl, phenylacetyl, and diphenylacetyl esters of 4-hydroxyquinuclidine and their methiodides have been prepared.2 4-Diphenylacetoxyquinuclidine methiodide has higher affinity for muscarinic receptors than 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP methiodide) but it is less selective. At 30 degrees C its affinity for receptors in ileum is about 5 times that for receptors in atria, a difference similar to that found with diphenylacetoxytrophine methiodide. With 4-DAMP methiodide affinity for receptors in the ileum is over 10 times that for receptors in atria.3 4-Diphenylacetoxyquinuclidine methiodide has higher affinity for muscarinic receptors than 3-diphenylacetoxyquinuclidine hydrochloride or its methiodide.4 4-Acetoxyquinuclidine hydrochloride has less than one-hundredth of the activity of 3-acetoxyquinuclidine hydrochloride (acecyclidine) on guinea-pig ileum, atria, and rat fundus: however, 4-acetoxyquinuclidine methiodide is consistently more active than its 3-isomer, though it is only about 1/25 times as active as acecyclidine.5 4-Acetoxyquinuclidine hydrochloride is only a poor substrate for electric eel acetylcholinesterase: its affinity is similar to that of acecyclidine but it is greatly reduced by methylation.6 The relations between the structure and activity of the agonists are very different from the relations between the structure and affinity of the antagonists, which supports the view that agonists and antagonists bind to different conformations of the muscarinic receptor.

Potent Mechanism-Based Inactivation Of Cytochrome P450 2B4 By 9-Ethynylphenanthrene: Implications For Allosteric Modulation Of Cytochrome P450 Catalysis1

PubMed Central

Zhang, Haoming; Gay, Sean C.; Shah, Manish; Foroozesh, Maryam; Liu, Jiawang; Osawa, Yoichi; Zhang, Qinghai; Stout, C. David; Halpert, James R.; Hollenberg, Paul F.

2013-01-01

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and kinact are 0.2 and 0.25 min−1, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and S50 of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by ESI-LC/MS, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to the Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206 leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low affinity site being the catalytic site and the high affinity site on the protein periphery. Computer-aided docking and MD simulations with one or two ligands bound revealed that the high affinity site is situated at the entrance of a substrate access channel surrounded by the F’ helix, β1/β2 loop and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302. PMID:23276288

Carbon Source Preference in Chemosynthetic Hot Spring Communities

PubMed Central

Urschel, Matthew R.; Kubo, Michael D.; Hoehler, Tori M.; Peters, John W.

2015-01-01

Rates of dissolved inorganic carbon (DIC), formate, and acetate mineralization and/or assimilation were determined in 13 high-temperature (>73°C) hot springs in Yellowstone National Park (YNP), Wyoming, in order to evaluate the relative importance of these substrates in supporting microbial metabolism. While 9 of the hot spring communities exhibited rates of DIC assimilation that were greater than those of formate and acetate assimilation, 2 exhibited rates of formate and/or acetate assimilation that exceeded those of DIC assimilation. Overall rates of DIC, formate, and acetate mineralization and assimilation were positively correlated with spring pH but showed little correlation with temperature. Communities sampled from hot springs with similar geochemistries generally exhibited similar rates of substrate transformation, as well as similar community compositions, as revealed by 16S rRNA gene-tagged sequencing. Amendment of microcosms with small (micromolar) amounts of formate suppressed DIC assimilation in short-term (<45-min) incubations, despite the presence of native DIC concentrations that exceeded those of added formate by 2 to 3 orders of magnitude. The concentration of added formate required to suppress DIC assimilation was similar to the affinity constant (Km) for formate transformation, as determined by community kinetic assays. These results suggest that dominant chemoautotrophs in high-temperature communities are facultatively autotrophic or mixotrophic, are adapted to fluctuating nutrient availabilities, and are capable of taking advantage of energy-rich organic substrates when they become available. PMID:25819970

Wrinkling and Folding on Patched Elastic Surfaces: Modulation of the Chemistry and Pattern Size of Microwrinkled Surfaces.

PubMed

Nogales, Aurora; Del Campo, Adolfo; Ezquerra, Tiberio A; Rodriguez-Hernández, Juan

2017-06-14

An unconventional strategy is proposed that takes advantage of localized high-deformation areas, referred to as folded wrinkles, to produce microstructured elastic surfaces with precisely controlled pattern dimensions and chemical distribution. For that purpose, elastic PDMS substrates were prestretched to a different extent and oxidized in particular areas using a mask. When the stretching was removed, the PDMS substrate exhibited out-of-plane deformations that largely depend on the applied prestretching. Prestretchings below 100% lead to affine deformations in which the treated areas are buckled. On the contrary, prestretchings above ε >100% prior to surface treatment induce the formation of folded wrinkles on those micrometer-size ultraviolet-ozone (UVO) treated areas upon relaxation. As a result, dual periodic wrinkles were formed due to the alternation of highly deformed (folded) and low deformed (buckled) areas. Our strategy is based on the surface treatment at precise positions upon prestretching of the elastic substrate (PDMS). Additionally, this approach can be used to template the formation of wrinkled surfaces by alternating lines of folded wrinkles (valleys) and low-deformed areas (hills). This effect allowed us to precisely tune the shape and distribution of the UVO exposed areas by varying the prestretching direction. Moreover, the wrinkle characteristics, including period and amplitude, exhibit a direct relation to the dimensions of the patterns present in the mask.

Productive Recognition of Factor IX by Factor XIa Exosites Requires Disulfide Linkage between Heavy and Light Chains of Factor XIa*

PubMed Central

Marcinkiewicz, Mariola M.; Sinha, Dipali; Walsh, Peter N.

2012-01-01

In the intrinsic pathway of blood coagulation factor XIa (FXIa) activates factor IX (FIX) by cleaving the zymogen at Arg145-Ala146 and Arg180-Val181 bonds releasing an 11-kDa activation peptide. FXIa and its isolated light chain (FXIa-LC) cleave S-2366 at comparable rates, but FXIa-LC is a very poor activator of FIX, possibly because FIX undergoes allosteric modification on binding to an exosite on the heavy chain of FXIa (FXIa-HC) required for optimal cleavage rates of the two scissile bonds of FIX. However preincubation of FIX with a saturating concentration of isolated FXIa-HC did not result in any potentiation in the rate of FIX cleavage by FXIa-LC. Furthermore, if FIX binding via the heavy chain exosite of FXIa determines the affinity of the enzyme-substrate interaction, then the isolated FXIa-HC should inhibit the rate of FIX activation by depleting the substrate. However, whereas FXIa/S557A inhibited FIX activation of by FXIa, FXIa-HC did not. Therefore, we examined FIX binding to FXIa/S557A, FXIa-HC, FXIa-LC, FXIa/C362S/C482S, and FXIa/S557A/C362S/C482S. The heavy and light chains are disulfide-linked in FXIa/S557A but not in FXIa/C362S/C482S and FXIa/S557A/C362S/C482S. In an ELISA assay only FXI/S557A ligated FIX with high affinity. Partial reduction of FXIa/S557A to produce heavy and light chains resulted in decreased FIX binding, and this function was regained upon reformation of the disulfide linkage between the heavy and the light chains. We therefore conclude that substrate recognition by the FXIa exosite(s) requires disulfide-linked heavy and light chains. PMID:22207756

DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

PubMed Central

Bernard, Mark A.; Wang, Leyu; Tachado, Souvenir D.

2015-01-01

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K m=74 nM) with substrate inhibition kinetics (K i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy. PMID:25793518

Differential niche dynamics among major marine invertebrate clades

PubMed Central

Hopkins, Melanie J; Simpson, Carl; Kiessling, Wolfgang

2014-01-01

The degree to which organisms retain their environmental preferences is of utmost importance in predicting their fate in a world of rapid climate change. Notably, marine invertebrates frequently show strong affinities for either carbonate or terrigenous clastic environments. This affinity is due to characteristics of the sediments as well as correlated environmental factors. We assessed the conservatism of substrate affinities of marine invertebrates over geological timescales, and found that niche conservatism is prevalent in the oceans, and largely determined by the strength of initial habitat preference. There is substantial variation in niche conservatism among major clades with corals and sponges being among the most conservative. Time-series analysis suggests that niche conservatism is enhanced during times of elevated nutrient flux, whereas niche evolution tends to occur after mass extinctions. Niche evolution is not necessarily elevated in genera exhibiting higher turnover in species composition. PMID:24313951

Integrated Affinity Biosensing Platforms on Screen-Printed Electrodes Electrografted with Diazonium Salts

PubMed Central

Yáñez-Sedeño, Paloma

2018-01-01

Adequate selection of the electrode surface and the strategies for its modification to enable subsequent immobilization of biomolecules and/or nanomaterials integration play a major role in the performance of electrochemical affinity biosensors. Because of the simplicity, rapidity and versatility, electrografting using diazonium salt reduction is among the most currently used functionalization methods to provide the attachment of an organic layer to a conductive substrate. This particular chemistry has demonstrated to be a powerful tool to covalently immobilize in a stable and reproducible way a wide range of biomolecules or nanomaterials onto different electrode surfaces. Considering the great progress and interesting features arisen in the last years, this paper outlines the potential of diazonium chemistry to prepare single or multianalyte electrochemical affinity biosensors on screen-printed electrodes (SPEs) and points out the existing challenges and future directions in this field. PMID:29495294

Integrated Affinity Biosensing Platforms on Screen-Printed Electrodes Electrografted with Diazonium Salts.

PubMed

Yáñez-Sedeño, Paloma; Campuzano, Susana; Pingarrón, José M

2018-02-24

Adequate selection of the electrode surface and the strategies for its modification to enable subsequent immobilization of biomolecules and/or nanomaterials integration play a major role in the performance of electrochemical affinity biosensors. Because of the simplicity, rapidity and versatility, electrografting using diazonium salt reduction is among the most currently used functionalization methods to provide the attachment of an organic layer to a conductive substrate. This particular chemistry has demonstrated to be a powerful tool to covalently immobilize in a stable and reproducible way a wide range of biomolecules or nanomaterials onto different electrode surfaces. Considering the great progress and interesting features arisen in the last years, this paper outlines the potential of diazonium chemistry to prepare single or multianalyte electrochemical affinity biosensors on screen-printed electrodes (SPEs) and points out the existing challenges and future directions in this field.

Isolation, one-step affinity purification, and characterization of a polyextremotolerant laccase from the halophilic bacterium Aquisalibacillus elongatus and its application in the delignification of sugar beet pulp.

PubMed

Rezaei, Shahla; Shahverdi, Ahmad Reza; Faramarzi, Mohammad Ali

2017-04-01

The aim of the present work was to study the ability of a halophilic bacterial laccase to efficient delignification in extreme conditions. Here, a highly stable extracellular laccase showing ligninolytic activity from halophilic Aquisalibacillus elongatus is described. The laccase production was strongly influenced by NaCl and CuSO 4 and under optimal conditions reached 4.8UmL -1 . The monomeric enzyme of 75kDa was purified by a synthetic affinity column with 68.2% yield and 99.8-fold purification. The enzyme showed some valuable features viz. stability against a wide range of organic solvents, salts, metals, inhibitors, and surfactants and specificity to a wide spectrum of substrates diverse in structure and redox potential. It retained more than 50% of the original activity at 25-75°C and pH 5.0-10.0. Furthermore, the enzyme was found to be effective in the delignification of sugar beet pulp in an ionic liquid that makes it useful for industrial applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle

PubMed Central

Mosadeghi, Ruzbeh; Reichermeier, Kurt M; Winkler, Martin; Schreiber, Anne; Reitsma, Justin M; Zhang, Yaru; Stengel, Florian; Cao, Junyue; Kim, Minsoo; Sweredoski, Michael J; Hess, Sonja; Leitner, Alexander; Aebersold, Ruedi; Peter, Matthias; Deshaies, Raymond J; Enchev, Radoslav I

2016-01-01

The COP9-Signalosome (CSN) regulates cullin–RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation. Both CSN and neddylated substrate undergo large conformational changes upon binding, with important roles played by the N-terminal domains of Csn2 and Csn4 and the RING domain of Rbx1 in enabling formation of a high affinity, fully active complex. The RING domain is crucial for deneddylation, and works in part through conformational changes involving insert-2 of Csn6. Nedd8 deconjugation and re-engagement of the active site zinc by the autoinhibitory Csn5 glutamate-104 diminish affinity for Cul1/Rbx1 by ~100-fold, resulting in its rapid ejection from the active site. Together, these mechanisms enable a dynamic deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network. DOI: http://dx.doi.org/10.7554/eLife.12102.001 PMID:27031283

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

PubMed

Halavaty, Andrei S; Rich, Rebecca L; Chen, Chao; Joo, Jeong Chan; Minasov, George; Dubrovska, Ievgeniia; Winsor, James R; Myszka, David G; Duban, Mark; Shuvalova, Ludmilla; Yakunin, Alexander F; Anderson, Wayne F

2015-05-01

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.

Versatile bio-ink for covalent immobilization of chimeric avidin on sol-gel substrates.

PubMed

Heikkinen, Jarkko J; Kivimäki, Liisa; Määttä, Juha A E; Mäkelä, Inka; Hakalahti, Leena; Takkinen, Kristiina; Kulomaa, Markku S; Hytönen, Vesa P; Hormi, Osmo E O

2011-10-15

A bio-ink for covalent deposition of thermostable, high affinity biotin-binding chimeric avidin onto sol-gel substrates was developed. The bio-ink was prepared from heterobifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide which was first reacted either with 3-aminopropyltriethoxysilane or 3-aminopropyldimethylethoxysilane to form silane linkers 6-maleimide-N-(3-(triethoxysilyl)propyl)hexanamide or -(ethoxydimethylsilyl)propyl)-hexanamide. C-terminal cysteine genetically engineered to chimeric avidin was reacted with the maleimide group of silane linker in methanol/PBS solution to form a suspension, which was printed on sol-gel modified PMMA film. Different concentrations of chimeric avidin and ratios between silane linkers were tested to find the best properties for the bio-ink to enable gravure or inkjet printing. Bio-ink prepared from 3-aminopropyltriethoxysilane was found to provide the highest amount of active immobilized chimeric avidin. The developed bio-ink was shown to be valuable for automated fabrication of avidin-functionalized polymer films. Copyright © 2011 Elsevier B.V. All rights reserved.

Polyphenol oxidase activity and antioxidant properties of Yomra apple (Malus communis L.) from Turkey.

PubMed

Can, Zehra; Dincer, Barbaros; Sahin, Huseyin; Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

2014-12-01

In this study, firstly, antioxidant and polyphenol oxidase (PPO) properties of Yomra apple were investigated. Seventeen phenolic constituents were measured by reverse phase-high-performance liquid chromatography (RP-HPLC). Total phenolic compounds (TPCs), ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activities were performed to measure antioxidant capacity. Some kinetic parameters (Km, Vmax), and inhibition behaviors against five different substrates were measured in the crude extract. Catechin and chlorogenic acid were found as the major components in the methanolic extract, while ferulic acid, caffeic acid, p-hydroxybenzoic acid, quercetin and p-coumaric acid were small quantities. Km values ranged from 0.70 to 10.10 mM in the substrates, and also 3-(4-hydroxyphenyl) propanoic acid (HPPA) and L-DOPA showed the highest affinity. The inhibition constant of Ki were ranged from 0.05 to 14.90 mM against sodium metabisulphite, ascorbic acid, sodium azide and benzoic acid, while ascorbic acid and sodium metabisulphite were the best inhibitors.

Ecophysiology of "halarsenatibacter silvermanii" strain SLAS-1T, gen. nov., sp. nov., a facultative chemoautotrophic arsenate respirer from salt-saturated Searles Lake, California

USGS Publications Warehouse

Blum, J.S.; Han, S.; Lanoil, B.; Saltikov, C.; Witte, B.; Tabita, F.R.; Langley, S.; Beveridge, T.J.; Jahnke, L.; Oremland, R.S.

2009-01-01

Searles Lake occupies a closed basin harboring salt-saturated, alkaline brines that have exceptionally high concentrations of arsenic oxyanions. Strain SLAS-1T was previously isolated from Searles Lake (R. S. Oremland, T. R. Kulp, J. Switzer Blum, S. E. Hoeft, S. Baesman, L. G. Miller, and J. F. Stolz, Science 308:1305-1308, 2005). We now describe this extremophile with regard to its substrate affinities, its unusual mode of motility, sequenced arrABD gene cluster, cell envelope lipids, and its phylogenetic alignment within the order Halanaero-bacteriales, assigning it the name "Halarsenatibacter silvermanii" strain SLAS-1T. We also report on the substrate dynamics of an anaerobic enrichment culture obtained from Searles Lake that grows under conditions of salt saturation and whose members include a novel sulfate reducer of the order Desulfovibriales, the archaeon Halorhabdus utahensis, as well as a close homolog of strain SLAS-1T. Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

Ecophysiology of "Halarsenatibacter silvermanii" strain SLAS-1T, gen. nov., sp. nov., a facultative chemoautotrophic arsenate respirer from salt-saturated Searles Lake, California.

PubMed

Blum, Jodi Switzer; Han, Sukkyun; Lanoil, Brian; Saltikov, Chad; Witte, Brian; Tabita, F Robert; Langley, Sean; Beveridge, Terry J; Jahnke, Linda; Oremland, Ronald S

2009-04-01

Searles Lake occupies a closed basin harboring salt-saturated, alkaline brines that have exceptionally high concentrations of arsenic oxyanions. Strain SLAS-1(T) was previously isolated from Searles Lake (R. S. Oremland, T. R. Kulp, J. Switzer Blum, S. E. Hoeft, S. Baesman, L. G. Miller, and J. F. Stolz, Science 308:1305-1308, 2005). We now describe this extremophile with regard to its substrate affinities, its unusual mode of motility, sequenced arrABD gene cluster, cell envelope lipids, and its phylogenetic alignment within the order Halanaerobacteriales, assigning it the name "Halarsenatibacter silvermanii" strain SLAS-1(T). We also report on the substrate dynamics of an anaerobic enrichment culture obtained from Searles Lake that grows under conditions of salt saturation and whose members include a novel sulfate reducer of the order Desulfovibriales, the archaeon Halorhabdus utahensis, as well as a close homolog of strain SLAS-1(T).

Beta-Lactamases Produced by a Pseudomonas aeruginosa Strain Highly Resistant to Carbenicillin

PubMed Central

Labia, Roger; Guionie, Marlène; Masson, Jean-Michel; Philippon, Alain; Barthelemy, Michel

1977-01-01

A Pseudomonas aeruginosa strain isolated at Besançon Hospital, France, proved to be highly resistant to carbenicillin and showed a high hydrolytic activity toward this antibiotic. We clearly demonstrated that two β-lactamases were synthetized: one of them, constitutive, has its enzymatic activity directed mainly toward penicillins, and carbenicillin appears to be its best substrate (higher Vmax); thus, this β-lactamase is a “carbenicillinase” that differs from the well-known “TEM-like” enzymes. The isoelectric point of this carbenicillinase is 5.30 ± 0.03. The other one is an inducible cephalosporinase, very similar to the cephalosporinases usually found in these organisms. Its isoelectric point is 8.66 ± 0.04. These two enzymes have been separated by affinity chromatography and isoelectric focusing. The kinetic constants were measured by computerized microacidimetry. Images PMID:406828

Cloning and functional characterization of the high-affinity K+ transporter HAK1 of pepper.

PubMed

Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

2004-10-01

High-affinity K+ uptake in plants plays a crucial role in K+ nutrition and different systems have been postulated to contribute to the high-affinity K+ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K+ uptake observed in roots. Pepper plants starved of K+ for 3 d showed high-affinity K+ uptake (Km of 6 microM K+) that was very sensitive to NH and their roots expressed a high-affinity K+ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast ( Saccharomyces cerevisiae ), CaHAK1 mediated high-affinity K+ and Rb+ uptake with Km values of 3.3 and 1.9 microM, respectively. Rb+ uptake was competitively inhibited by micromolar concentrations of NH and Cs+, and by millimolar concentrations of Na+.

Probing Allosteric Inhibition Mechanisms of the Hsp70 Chaperone Proteins Using Molecular Dynamics Simulations and Analysis of the Residue Interaction Networks.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2016-08-22

Although molecular mechanisms of allosteric regulation in the Hsp70 chaperones have been extensively studied at both structural and functional levels, the current understanding of allosteric inhibition of chaperone activities by small molecules is still lacking. In the current study, using a battery of computational approaches, we probed allosteric inhibition mechanisms of E. coli Hsp70 (DnaK) and human Hsp70 proteins by small molecule inhibitors PET-16 and novolactone. Molecular dynamics simulations and binding free energy analysis were combined with network-based modeling of residue interactions and allosteric communications to systematically characterize and compare molecular signatures of the apo form, substrate-bound, and inhibitor-bound chaperone complexes. The results suggested a mechanism by which the allosteric inhibitors may leverage binding energy hotspots in the interaction networks to stabilize a specific conformational state and impair the interdomain allosteric control. Using the network-based centrality analysis and community detection, we demonstrated that substrate binding may strengthen the connectivity of local interaction communities, leading to a dense interaction network that can promote an efficient allosteric communication. In contrast, binding of PET-16 to DnaK may induce significant dynamic changes and lead to a fractured interaction network and impaired allosteric communications in the DnaK complex. By using a mechanistic-based analysis of distance fluctuation maps and allosteric propensities of protein residues, we determined that the allosteric network in the PET-16 complex may be small and localized due to the reduced communication and low cooperativity of the substrate binding loops, which may promote the higher rates of substrate dissociation and the decreased substrate affinity. In comparison with the significant effect of PET-16, binding of novolactone to HSPA1A may cause only moderate network changes and preserve allosteric coupling between the allosteric pocket and the substrate binding region. The impact of novolactone on the conformational dynamics and allosteric communications in the HSPA1A complex was comparable to the substrate effect, which is consistent with the experimental evidence that PET-16, but not novolactone binding, can significantly decrease substrate affinity. We argue that the unique dynamic and network signatures of PET-16 and novolactone may be linked with the experimentally observed functional effects of these inhibitors on allosteric regulation and substrate binding.

Analysis of Substrates of Protein Kinase C Isoforms in Human Breast Cells By The Traceable Kinase Method

PubMed Central

Chen, Xiangyu; Zhao, Xin; Abeyweera, Thushara P.; Rotenberg, Susan A.

2012-01-01

A previous report (Biochemistry 46: 2364–2370, 2007) described the application of The Traceable Kinase Method to identify substrates of PKCα in non-transformed human breast MCF-10A cells. Here, a non-radioactive variation of this method compared the phospho-protein profiles of three traceable PKC isoforms (α, δ and ζ) for the purpose of identifying novel, isoform-selective substrates. Each FLAG-tagged traceable kinase was expressed and co-immunoprecipitated along with high affinity substrates. The isolated kinase and its associated substrates were subjected to an in vitro phosphorylation reaction with traceable kinase-specific N6-phenyl-ATP, and the resulting phospho-proteins were analyzed by Western blot with an antibody that recognizes the phosphorylated PKC consensus site. Phospho-protein profiles generated by PKC-α and -δ were similar and differed markedly from that of PKC-ζ. Mass spectrometry of selected bands revealed known PKC substrates and several potential substrates that included the small GTPase-associated effector protein Cdc42 effector protein-4 (CEP4). Of those potential substrates tested, only CEP4 was phosphorylated by pure PKC-α, –δ, and −ζ isoforms in vitro, and by endogenous PKC isoforms in MCF-10A cells treated with DAG-lactone, a membrane permeable PKC activator. Under these conditions, the stoichiometry of CEP4 phosphorylation was 3.2 ± 0.5 (mol phospho-CEP4/mol CEP4). Following knock-down with isoform-specific shRNA-encoding plasmids, phosphorylation of CEP4 was substantially decreased in response to silencing of each of the three isoforms (PKC–α, –δ, or –ζ), whereas testing of kinase-dead mutants supported a role for only PKC-α and –δ in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms. PMID:22897107

Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases

PubMed Central

Viigand, Katrin; Visnapuu, Triinu; Mardo, Karin; Aasamets, Anneli

2016-01-01

Abstract Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α‐methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α‐glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an α‐glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose‐like substrates (α‐1,4‐glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate‐binding pocket of MAL1 has three subsites (–1, +1 and +2) and that binding is strongest at the –1 subsite. The DSF assay results were in good accordance with affinity (K m) and inhibition (K i) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α‐glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:26919272

Photochemical grafting of methyl groups on a Si(111) surface using a Grignard reagent.

PubMed

Herrera, Marvin Ustaris; Ichii, Takashi; Murase, Kuniaki; Sugimura, Hiroyuki

2013-12-01

The photochemical grafting of methyl groups onto an n-type Si(111) substrate was successfully achieved using a Grignard reagent. The preparation involved illuminating a hydrogen-terminated Si(111) that was immersed in a CH3MgBr-THF solution. The success was attributed to the ability of the n-type hydrogenated substrate to produce holes on its surface when illuminated. The rate of grafting methyl groups onto the silicon surface was higher when a larger illumination intensity or when a substrate with lower dopant concentration was used. In addition, the methylated layer has an atomically flat structure, has a hydrophobic surface, and has electron affinity that was lower than the bulk Si. Copyright © 2013 Elsevier Inc. All rights reserved.

De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.

PubMed

Hamilton, P J; Campbell, N G; Sharma, S; Erreger, K; Herborg Hansen, F; Saunders, C; Belovich, A N; Sahai, M A; Cook, E H; Gether, U; McHaourab, H S; Matthies, H J G; Sutcliffe, J S; Galli, A

2013-12-01

De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster, the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.

The functional divergence of short-chain dehydrogenases involved in tropinone reduction.

PubMed

Brock, Andrea; Brandt, Wolfgang; Dräger, Birgit

2008-05-01

Tropane alkaloids typically occur in the Solanaceae and are also found in Cochlearia officinalis, a member of the Brassicaceae. Tropinone reductases are key enzymes of tropane alkaloid metabolism. Two different tropinone reductases form one stereoisomeric product each, either tropine for esterified alkaloids or pseudotropine that is converted to calystegines. A cDNA sequence with similarity to known tropinone reductases (TR) was cloned from C. officinalis. The protein was expressed in Escherichia coli, and found to catalyze the reduction of tropinone. The enzyme is a member of the short-chain dehydrogenase enzyme family and shows broad substrate specificity. Several synthetic ketones were accepted as substrates, with higher affinity and faster enzymatic turnover than observed for tropinone. C. officinalis TR produced both the isomeric alcohols tropine and pseudotropine from tropinone using NADPH + H(+) as co-substrate. Tropinone reductases of the Solanaceae, in contrast, are strictly stereospecific and form one tropane alcohol only. The Arabidopsis thaliana homologue of C. officinalis TR showed high sequence similarity, but did not reduce tropinone. A tyrosine residue was identified in the active site of C. officinalis TR that appeared responsible for binding and orientation of tropinone. Mutagenesis of the tyrosine residue yielded an active reductase, but with complete loss of TR activity. Thus C. officinalis TR presents an example of an enzyme with relaxed substrate specificity, like short-chain dehydrogenases, that provides favorable preconditions for the evolution of novel functions in biosynthetic sequences.

Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.

PubMed

Erickson, Jeffrey D

2017-07-01

Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC 50 1.3 ± 0.5 μM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K + -stimulated MeAIB transport displays an affinity (K m ) for MeAIB of 37 ± 1.2 μM, saturates at ~ 200 μM, is dependent on extracellular Ca 2+ , and is blocked by inhibition of voltage-gated Ca 2+ channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca 2+ and voltage-gated calcium channels, but is also blocked by the Na + channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca 2+ , but on Na + ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K + -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805. © 2017 International Society for Neurochemistry.

Functional characterization of Penicillium occitanis Pol6 and Penicillium funiculosum GH11 xylanases.

PubMed

Driss, Dorra; Berrin, Jean Guy; Juge, Nathalie; Bhiri, Fatma; Ghorbel, Raoudha; Chaabouni, Semia Ellouz

2013-08-01

Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of heteroxylans constituting the lignocellulosic plant cell wall. Xylanases from the GH11 family are considered as true xylanases because of their high substrate specificity. In order to study in depth a crucial difference in the thumb region between two closely related xylanases from Penicillium in terms of kinetic parameters and inhibition sensitivity, the GH11 xylanases from Penicillium occitanis Pol6 (PoXyn3) and from Penicillium funiculosum (PfXynC) were heterologously expressed in Pichia pastoris. The PoXyn3 and PfXynC cDNAs encoding mature xylanases were cloned into pGAPZαA vectors and integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase constitutive promoter. PfXynC was expressed as a His-tagged recombinant protein and purified from the supernatant homogeneity by a one-step purification protocol using immobilized metal affinity chromatography. The recombinant PoXyn3 was purified using a single anion-exchange chromatography. The purified recombinant enzymes were optimally active at 45°C and pH 4.0 for PoXyn3 and 40°C and pH 3.0 for PfXynC. The measured kinetic parameters (k(cat) and Vmax) showed that PfXynC was five times more active than PoXyn3 irrespective of the substrate whereas the apparent affinity (K(m)) was similar. The recombinant enzymes showed distinct sensitivity to the Triticum aestivum xylanase inhibitor TAXI-I. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular mechanisms for generating transmembrane proton gradients

PubMed Central

Gunner, M.R.; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

2013-01-01

Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side. PMID:23507617

Metal Dependence of the Xylose Isomerase from Piromyces sp. E2 Explored by Activity Profiling and Protein Crystallography

PubMed Central

2017-01-01

Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn2+ is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn2+, which was established by measuring the activation constants (Kact) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity. PMID:29045784

Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferasel identified using phage display and biopanning

USDA-ARS?s Scientific Manuscript database

The role of PROTEIN ISOASPARTYL-METHYLTRANSFERASE (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of specific PIMT target proteins in p...

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

PubMed Central

Liu, K H; Huang, C Y; Tsay, Y F

1999-01-01

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis. PMID:10330471

Observation of positive and small electron affinity of Si-doped AlN films grown by metalorganic chemical vapor deposition on n-type 6H-SiC

NASA Astrophysics Data System (ADS)

Feng, Liang; Ping, Chen; De-Gang, Zhao; De-Sheng, Jiang; Zhi-Juan, Zhao; Zong-Shun, Liu; Jian-Jun, Zhu; Jing, Yang; Wei, Liu; Xiao-Guang, He; Xiao-Jing, Li; Xiang, Li; Shuang-Tao, Liu; Hui, Yang; Li-Qun, Zhang; Jian-Ping, Liu; Yuan-Tao, Zhang; Guo-Tong, Du

2016-05-01

We have investigated the electron affinity of Si-doped AlN films (N Si = 1.0 × 1018-1.0 × 1019 cm-3) with thicknesses of 50, 200, and 400 nm, synthesized by metalorganic chemical vapor deposition (MOCVD) under low pressure on the n-type (001)6H-SiC substrates. The positive and small electron affinity of AlN films was observed through the ultraviolet photoelectron spectroscopy (UPS) analysis, where an increase in electron affinity appears with the thickness of AlN films increasing, i.e., 0.36 eV for the 50-nm-thick one, 0.58 eV for the 200-nm-thick one, and 0.97 eV for the 400-nm-thick one. Accompanying the x-ray photoelectron spectroscopy (XPS) analysis on the surface contaminations, it suggests that the difference of electron affinity between our three samples may result from the discrepancy of surface impurity contaminations. Project supported by the National Natural Science Foundation of China (Grant Nos. 61574135, 61574134, 61474142, 61474110, 61377020, 61376089, 61223005, and 61321063), the One Hundred Person Project of the Chinese Academy of Sciences, and the Basic Research Project of Jiangsu Province, China (Grant No. BK20130362).

Mutagenesis of threonine to serine in the active site of Mycobacterium tuberculosis fructose-1,6-bisphosphatase (Class II) retains partial enzyme activity.

PubMed

Bondoc, Jasper Marc G; Wolf, Nina M; Ndichuck, Michael; Abad-Zapatero, Celerino; Movahedzadeh, Farahnaz

2017-09-01

The glpX gene encodes for the Class II fructose-1,6-bisphosphatase enzyme in Mycobacterium tuberculosis ( Mt ), an essential enzyme for pathogenesis. We have performed site directed mutagenesis to introduce two mutations at residue Thr84, T84A and T84S, to explore the binding affinity of the substrate and the catalytic mechanism. The T84A mutant fully abolishes enzyme activity while retaining substrate binding affinity. In contrast, the T84S mutant retains some activity having a 10 times reduction in V max and exhibited similar sensitivity to lithium when compared to the wildtype. Homology modeling using the Escherichia coli enzyme structure suggests that the replacement of the critical nucleophile OH - in the Thr84 residue of the wildtype of Mt FBPase by Ser84 results in subtle alterations of the position and orientation that reduce the catalytic efficiency. This mutant could be used to trap reaction intermediates, through crystallographic methods, facilitating the design of potent inhibitors via structure-based drug design.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

PubMed Central

Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

2014-01-01

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

Artificial light-regulation of an allosteric bi-enzyme complex by a photosensitive ligand.

PubMed

Kneuttinger, Andrea C; Winter, Martin; Simeth, Nadja A; Heyn, Kristina; Merkl, Rainer; König, Burkhard; Sterner, Reinhard

2018-05-29

The artificial regulation of proteins by light is an emerging sub-discipline of synthetic biology. Here, we used this concept in order to photo-control both catalysis and allostery within the heterodimeric enzyme complex imidazole glycerol phosphate synthase (ImGP-S). The ImGP-S consists of the cyclase subunit HisF and the glutaminase subunit HisH, which is allosterically stimulated by substrate binding to HisF. We show that a light-sensitive diarylethene (DTE)-based competitive inhibitor in its ring-open state binds with low micromolar affinity to the cyclase subunit and displaces its substrate from the active site. As a consequence, catalysis by HisF and allosteric stimulation of HisH are impaired. Following UV-light irradiation, the DTE-ligand adopts its ring-closed state and loses affinity for HisF, restoring activity and allostery. Our approach allows for the switching of ImGP-S activity and allostery during catalysis and appears to be generally applicable for the light-regulation of other multi-enzyme complexes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Anatomy of F1-ATPase powered rotation.

PubMed

Martin, James L; Ishmukhametov, Robert; Hornung, Tassilo; Ahmad, Zulfiqar; Frasch, Wayne D

2014-03-11

F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank).

Anatomy of F1-ATPase powered rotation

PubMed Central

Martin, James L.; Ishmukhametov, Robert; Hornung, Tassilo; Ahmad, Zulfiqar; Frasch, Wayne D.

2014-01-01

F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank). PMID:24567403

Unusual dewetting of thin polymer films in liquid media containing a poor solvent and a nonsolvent.

PubMed

Xu, Lin; Sharma, Ashutosh; Joo, Sang Woo; Liu, Hui; Shi, Tongfei

2014-12-16

We investigate the control of pattern size and kinetics in spontaneous dewetting of thin polymer films (polystyrene) that are stable to thermal annealing by annealing in a poor solvent (acetone)/nonsolvent (ethanol or n-hexane) liquid mixture. Dewetting occurs by the formation and growth of circular holes that coalesce to form droplets. The influence of the nature and the volume fraction of the nonsolvents on the contact angle of polymer droplets, number density of holes, and the kinetics of holes formation and growth is studied. Addition of ethanol greatly increases the hole density and slows down the kinetics substantially, while affecting only a small change in wettability. n-Hexane addition shows an interesting nonmonotonic response in decreasing the hole density and contact angle in the volume fraction range of 0-0.3 but an opposite effect beyond that. Although the two nonsolvents chosen cannot by themselves induce dewetting, their relative affinity for the solid substrate vis-à-vis acetone can strongly influence the observed dewetting scenarios that are not understood by the existing theoretical considerations. n-Hexane, for example, has great affinity for silicon substrate. In addition to the changes in wettability, viscosity, and film interfacial tension engendered by the nonsolvents, the possibility of the formation of adsorbed liquid layers at the substrate-polymer interface, which can modify the interfacial friction and slippage, needs to be considered.

Amorphous oxides as electron transport layers in Cu(In,Ga)Se 2 superstrate devices: Amorphous oxides in Cu(In,Ga)Se 2 superstrate devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heinemann, M. D.; van Hest, M. F. A. M.; Contreras, M.

Cu(In,Ga)Se2 (CIGS) solar cells in superstrate configuration promise improved light management and higher stability compared to substrate devices, but they have yet to deliver comparable power conversion efficiencies (PCEs). Chemical reactions between the CIGS layer and the front contact were shown in the past to deteriorate the p-n junction in superstrate devices, which led to lower efficiencies compared to the substrate-type devices. This work aims to solve this problem by identifying a buffer layer between the CIGS layer and the front contact, acting as the electron transport layer, with an optimized electron affinity, doping density and chemical stability. Using combinatorialmore » material exploration we identified amorphous gallium oxide (a-GaOx) as a potentially suitable buffer layer material. The best results were obtained for a-GaOx with an electron affinity that was found to be comparable to that of CIGS. Based on the results of device simulations, it is assumed that detrimental interfacial acceptor states are present at the interface between CIGS and a-GaOx. However, these initial experiments indicate the potential of a-GaOx in this application, and how to reach performance parity with substrate devices, by further increase of its n-type doping density.« less

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli.

PubMed

Botou, Maria; Lazou, Panayiota; Papakostas, Konstantinos; Lambrinidis, George; Evangelidis, Thomas; Mikros, Emmanuel; Frillingos, Stathis

2018-04-01

The uracil permease UraA of Escherichia coli is a structurally known prototype for the ubiquitous Nucleobase-Ascorbate Transporter (NAT) or Nucleobase-Cation Symporter-2 (NCS2) family and represents a well-defined subgroup of bacterial homologs that remain functionally unstudied. Here, we analyze four of these homologs, including RutG of E. coli which shares 35% identity with UraA and is encoded in the catabolic rut (pyrimidine utilization) operon. Using amplified expression in E. coli K-12, we show that RutG is a high-affinity permease for uracil, thymine and, at low efficiency, xanthine and recognizes also 5-fluorouracil and oxypurinol. In contrast, UraA and the homologs from Acinetobacter calcoaceticus and Aeromonas veronii are permeases specific for uracil and 5-fluorouracil. Molecular docking indicates that thymine is hindered from binding to UraA by a highly conserved Phe residue which is absent in RutG. Site-directed replacement of this Phe with Ala in the three uracil-specific homologs allows high-affinity recognition and/or transport of thymine, emulating the RutG profile. Furthermore, all RutG orthologs from enterobacteria retain an Ala at this position, implying that they can use both uracil and thymine and, possibly, xanthine as substrates and provide the bacterial cell with a range of catabolizable nucleobases. © 2018 John Wiley & Sons Ltd.

Quantitative insight into the design of compounds recognized by the L-type amino acid transporter 1 (LAT1).

PubMed

Ylikangas, Henna; Malmioja, Kalle; Peura, Lauri; Gynther, Mikko; Nwachukwu, Emmanuel O; Leppänen, Jukka; Laine, Krista; Rautio, Jarkko; Lahtela-Kakkonen, Maija; Huttunen, Kristiina M; Poso, Antti

2014-12-01

L-Type amino acid transporter 1 (LAT1) is a transmembrane protein expressed abundantly at the blood-brain barrier (BBB), where it ensures the transport of hydrophobic acids from the blood to the brain. Due to its unique substrate specificity and high expression at the BBB, LAT1 is an intriguing target for carrier-mediated transport of drugs into the brain. In this study, a comparative molecular field analysis (CoMFA) model with considerable statistical quality (Q(2) =0.53, R(2) =0.75, Q(2) SE=0.77, R(2) SE=0.57) and good external predictivity (CCC=0.91) was generated. The model was used to guide the synthesis of eight new prodrugs whose affinity for LAT1 was tested by using an in situ rat brain perfusion technique. This resulted in the creation of a novel LAT1 prodrug with L-tryptophan as the promoiety; it also provided a better understanding of the molecular features of LAT1-targeted high-affinity prodrugs, as well as their promoiety and parent drug. The results obtained will be beneficial in the rational design of novel LAT1-binding prodrugs and other compounds that bind to LAT1. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

PubMed

Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

2011-02-01

A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry. Copyright © 2010 Elsevier Ltd. All rights reserved.

Characterization of substrate binding of the WW domains in human WWP2 protein.

PubMed

Jiang, Jiahong; Wang, Nan; Jiang, Yafei; Tan, Hongwei; Zheng, Jimin; Chen, Guangju; Jia, Zongchao

2015-07-08

WW domains harbor substrates containing proline-rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human WWP2 (hWWP2). Herein we have employed multiple techniques to investigate the second WW domain (WW2) in hWWP2. Our results show that hWWP2 is a specialized E3 for PPxY motif-containing substrates only and does not recognize other amino acids and phospho-residues. The strongest binding affinity of WW2, and the incompatibility between each WW domain, imply a novel relationship, and our SPR experiment reveals a dynamic binding mode in Class-I WW domains for the first time. The results from alanine-scanning mutagenesis and modeling further point to functionally conserved residues in WW2. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

PubMed

Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

2017-09-01

The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probing Substrate Interactions in the Active Tunnel of a Catalytically Deficient Cellobiohydrolase (Cel7)*

PubMed Central

Colussi, Francieli; Sørensen, Trine H.; Alasepp, Kadri; Kari, Jeppe; Cruys-Bagger, Nicolaj; Windahl, Michael S.; Olsen, Johan P.; Borch, Kim; Westh, Peter

2015-01-01

Cellobiohydrolases break down cellulose sequentially by sliding along the crystal surface with a single cellulose strand threaded through the catalytic tunnel of the enzyme. This so-called processive mechanism relies on a complex pattern of enzyme-substrate interactions, which need to be addressed in molecular descriptions of processivity and its driving forces. Here, we have used titration calorimetry to study interactions of cellooligosaccharides (COS) and a catalytically deficient variant (E212Q) of the enzyme Cel7A from Trichoderma reesei. This enzyme has ∼10 glucopyranose subsites in the catalytic tunnel, and using COS ligands with a degree of polymerization (DP) from 2 to 8, different regions of the tunnel could be probed. For COS ligands with a DP of 2–3 the binding constants were around 105 m−1, and for longer ligands (DP 5–8) this value was ∼107 m−1. Within each of these groups we did not find increased affinity as the ligands got longer and potentially filled more subsites. On the contrary, we found a small but consistent affinity loss as DP rose from 6 to 8, particularly at the higher investigated temperatures. Other thermodynamic functions (ΔH, ΔS, and ΔCp) decreased monotonously with both temperature and DP. Combined interpretation of these thermodynamic results and previously published structural data allowed assessment of an affinity profile along the length axis of the active tunnel. PMID:25477511

Purification, Characterization, and Sensitivity to Pesticides of Carboxylesterase From Dendrolimus superans (Lepidoptera: Lasiocampidae)

PubMed Central

Zou, Chuan-shan; Cao, Chuan-wang; Zhang, Guo-cai; Wang, Zhi-ying

2014-01-01

Abstract Through a combination of steps including centrifugation, ammonium sulfate gradient precipitation, sephadex G-25 gel chromatography, diethylaminoethyl cellulose 52 ion-exchange chromatography and hydroxyapatite affinity chromatography, carboxylesterase (CarE, EC3.1.1.1) from sixth instar larch caterpillar moth, Dendrolimus superans (Lepidoptera: Lasiocampidae) larvae was purified and its biochemical properties were compared between crude homogenate and purified CarE. The final purified CarE after hydroxyapatite chromatography had a specific activity of 52.019 μmol/(min·mg protein), 138.348-fold of crude homogenate, and the yield of 2.782%. The molecular weight of the purified CarE was approximately 84.78 kDa by SDS-PAGE. Three pesticides (dichlorvos, lambda-cyhalothrin, and avermectins) showed different inhibition to crude CarE and purified CarE, respectively. In vitro median inhibitory concentration indicated that the sensitivity of CarE (both crude homogenate and final purified CarE) to pesticides was in decreasing order of dichlorvos > avermectins > lambda-cyhalothrin. By the kinetic analysis, the substrates alpha-naphthyl acetate (α-NA) and beta-naphthyl acetate (β-NA) showed lesser affinity to crude extract than purified CarE. The results also indicated that both crude homogenate and purified CarE had more affinity to α-NA than to β-NA, and the Kcat and Vmax values of crude extract were lower than purified CarE using α-NA or β-NA as substrate. PMID:25525114

Regulation of the HscA ATPase reaction cycle by the co-chaperone HscB and the iron-sulfur cluster assembly protein IscU.

PubMed

Silberg, Jonathan J; Tapley, Tim L; Hoff, Kevin G; Vickery, Larry E

2004-12-24

The ATPase activity of HscA, a specialized hsp70 molecular chaperone from Escherichia coli, is regulated by the iron-sulfur cluster assembly protein IscU and the J-type co-chaperone HscB. IscU behaves as a substrate for HscA, and HscB enhances the binding of IscU to HscA. To better understand the mechanism by which HscB and IscU regulate HscA, we examined binding of HscB to the different conformational states of HscA and the effects of HscB and IscU on the kinetics of the individual steps of the HscA ATPase reaction cycle. Affinity sensor studies revealed that whereas IscU binds both ADP (R-state) and ATP (T-state) HscA complexes, HscB interacts only with an ATP-bound state. Studies of ATPase activity under single-turnover and rapid mixing conditions showed that both IscU and HscB interact with the low peptide affinity T-state of HscA (HscA++.ATP) and that both modestly accelerate (3-10-fold) the rate-determining steps in the HscA reaction cycle, k(hyd) and k(T-->R). When present together, IscU and HscB synergistically stimulate both k(hyd) (approximately = 500-fold) and k(T-->R) (approximately = 60-fold), leading to enhanced formation of the HscA.ADP-IscU complex (substrate capture). Following ADP/ATP exchange, IscU also stimulates k(R-->T) (approximately = 50-fold) and thereby accelerates the rate at which the low peptide affinity HscA++.ATP T-state is regenerated. Because HscA nucleotide exchange is fast, the overall rate of the chaperone cycle in vivo will be determined by the availability of the IscU-HscB substrate-co-chaperone complex.

A novel intermembrane space–targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding

PubMed Central

Sideris, Dionisia P.; Petrakis, Nikos; Katrakili, Nitsa; Mikropoulou, Despina; Gallo, Angelo; Ciofi-Baffoni, Simone; Banci, Lucia; Bertini, Ivano

2009-01-01

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step–specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys. PMID:20026652

Magnetite-doped polydimethylsiloxane (PDMS) for phosphopeptide enrichment.

PubMed

Sandison, Mairi E; Jensen, K Tveen; Gesellchen, F; Cooper, J M; Pitt, A R

2014-10-07

Reversible phosphorylation plays a key role in numerous biological processes. Mass spectrometry-based approaches are commonly used to analyze protein phosphorylation, but such analysis is challenging, largely due to the low phosphorylation stoichiometry. Hence, a number of phosphopeptide enrichment strategies have been developed, including metal oxide affinity chromatography (MOAC). Here, we describe a new material for performing MOAC that employs a magnetite-doped polydimethylsiloxane (PDMS), that is suitable for the creation of microwell array and microfluidic systems to enable low volume, high throughput analysis. Incubation time and sample loading were explored and optimized and demonstrate that the embedded magnetite is able to enrich phosphopeptides. This substrate-based approach is rapid, straightforward and suitable for simultaneously performing multiple, low volume enrichments.

Catalytic activity of enzymes immobilized on AlGaN /GaN solution gate field-effect transistors

NASA Astrophysics Data System (ADS)

Baur, B.; Howgate, J.; von Ribbeck, H.-G.; Gawlina, Y.; Bandalo, V.; Steinhoff, G.; Stutzmann, M.; Eickhoff, M.

2006-10-01

Enzyme-modified field-effect transistors (EnFETs) were prepared by immobilization of penicillinase on AlGaN /GaN solution gate field-effect transistors. The influence of the immobilization process on enzyme functionality was analyzed by comparing covalent immobilization and physisorption. Covalent immobilization by Schiff base formation on GaN surfaces modified with an aminopropyltriethoxysilane monolayer exhibits high reproducibility with respect to the enzyme/substrate affinity. Reductive amination of the Schiff base bonds to secondary amines significantly increases the stability of the enzyme layer. Electronic characterization of the EnFET response to penicillin G indicates that covalent immobilization leads to the formation of an enzyme (sub)monolayer.

High Surface-Enhanced Raman Scattering (SERS) Amplification Factor Obtained with Silver Printed Circuit Boards and the Influence of Phenolic Resins for the Characterization of the Pesticide Thiram.

PubMed

Silva de Almeida, Francylaine; Bussler, Larissa; Marcio Lima, Sandro; Fiorucci, Antonio Rogério; da Cunha Andrade, Luis Humberto

2016-07-01

In this work, low-cost substrates with rough silver surfaces were prepared from commercial copper foil-covered phenolic board (CPB) and an aqueous solution of AgNO3, and were used for surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) measurements. A maximum SERS amplification factor of 1.2 × 10(7) was obtained for Rhodamine 6G (R6G), and use of the CPB resulted in a detection limit for Thiram pesticide of 0.5 µmol L(-1) The minimum detection level was limited by residual traces of phenolic groups that originated from the substrate resin, which became solubilized in the aqueous Ag(+) solution. It was found that the bands corresponding to the impurities had less influence in the Thiram analysis, which could be explained by the high affinity of sulfur for Ag surfaces. The influence of impurities in the SERS analyses therefore depended on the linkage between the rough silver surface and the analyte. The findings demonstrated the ease and effectiveness of using CPB to prepare a nanostructured surface for SERS. © The Author(s) 2016.

The disorderly conduct of Hsc70 and its interaction with the Alzheimer's related Tau protein.

PubMed

Taylor, Isabelle R; Ahmad, Atta; Wu, Taia; Nordhues, Bryce A; Bhullar, Anup; Gestwicki, Jason E; Zuiderweg, Erik R P

2018-05-15

Hsp70 chaperones bind to various protein substrates for folding, trafficking, and degradation. Considerable structural information is available about how prokaryotic Hsp70 (DnaK) binds substrates, but less is known about mammalian Hsp70s, of which there are 13 isoforms encoded in the human genome. Here, we report the interaction between the human Hsp70 isoform heat shock cognate 71 KDa protein (Hsc70 or HSPA8) and peptides derived from the microtubule-associated protein tau, which is linked to Alzheimer's disease. For structural studies, we used an Hsc70 construct (called BETA) comprising the substrate-binding domain, but lacking the lid. Importantly, we found that truncating the lid does not significantly impair Hsc70's chaperone activity or allostery in vitro. Using NMR, we show that BETA is partially dynamically disordered in the absence of substrate and that binding of the tau sequence GKVQIINKKG (with a KD = 500 nM) causes dramatic rigidification of BETA. Nuclear Overhauser effect distance measurements revealed that tau binds to the canonical substrate-binding cleft, similar to the binding observed with DnaK. To further develop BETA as a tool for studying Hsc70 interactions, we also measured BETA binding in NMR and fluorescent competition assays to peptides derived from huntingtin, insulin, a second tau-recognition sequence, and a KFERQ-like sequence linked to chaperone-mediated autophagy. We found that the insulin C-peptide binds BETA with high affinity (KD < 100 nM), whereas the others do not (KD > 100 μM). Together, our findings reveal several similarities and differences in how prokaryotic and mammalian Hsp70 isoforms interact with different substrate peptides. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

PubMed Central

Yang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-Qun

2013-01-01

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca2+ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca2+ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth. PMID:23527883

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms.

PubMed

Yang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-Qun

2014-01-01

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure-function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca(2+) removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca(2+) removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein-ligand binding, including the concept of the free energy landscape (FEL) of the protein-solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

PubMed

Krall, Jacob; Jensen, Claus Hatt; Bavo, Francesco; Falk-Petersen, Christina Birkedahl; Haugaard, Anne Stæhr; Vogensen, Stine Byskov; Tian, Yongsong; Nittegaard-Nielsen, Mia; Sigurdardóttir, Sara Björk; Kehler, Jan; Kongstad, Kenneth Thermann; Gloriam, David E; Clausen, Rasmus Prætorius; Harpsøe, Kasper; Wellendorph, Petrine; Frølund, Bente

2017-11-09

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar K i ) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

Structural and In Vivo Studies on Trehalose-6-Phosphate Synthase from Pathogenic Fungi Provide Insights into Its Catalytic Mechanism, Biological Necessity, and Potential for Novel Antifungal Drug Design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miao, Yi; Tenor, Jennifer L.; Toffaletti, Dena L.

ABSTRACT The disaccharide trehalose is critical to the survival of pathogenic fungi in their human host. Trehalose-6-phosphate synthase (Tps1) catalyzes the first step of trehalose biosynthesis in fungi. Here, we report the first structures of eukaryotic Tps1s in complex with substrates or substrate analogues. The overall structures of Tps1 fromCandida albicansandAspergillus fumigatusare essentially identical and reveal N- and C-terminal Rossmann fold domains that form the glucose-6-phosphate and UDP-glucose substrate binding sites, respectively. These Tps1 structures with substrates or substrate analogues reveal key residues involved in recognition and catalysis. Disruption of these key residues severely impaired Tps1 enzymatic activity. Subsequent cellularmore » analyses also highlight the enzymatic function of Tps1 in thermotolerance, yeast-hypha transition, and biofilm development. These results suggest that Tps1 enzymatic functionality is essential for the fungal stress response and virulence. Furthermore, structures of Tps1 in complex with the nonhydrolyzable inhibitor, validoxylamine A, visualize the transition state and support an internal return-like catalytic mechanism that is generalizable to other GT-B-fold retaining glycosyltransferases. Collectively, our results depict key Tps1-substrate interactions, unveil the enzymatic mechanism of these fungal proteins, and pave the way for high-throughput inhibitor screening buttressed and guided by the current structures and those of high-affinity ligand-Tps1 complexes. IMPORTANCEInvasive fungal diseases have emerged as major threats, resulting in more than 1.5 million deaths annually worldwide. This epidemic has been further complicated by increasing resistance to all major classes of antifungal drugs in the clinic. Trehalose biosynthesis is essential for the fungal stress response and virulence. Critically, this biosynthetic pathway is absent in mammals, and thus, the two enzymes that carry out trehalose biosynthesis, namely, trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2), are prominent targets for antifungal intervention. Here, we report the first eukaryotic Tps1 structures from the pathogenic fungiCandida albicansandAspergillus fumigatusin complex with substrates, substrate analogues, and inhibitors. These structures reveal key protein-substrate interactions, providing atomic-level scaffolds for structure-guided drug design of novel antifungals that target Tps1.« less

Structural and In Vivo Studies on Trehalose-6-Phosphate Synthase from Pathogenic Fungi Provide Insights into Its Catalytic Mechanism, Biological Necessity, and Potential for Novel Antifungal Drug Design.

PubMed

Miao, Yi; Tenor, Jennifer L; Toffaletti, Dena L; Maskarinec, Stacey A; Liu, Jiuyu; Lee, Richard E; Perfect, John R; Brennan, Richard G

2017-07-25

The disaccharide trehalose is critical to the survival of pathogenic fungi in their human host. Trehalose-6-phosphate synthase (Tps1) catalyzes the first step of trehalose biosynthesis in fungi. Here, we report the first structures of eukaryotic Tps1s in complex with substrates or substrate analogues. The overall structures of Tps1 from Candida albicans and Aspergillus fumigatus are essentially identical and reveal N- and C-terminal Rossmann fold domains that form the glucose-6-phosphate and UDP-glucose substrate binding sites, respectively. These Tps1 structures with substrates or substrate analogues reveal key residues involved in recognition and catalysis. Disruption of these key residues severely impaired Tps1 enzymatic activity. Subsequent cellular analyses also highlight the enzymatic function of Tps1 in thermotolerance, yeast-hypha transition, and biofilm development. These results suggest that Tps1 enzymatic functionality is essential for the fungal stress response and virulence. Furthermore, structures of Tps1 in complex with the nonhydrolyzable inhibitor, validoxylamine A, visualize the transition state and support an internal return-like catalytic mechanism that is generalizable to other GT-B-fold retaining glycosyltransferases. Collectively, our results depict key Tps1-substrate interactions, unveil the enzymatic mechanism of these fungal proteins, and pave the way for high-throughput inhibitor screening buttressed and guided by the current structures and those of high-affinity ligand-Tps1 complexes. IMPORTANCE Invasive fungal diseases have emerged as major threats, resulting in more than 1.5 million deaths annually worldwide. This epidemic has been further complicated by increasing resistance to all major classes of antifungal drugs in the clinic. Trehalose biosynthesis is essential for the fungal stress response and virulence. Critically, this biosynthetic pathway is absent in mammals, and thus, the two enzymes that carry out trehalose biosynthesis, namely, trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2), are prominent targets for antifungal intervention. Here, we report the first eukaryotic Tps1 structures from the pathogenic fungi Candida albicans and Aspergillus fumigatus in complex with substrates, substrate analogues, and inhibitors. These structures reveal key protein-substrate interactions, providing atomic-level scaffolds for structure-guided drug design of novel antifungals that target Tps1. Copyright © 2017 Miao et al.

Effect of atomic layer deposition temperature on current conduction in Al2O3 films formed using H2O oxidant

NASA Astrophysics Data System (ADS)

Hiraiwa, Atsushi; Matsumura, Daisuke; Kawarada, Hiroshi

2016-08-01

To develop high-performance, high-reliability gate insulation and surface passivation technologies for wide-bandgap semiconductor devices, the effect of atomic layer deposition (ALD) temperature on current conduction in Al2O3 films is investigated based on the recently proposed space-charge-controlled field emission model. Leakage current measurement shows that Al2O3 metal-insulator-semiconductor capacitors formed on the Si substrates underperform thermally grown SiO2 capacitors at the same average field. However, using equivalent oxide field as a more practical measure, the Al2O3 capacitors are found to outperform the SiO2 capacitors in the cases where the capacitors are negatively biased and the gate material is adequately selected to reduce virtual dipoles at the gate/Al2O3 interface. The Al2O3 electron affinity increases with the increasing ALD temperature, but the gate-side virtual dipoles are not affected. Therefore, the leakage current of negatively biased Al2O3 capacitors is approximately independent of the ALD temperature because of the compensation of the opposite effects of increased electron affinity and permittivity in Al2O3. By contrast, the substrate-side sheet of charge increases with increasing ALD temperature above 210 °C and hence enhances the current of positively biased Al2O3 capacitors more significantly at high temperatures. Additionally, an anomalous oscillatory shift of the current-voltage characteristics with ALD temperature was observed in positively biased capacitors formed by low-temperature (≤210 °C) ALD. This shift is caused by dipoles at the Al2O3/underlying SiO2 interface. Although they have a minimal positive-bias leakage current, the low-temperature-grown Al2O3 films cause the so-called blisters problem when heated above 400 °C. Therefore, because of the absence of blistering, a 450 °C ALD process is presently the most promising technology for growing high-reliability Al2O3 films.

Electrophoretically deposited reduced graphene oxide platform for food toxin detection

NASA Astrophysics Data System (ADS)

Srivastava, Saurabh; Kumar, Vinod; Ali, Md Azahar; Solanki, Pratima R.; Srivastava, Anchal; Sumana, Gajjala; Saxena, Preeti Suman; Joshi, Amish G.; Malhotra, B. D.

2013-03-01

Reduced graphene oxide (RGO) due to its excellent electrochemical properties and large surface area, has recently aroused much interest for electrochemical biosensing application. Here, the chemically active RGO has been synthesized and deposited onto an indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. This novel platform has been utilized for covalent attachment of the monoclonal antibodies of aflatoxin B1 (anti-AFB1) for food toxin (AFB1) detection. The electron microscopy, X-ray diffraction, and UV-visible studies reveal successful synthesis of reduced graphene oxide while the XPS and FTIR studies suggest its carboxylic functionalized nature. The electrochemical sensing results of the anti-AFB1/RGO/ITO based immunoelectrode obtained as a function of aflatoxin concentration show high sensitivity (68 μA ng-1 mL cm-2) and improved detection limit (0.12 ng mL-1). The association constant (ka) for antigen-antibody interaction obtained as 5 × 10-4 ng mL-1 indicates high affinity of antibodies toward the antigen (AFB1).Reduced graphene oxide (RGO) due to its excellent electrochemical properties and large surface area, has recently aroused much interest for electrochemical biosensing application. Here, the chemically active RGO has been synthesized and deposited onto an indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. This novel platform has been utilized for covalent attachment of the monoclonal antibodies of aflatoxin B1 (anti-AFB1) for food toxin (AFB1) detection. The electron microscopy, X-ray diffraction, and UV-visible studies reveal successful synthesis of reduced graphene oxide while the XPS and FTIR studies suggest its carboxylic functionalized nature. The electrochemical sensing results of the anti-AFB1/RGO/ITO based immunoelectrode obtained as a function of aflatoxin concentration show high sensitivity (68 μA ng-1 mL cm-2) and improved detection limit (0.12 ng mL-1). The association constant (ka) for antigen-antibody interaction obtained as 5 × 10-4 ng mL-1 indicates high affinity of antibodies toward the antigen (AFB1). Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr32242d

Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha.

PubMed

De Marco, L; Mazzucato, M; Masotti, A; Ruggeri, Z M

1994-03-04

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

Unusual Genomic Traits Suggest Methylocystis bryophila S285 to Be Well Adapted for Life in Peatlands

PubMed Central

Han, Dongfei; Dedysh, Svetlana N

2018-01-01

Abstract The genus Methylocystis belongs to the class Alphaproteobacteria, the family Methylocystaceae, and encompasses aerobic methanotrophic bacteria with the serine pathway of carbon assimilation. All Methylocystis species are able to fix dinitrogen and several members of this genus are also capable of using acetate or ethanol in the absence of methane, which explains their wide distribution in various habitats. One additional trait that enables their survival in the environment is possession of two methane-oxidizing isozymes, the conventional particulate methane monooxygenase (pMMO) with low-affinity to substrate (pMMO1) and the high-affinity enzyme (pMMO2). Here, we report the finished genome sequence of Methylocystis bryophila S285, a pMMO2-possessing methanotroph from a Sphagnum-dominated wetland, and compare it to the genome of Methylocystis sp. strain SC2, which is the first methanotroph with confirmed high-affinity methane oxidation potential. The complete genome of Methylocystis bryophila S285 consists of a 4.53 Mb chromosome and one plasmid, 175 kb in size. The genome encodes two types of particulate MMO (pMMO1 and pMMO2), soluble MMO and, in addition, contains a pxmABC-like gene cluster similar to that present in some gammaproteobacterial methanotrophs. The full set of genes related to the serine pathway, the tricarboxylic acid cycle as well as the ethylmalonyl-CoA pathway is present. In contrast to most described methanotrophs including Methylocystis sp. strain SC2, two different types of nitrogenases, that is, molybdenum–iron and vanadium–iron types, are encoded in the genome of strain S285. This unique combination of genome-based traits makes Methylocystis bryophila well adapted to the fluctuation of carbon and nitrogen sources in wetlands. PMID:29390143

Structural Transformation Detection Contributes to Screening of Behaviorally Active Compounds: Dynamic Binding Process Analysis of DhelOBP21 from Dastarcus helophoroides.

PubMed

Yang, Rui-Nan; Li, Dong-Zhen; Yu, Guangqiang; Yi, Shan-Cheng; Zhang, Yinan; Kong, De-Xin; Wang, Man-Qun

2017-12-01

In light of reverse chemical ecology, the fluorescence competitive binding assays of functional odorant binding proteins (OBPs) is a recent advanced approach for screening behaviorally active compounds of insects. Previous research on Dastareus helophoroides identified a minus-C OBP, DhelOBP21, which preferably binds to several ligands. In this study, only (+)-β-pinene proved attractive to unmated adult beetles. To obtain a more in-depth explanation of the lack of behavioral activity of other ligands we selected compounds with high (camphor) and low (β-caryophyllene) binding affinities. The structural transformation of OBPs was investigated using well-established approaches for studying binding processes, such as fluorescent quenching assays, circular dichroism, and molecular dynamics. The dynamic binding process revealed that the flexibility of DhelOBP21 seems conducive to binding specific ligands, as opposed to broad substrate binding. The compound (+)-β-pinene and DhelOBP21 formed a stable complex through a secondary structural transformation of DhelOBP21, in which its amino-terminus transformed from random coil to an α-helix to cover the binding pocket. On the other hand, camphor could not efficiently induce a stable structural transformation, and its high binding affinities were due to strong hydrogen-bonding, compromising the structure of the protein. The other compound, β-caryophyllene, only collided with DhelOBP21 and could not be positioned in the binding pocket. Studying structural transformation of these proteins through examining the dynamic binding process rather than using approaches that just measure binding affinities such as fluorescence competitive binding assays can provide a more efficient and reliable approach for screening behaviorally active compounds.

Sequence intrinsic somatic mutation mechanisms contribute to affinity maturation of VRC01-class HIV-1 broadly neutralizing antibodies

PubMed Central

Hwang, Joyce K.; Wang, Chong; Du, Zhou; Meyers, Robin M.; Kepler, Thomas B.; Neuberg, Donna; Kwong, Peter D.; Mascola, John R.; Joyce, M. Gordon; Bonsignori, Mattia; Haynes, Barton F.; Yeap, Leng-Siew; Alt, Frederick W.

2017-01-01

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1–infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms. PMID:28747530

Tyrosine phosphorylation and association of Syk with Fc gamma RII in monocytic THP-1 cells.

PubMed Central

Ghazizadeh, S; Bolen, J B; Fleit, H B

1995-01-01

Although the cytoplasmic portion of the low-affinity receptor for immunoglobulin G, Fc gamma RII, does not contain a kinase domain, rapid tyrosine phosphorylation of intracellular substrates occurs in response to aggregation of the receptor. The use of specific tyrosine kinase inhibitors has suggested that these phosphorylations are required for subsequent cellular responses. We previously demonstrated the coprecipitation of a tyrosine kinase activity with Fc gamma RII, suggesting that non-receptor tyrosine kinases might associate with the cytoplasmic domain of Fc gamma RII. Anti-receptor immune complex kinase assays revealed the coprecipitation of several phosphoproteins, most notably p56/53lyn, an Src-family protein tyrosine kinase (PTK), and a 72 kDa phosphoprotein. Here we identify the 72 kDa Fc gamma RII-associated protein as p72syk (Syk), a member of a newly described family of non-receptor PTKs. A rapid and transient tyrosine phosphorylation of Syk was observed following Fc gamma RII activation. Syk was also tyrosyl-phosphorylated following aggregation of the high-affinity Fc gamma receptor, Fc gamma RI. The Fc gamma RI activation did not result in association of Syk with Fc gamma RII, implying that distinct pools of Syk are activated upon aggregation of each receptor in a localized manner. These results demonstrate a physical association between Syk and Fc gamma RII and suggest that the molecules involved in Fc gamma RII signalling are very similar to the ones utilized by multichain immune recognition receptors such as the B-cell antigen receptor and the high-affinity IgE receptor. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7530449

Comparison of the catalytic properties of the botulinum neurotoxin subtypes A1 and A5.

PubMed

Wang, Dongxia; Krilich, Joan; Pellett, Sabine; Baudys, Jakub; Tepp, William H; Barr, John R; Johnson, Eric A; Kalb, Suzanne R

2013-12-01

Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening disease botulism through the inhibition of neurotransmitter release by cleaving essential SNARE proteins. There are seven serologically distinctive types of BoNTs and many subtypes within a serotype have been identified. BoNT/A5 is a recently discovered subtype of type A botulinum neurotoxin which possesses a very high degree of sequence similarity and identity to the well-studied A1 subtype. In the present study, we examined the endopeptidase activity of these two BoNT/A subtypes and our results revealed significant differences in substrate binding and cleavage efficiency between subtype A5 and A1. Distinctive hydrolysis efficiency was observed between the two toxins during cleavage of the native substrate SNAP-25 versus a shortened peptide mimic. N-terminal truncation studies demonstrated that a key region of the SNAP-25, including the amino acid residues at 151 through 154 located in the remote binding region of the substrate, contributed to the differential catalytic properties between A1 and A5. Elevated binding affinity of the peptide substrate resulted from including these important residues and enhanced BoNT/A5's hydrolysis efficiency. In addition, mutations of these amino acid residues affect the proteolytic performance of the two toxins in different ways. This study provides a better understanding of the biological activity of these toxins, their performance characteristics in the Endopep-MS assay to detect BoNT in clinical samples and foods, and is useful for the development of peptide substrates. © 2013. Published by Elsevier B.V. All rights reserved.

Junction-based field emission structure for field emission display

DOEpatents

Dinh, Long N.; Balooch, Mehdi; McLean, II, William; Schildbach, Marcus A.

2002-01-01

A junction-based field emission display, wherein the junctions are formed by depositing a semiconducting or dielectric, low work function, negative electron affinity (NEA) silicon-based compound film (SBCF) onto a metal or n-type semiconductor substrate. The SBCF can be doped to become a p-type semiconductor. A small forward bias voltage is applied across the junction so that electron transport is from the substrate into the SBCF region. Upon entering into this NEA region, many electrons are released into the vacuum level above the SBCF surface and accelerated toward a positively biased phosphor screen anode, hence lighting up the phosphor screen for display. To turn off, simply switch off the applied potential across the SBCF/substrate. May be used for field emission flat panel displays.

Properties and substrate specificity of the leucyl-, the threonyl- and the valyl-transfer-ribonucleic acid synthetases from Aesculus species

PubMed Central

Anderson, J. W.; Fowden, L.

1970-01-01

1. Leucyl- and threonyl-tRNA synthetases were partially purified up to 100-fold and 30-fold respectively from cotyledons of Aesculus hippocastanum and were largely separated from the other aminoacyl-tRNA synthetases. Valyl-tRNA synthetase was purified 25-fold from cotyledons of Aesculus californica. 2. Some properties are reported for the three enzymes when assayed by the [32P]pyrophosphate-ATP exchange technique. 3. β-(Methylenecyclopropyl)alanine, isoleucine, azaleucine, norleucine and γ-hydroxynorvaline acted as alternative substrates for the leucyl-tRNA synthetase; the enzyme's affinity for β-(methylenecyclopropyl)-alanine and for isoleucine was about 80-fold less than that exhibited for leucine. 4. α-Cyclopropylglycine and α-cyclobutylglycine acted as alternative substrates for the valyl-tRNA synthetase. PMID:5493505

Simulation studies of substrate recognition by the exocellulase CelF from Clostridium cellulolyticum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Mo; Himmel, Michael E.; Wilson, David B.

Molecular dynamics (MD) simulations were used to study substrate recognition by the family 48 exocellulase CelF from Clostridium cellulolyticum. It was hypothesized that residues around the entrance of the active site tunnel of this enzyme might serve to recognize and bind the substrate through an affinity for the cellulose monomer repeat unit, ..beta..-d-glucopyranose. Simulations were conducted of the catalytic domain of this enzyme surrounded by a concentrated solution of ..beta..-d-glucopyranose, and the full three-dimensional probability distribution for finding sugar molecules adjacent to the enzyme was calculated from the trajectory. A significant probability of finding the sugar stacked against the planarmore » faces of Trp 310 and Trp 312 at the entrance of the active site tunnel was observed.« less

The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae.

PubMed

Bracher, Jasmine M; Verhoeven, Maarten D; Wisselink, H Wouter; Crimi, Barbara; Nijland, Jeroen G; Driessen, Arnold J M; Klaassen, Paul; van Maris, Antonius J A; Daran, Jean-Marc G; Pronk, Jack T

2018-01-01

l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2 , had been deleted. Sugar transport assays indicated that this fungal transporter, designated as Pc AraT, is a high-affinity ( K m  = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10 -3 and 1.8 g L -1 , respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of Pc AraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L -1 l-arabinose and 20 g L -1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make Pc AraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

Mutational analysis of microbial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) towards enhancement of binding affinity: A computational approach.

PubMed

Kumar, Pravin; Ghosh Sachan, Shashwati; Poddar, Raju

2017-10-01

Improving the industrial enzyme for better yield of the product is important and a challenging task. One of such important industrial enzymes is microbial Hydroxycinnamoyl-CoA hydratase-lyase (HCHL). It converts feruloyl-CoA to vanillin. We place our efforts towards the improvement of its catalytic activity with comprehensive computational investigation. Catalytic core of the HCHL was explored with molecular modeling and docking approaches. Site-directed mutations were introduced in the catalytic site of HCHL in a sequential manner to generate different mutants of HCHL. Basis of mutation is to increase the interaction between HCHL and substrate feruloyl-CoA through interatomic forces and hydrogen bond formation. A rigorous molecular dynamics (MD) simulation was performed to check the stability of mutant's structure. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), dynamic cross correlation (DCCM) and principal component analysis (PCA) were also performed to analyze flexibility and stability of structures. Docking studies were carried out between different mutants of HCHL and feruloyl-CoA. Investigation of the different binding sites and the interactions with mutant HCHLs and substrate allowed us to highlight the improved performance of mutants than wild type HCHL. This was further validated with MD simulation of complex consisting of different mutants and substrate. It further confirms all the structures are stable. However, mutant-2 showed better affinity towards substrate by forming hydrogen bond between active site and feruloyl-CoA. We propose that increase in hydrogen bond formation might facilitate in dissociation of vanillin from feruloyl-CoA. The current work may be useful for the future development of 'tailor-made' enzymes for better yield of vanillin. Copyright © 2017 Elsevier Inc. All rights reserved.

Kinetic, mechanistic, and structural modeling studies of truncated wild-type leucine-rich repeat kinase 2 and the G2019S mutant.

PubMed

Liu, Min; Kang, Stephanie; Ray, Soumya; Jackson, Justin; Zaitsev, Alexandra D; Gerber, Scott A; Cuny, Gregory D; Glicksman, Marcie A

2011-11-01

Leucine-rich repeat kinase 2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson's disease (PD). Here, we characterize the kinetic and catalytic mechanisms of truncated wild-type (t-wt) LRRK2 and its most common mutant, G2019S (t-G2019S), with a structural interpretation of the kinase domain. First, the substitution of threonine with serine in the LRRKtide peptide results in a much less efficient substrate as demonstrated by a 26-fold decrease in k(cat) and a 6-fold decrease in binding affinity. The significant decrease in k(cat) is attributed to a slow chemical transfer step as evidenced by the inverse solvent kinetic isotope effect in the proton inventory and pL (pH or pD)-dependent studies. The shape of the proton inventory and pL profile clearly signals the involvement of a general base (pK(a) = 7.5) in the catalysis with a low fractionation factor in the ground state. We report for the first time that the increased kinase activity of the G2019S mutant is substrate-dependent. Homology modeling of the kinase domain (open and closed forms) and structural analysis of the docked peptide substrates suggest that electrostatic interactions play an important role in substrate recognition, which is affected by G2019S and may directly influence the kinetic properties of the enzyme. Finally, the GTPase activity of the t-G2019S mutant was characterized, and the mutation modestly decreases GTPase activity without significantly affecting GTP binding affinity.

Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

PubMed Central

Orcutt, Kelly Davis; Slusarczyk, Adrian L; Cieslewicz, Maryelise; Ruiz-Yi, Benjamin; Bhushan, Kumar R; Frangioni, John V; Wittrup, K Dane

2014-01-01

Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals. PMID:21315278

Revisiting nitrification in the Eastern Tropical South Pacific: A focus on controls

NASA Astrophysics Data System (ADS)

Peng, Xuefeng; Fuchsman, Clara A.; Jayakumar, Amal; Warner, Mark J.; Devol, Allan H.; Ward, Bess B.

2016-03-01

Nitrification, the oxidation of ammonium (NH4+) to nitrite (NO2-) and to nitrate (NO3-), is a component of the nitrogen (N) cycle internal to the fixed N pool. In oxygen minimum zones (OMZs), which are hotspots for oceanic fixed N loss, nitrification plays a key role because it directly supplies substrates for denitrification and anaerobic ammonia oxidation (anammox), and may compete for substrates with these same processes. However, the control of oxygen and substrate concentrations on nitrification are not well understood. We performed onboard incubations with 15N-labeled substrates to measure rates of NH4+ and NO2- oxidation in the eastern tropical South Pacific (ETSP). The spatial and depth distributions of NH4+ and NO2- oxidation rates were primarily controlled by NH4+ and NO2- availability, oxygen concentration, and light. In the euphotic zone, nitrification was partially photoinhibited. In the anoxic layer, NH4+ oxidation was negligible or below detection, but high rates of NO2- oxidation were observed. NH4+ oxidation displayed extremely high affinity for both NH4+ and oxygen. The positive linear correlations between NH4+ oxidation rates and in situ NH4+ concentrations and ammonia monooxygenase subunit A (amoA) gene abundances in the upper oxycline indicate that the natural assemblage of ammonia oxidizers responds to in situNH4+ concentrations or supply by adjusting their population size, which determines the NH4+ oxidation potential. The depth distribution of archaeal and bacterial amoA gene abundances and N2O concentration, along with independently reported simultaneous direct N2O production rate measurements, suggests that AOA were predominantly responsible for NH4+ oxidation, which was a major source of N2O production at oxygen concentrations > 5 µM.

Cysteine Scanning Mutagenesis of Transmembrane Domain 10 in Organic Anion Transporting Polypeptide 1B1

PubMed Central

2015-01-01

Organic anion transporting polypeptide (OATP) 1B1 is an important drug transporter expressed in human hepatocytes. Previous studies have indicated that transmembrane (TM) domain 2, 6, 8, 9, and in particular 10 might be part of the substrate binding site/translocation pathway. To explore which amino acids in TM10 are important for substrate transport, we mutated 34 amino acids individually to cysteines, expressed them in HEK293 cells, and determined their surface expression. Transport activity of the two model substrates estrone-3-sulfate and estradiol-17β-glucuronide as well as of the drug substrate valsartan for selected mutants was measured. Except for F534C and F537C, all mutants were expressed at the plasma membrane of HEK293 cells. Mutants Q541C and A549C did not transport estradiol-17β-glucuronide and showed negligible estrone-3-sulfate transport. However, A549C showed normal valsartan transport. Pretreatment with the anionic and cell impermeable sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) affected the transport of each substrate differently. Pretreatment of L545C abolished estrone-3-sulfate uptake almost completely, while it stimulated estradiol-17β-glucuronide uptake. Further analyses revealed that mutant L545C in the absence of MTSES showed biphasic kinetics for estrone-3-sulfate that was converted to monophasic kinetics with a decreased apparent affinity, explaining the previously seen inhibition. In contrast, the apparent affinity for estradiol-17β-glucuronide was not changed by MTSES treatment, but the Vmax value was increased about 4-fold, explaining the previously seen stimulation. Maleimide labeling of L545C was affected by preincubation with estrone-3-sulfate but not with estradiol-17β-glucuronide. These results strongly suggest that L545C is part of the estrone-3-sulfate binding site/translocation pathway but is not directly involved in binding/translocation of estradiol-17β-glucuronide. PMID:24673529

ATP-dependent transport of statins by human and rat MRP2/Mrp2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ellis, Lucy C.J., E-mail: Luc_ellis@yahoo.co.uk; Hawksworth, Gabrielle M.; Weaver, Richard J.

2013-06-01

Multidrug resistance associated protein-2, MRP2 (human), Mrp2 (rat) are an efflux transporter, responsible for the transport of numerous endogenous and xenobiotic compounds including taurocholate, methotrexate and carboxydichlorofluorescein (CDF). The present study aims to characterise transport of statins by human and rat MRP2/Mrp2 using membrane and vesicle preparations. All statins tested (simvastatin, pravastatin, pitavastatin, fluvastatin, atorvastatin, lovastatin and rosuvastatin) stimulated vanadate-sensitive ATPase activity in membranes expressing human or rat MRP2/Mrp2, suggesting that all statins are substrates of human and rat MRP2/Mrp2. The substrate affinity (Km) of all statins for MRP2/Mrp2 was comparable and no correlation between lipophilicity (logD{sub 7.0}) and Kmmore » was seen. All statins also inhibited uptake of the fluorescent Mrp2 substrate, CDF (1 μM) into vesicles expressing human or rat MRP2/Mrp2 with similar IC{sub 50} values. Fitting of the inhibitory data to the hill slope equation, gave hill coefficients (h) of greater than one, suggesting that transport involved more than one binding site for inhibitors of MPR2 and Mrp2. We conclude that statins were transported by both human and rat MRP2/Mrp2 with similar affinity. Statins were also shown to compete with other substrates for transport by MRP2/Mrp2 and that this transport involved more than one binding site on the Mrp2/MRP2 protein. - Highlights: • We characterised MRP2 (human)/Mrp2 (rat)-mediated transport of statins. • We show statins were transported by human and rat MRP2/Mrp2. • Statins competed with a known substrate for transport by MRP2/Mrp2. • Competition involved more than one binding site on the MRP2/Mrp2 protein.« less

Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants.

PubMed Central

Wang, R; Crawford, N M

1996-01-01

Two mutations have been found in a gene (NRT2) of Arabidopsis thaliana that specifically impair constitutive, high-affinity nitrate uptake. These mutants were selected for resistance to 0.1 mM chlorate in the absence of nitrate. Progency from one of the backcrossed mutants showed no constitutive uptake of nitrate below 0.5 mM at pH 7.0 in liquid culture (that is, within 30 min of initial exposure to nitrate). All other uptake activities measured (high-affinity phosphate and sulfate uptake, inducible high-affinity nitrate uptake, and constitutive low-affinity nitrate uptake) were present or nearly normal in the backcrossed mutant. Electrophysiological analysis of individual root cells showed that the nrt2 mutant showed little response to 0.25 mM of nitrate, whereas NRT2 wild-type cells showed an initial depolarization followed by recovery. At 10 mM of nitrate both the mutant and wild-type cells displayed similar, strong electrical responses. These results indicate that NRT2 is a critical and perhaps necessary gene for constitutive, high-affinity nitrate uptake in Arabidopsis, but not for inducible, high-affinity nor constitutive, low-affinity nitrate uptake. Thus, these systems are genetically distinct. PMID:8799195

Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions

PubMed Central

Shukla, Suneet; Abel, Biebele; Chufan, Eduardo E.; Ambudkar, Suresh V.

2017-01-01

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10–40 nm range. Similarly, a 30–150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment. PMID:28283574

Transport mechanism for lovastatin acid in bovine kidney NBL-1 cells: kinetic evidences imply involvement of monocarboxylate transporter 4.

PubMed

Nagasawa, Kazuki; Nagai, Katsuhito; Ishimoto, Atsushi; Fujimoto, Sadaki

2003-08-27

We previously indicated that lovastatin acid, a 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, was transported by a monocarboxylate transporter (MCT) in cultured rat mesangial cells. In this study, to identify the MCT isoform(s) responsible for the lovastatin acid uptake, the transport mechanism was investigated using bovine kidney NBL-1 cells, which have been reported to express only MCT4 at the protein level. On RT-PCR analysis, the message of mRNAs for MCT1 and MCT4 was detected in the NBL-1 cells used in this study, which was confirmed by kinetic analysis of [14C]L-lactic acid uptake, consisting of high- and low-affinity components corresponding to MCT1 and MCT4, respectively. The lovastatin acid uptake depended on an inwardly directed H+-gradient, and was inhibited by representative monocarboxylates, but not by inhibitors/substrates for organic anion transporting polypeptides and organic anion transporters. In addition, L-lactic acid competitively inhibited the uptake of lovastatin acid and lovastatin acid inhibited the low affinity component of [14C]L-lactic acid uptake dose dependently. The inhibition constant of L-lactic acid for lovastatin acid uptake was almost the same as the Michaelis constant for [14C]L-lactic acid uptake by the low-affinity component. These kinetic evidences imply that lovastatin acid was taken up into NBL-1 cells via MCT4.

Automethylation of Protein Arginine Methyltransferase 8 (PRMT8) Regulates Activity by Impeding S-Adenosylmethionine Sensitivity*

PubMed Central

Dillon, Myles B. C.; Rust, Heather L.; Thompson, Paul R.; Mowen, Kerri A.

2013-01-01

Protein arginine methyltransferase (PRMT) 8 is unique among the PRMTs, as it has a highly restricted tissue expression pattern and an N terminus that contains two automethylation sites and a myristoylation site. PRMTs catalyze the transfer of a methyl group from S-adenosylmethionine (AdoMet) to a peptidylarginine on a protein substrate. Currently, the physiological roles, regulation, and cellular substrates of PRMT8 are poorly understood. However, a thorough understanding of PRMT8 kinetics should provide insights into each of these areas, thereby enhancing our understanding of this unique enzyme. In this study, we determined how automethylation regulates the enzymatic activity of PRMT8. We found that preventing automethylation with lysine mutations (preserving the positive charge of the residue) increased the turnover rate and decreased the Km of AdoMet but did not affect the Km of the protein substrate. In contrast, mimicking automethylation with phenylalanine (i.e. mimicking the increased hydrophobicity) decreased the turnover rate. The inhibitory effect of the PRMT8 N terminus could be transferred to PRMT1 by creating a chimeric protein containing the N terminus of PRMT8 fused to PRMT1. Thus, automethylation of the N terminus likely regulates PRMT8 activity by decreasing the affinity of the enzyme for AdoMet. PMID:23946480

Changes in small angle X-ray scattering parameters observed upon ligand binding to rabbit muscle pyruvate kinase are not correlated with allosteric transitions†

PubMed Central

Fenton, Aron W.; Williams, Rachel; Trewhella, Jill

2010-01-01

Protein fluorescence and small-angle X-ray scattering (SAXS) have been used to monitor effector affinity and conformational changes previously associated with allosteric regulation in rabbit muscle pyruvate kinase (M1-PYK). In the absence of substrate (phosphoenolpyruvate; PEP), SAXS-monitored conformational changes in M1-PYK elicited by the binding of phenylalanine (an allosteric inhibitor that reduces the affinity of M1-PYK for PEP) are similar to those observed upon binding of alanine or 2-aminobutyric acid. Under the current assay conditions, these small amino acids bind to the protein, but elicit a minimal change in the affinity of the protein for PEP. Therefore, if changes in scattering signatures represent cleft closure via domain rotation as previously interpreted, it can be concluded that these motions are not sufficient to elicit allosteric inhibition. Additionally, although PEP has similar affinities for the free enzyme and the M1-PYK/small-amino-acid complexes (i.e. the small amino acids have minimal allosteric effects), PEP binding elicits different changes in the SAXS signature of the free enzyme vs. the M1-PYK/small-amino-acid complexes. PMID:20712377

The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

PubMed Central

Fenton, Aron W.; Hutchinson, Myra

2009-01-01

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37°C. As a function of increasing pH, hL-PYK's affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and Ala slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to Ala is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for Ala inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response. PMID:19467627

Investigation of molybdenum-crosslinker interfaces for affinity based electrochemical biosensing applications

NASA Astrophysics Data System (ADS)

Kamakoti, Vikramshankar; Shanmugam, Nandhinee Radha; Tanak, Ambalika Sanjeev; Jagannath, Badrinath; Prasad, Shalini

2018-04-01

Molybdenum (Mo) has been investigated for implementation as an electrode material for affinity based biosensing towards devloping flexibe electronic biosensors. Treatment of the native oxide of molybdenum was investigated through two surface treatment strategies namely thiol and carbodiimide crosslinking methods. The binding interaction between cross-linker molecules and Mo electrode surface has been characterized using Fourier Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and optical microscopy. The efficacy of treatment of Mo with its native oxide using carbodiimide cross linking methodology was established. The carbodiimide cross-linking chemistry was found to possess better surface coverage and binding affinity with Molybdenum electrode surface when compared to thiol cross-linking chemistry.Electrochemical characterization of Mo electrode using Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltametry (CV) techniques was performed to evaluate the effect of ionic properties of solution buffer on the Mo electrode's performance. Affinity based biosensing of C-Reactive Protein (CRP) has been demonstrated on a flexible nanoporous polymeric substrate with detection threshold of 100 pg/ml in synthetic urine buffer medium. The biosensor has been evaluated to be developed as a dipstick based point of care device for detection of biomarkers in urine.

Competitive horseradish peroxidase-linked aptamer assay for sensitive detection of Aflatoxin B1.

PubMed

Sun, Linlin; Zhao, Qiang

2018-03-01

Aflatoxin B1 (AFB1) is one of highly toxic mycotoxins and a known human carcinogen. The frequent contamination of AFB1 in food products and large health risk of AFB1 have raised global concerns. Sensitive detection of AFB1 is of vital importance and highly demanded. Herein, we reported a competitive horseradish peroxidase (HRP)-linked aptamer assay for AFB1, combining the advantages of aptamer for affinity binding and enzyme label for signal amplification. In this assay, free AFB1 in solution competed with a covalent conjugate of bovine serum albumin-AFB1 (BSA-AFB1) coated on the wells of microplate in binding to the HRP-labeled aptamer probe. HRP attached on BSA-AFB1 in the wells catalyzed the conversion of substrates into products, allowing the final detection of AFB1 through measurement of the generated products. When TMB (3,3',5,5'-tetramethylbenzidine dihydrochloride) was used as substrate, absorbance analysis of the product of enzyme reaction enabled the detection of AFB1 at 0.2nM. We further lowered the detection limit of AFB1 to 0.01nM through chemiluminescence analysis by using chemiluminescence substrate of HRP. This assay enabled the detection of AFB1 in complex sample matrix, such as diluted white wine and maize flour. This assay provides a simple, sensitive and rapid method for AFB1 determination. Copyright © 2017 Elsevier B.V. All rights reserved.

Poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): a bioactive copolymer for specific targeting to folate receptor-positive cancer cells.

PubMed

Chen, Yin; Cao, Wenbin; Zhou, Junli; Pidhatika, Bidhari; Xiong, Bin; Huang, Lu; Tian, Qian; Shu, Yiwei; Wen, Weijia; Hsing, I-Ming; Wu, Hongkai

2015-02-04

In this study, we present the preparation, characterization and application of a novel bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), which has a specific interaction with folate receptor (FR)-positive cancer cells. Glass surface immobilized with PLL-g-PMOXA-c-FA was demonstrated to be adhesive to FR-positive cancer cells (HeLa, JEG-3) while nonadhesive to FR-negative ones (MCF-7, HepG2) in 3 h. The specific interaction between conjugated FA on the substrate and FRs on the cells could hardly be inhibited unless a high concentration (5 mM) of free FA was used due to the multivalent nature of it. The FA functionality ratio of the copolymer on the substrate had a significant influence on the adhesion of HeLa cells, and our experiments revealed that the affinity of the substrate to the cells declined dramatically with the decrease of functionality ratio. This was believed to be caused by the polydispersity of PMOXA tethers, as supported by GPC and ToF-SIMS data. As a proof of concept in the application of our material, we demonstrated successful recovery of HeLa cells from mixture with MCF-7 (1:100) on the copolymer-coated glass, and our results showed that both high sensitivity (95.6 ± 13.3%) and specificity (24.3 ± 8.6%) were achieved.

Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity.

PubMed

Segawa, H; Fukasawa, Y; Miyamoto, K; Takeda, E; Endou, H; Kanai, Y

1999-07-09

We have isolated a cDNA from rat small intestine that encodes a novel Na+-independent neutral amino acid transporter with distinctive characteristics in substrate selectivity and transport property. The encoded protein, designated L-type amino acid transporter-2 (LAT-2), shows amino acid sequence similarity to the system L Na+-independent neutral amino acid transporter LAT-1 (Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., and Endou, H. (1998) J. Biol. Chem. 273, 23629-23632) (50% identity) and the system y+L transporters y+LAT-1 (47%) and KIAA0245/y+LAT-2 (45%) (Torrents, D., Estevez, R., Pineda, M., Fernandez, E., Lloberas, J., Shi, Y.-B., Zorzano, A., and Palacin, M. (1998) J. Biol. Chem. 273, 32437-32445). LAT-2 is a nonglycosylated membrane protein. It requires 4F2 heavy chain, a type II membrane glycoprotein, for its functional expression in Xenopus oocytes. LAT-2-mediated transport is not dependent on Na+ or Cl- and is inhibited by a system L-specific inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), indicating that LAT-2 is a second isoform of the system L transporter. Compared with LAT-1, which prefers large neutral amino acids with branched or aromatic side chains, LAT-2 exhibits remarkably broad substrate selectivity. It transports all of the L-isomers of neutral alpha-amino acids. LAT-2 exhibits higher affinity (Km = 30-50 microM) to Tyr, Phe, Trp, Thr, Asn, Ile, Cys, Ser, Leu, Val, and Gln and relatively lower affinity (Km = 180-300 microM) to His, Ala, Met, and Gly. In addition, LAT-2 mediates facilitated diffusion of substrate amino acids, as distinct from LAT-1, which mediates amino acid exchange. LAT-2-mediated transport is increased by lowering the pH level, with peak activity at pH 6.25, because of the decrease in the Km value without changing the Vmax value. Because of these functional properties and a high level of expression of LAT-2 in the small intestine, kidney, placenta, and brain, it is suggested that the heterodimeric complex of LAT-2 and 4F2 heavy chain is involved in the trans-cellular transport of neutral amino acids in epithelia and blood-tissue barriers.

Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

PubMed

Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

2015-07-01

Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. Copyright © 2015 Elsevier B.V. All rights reserved.

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

Engineering nanomaterials with a combined electrochemical and molecular biomimetic approach

NASA Astrophysics Data System (ADS)

Dai, Haixia

Biocomposite materials, such as bones, teeth, and shells, are created using mild aqueous solution-based processes near room temperature. Proteins add flexibility to these processes by facilitating the nucleation, growth, and ordering of specific inorganic materials into hierarchical structures. We aim to develop a biomimetic strategy for engineering technologically relevant inorganic materials with controlled compositions and structures, as Nature does, using proteins to orchestrate material formation and assembly. This approach involves three basic steps: (i) preparation of inorganic substrates compatible with combinatorial polypeptide screening; (ii) identification of inorganic-binding polypeptides and their engineering into inorganic-binding proteins; and (iii) protein-mediated inorganic nucleation and organization. Cuprous oxide (Cu2O), a p-type semiconductor, has been used to demonstrate all three steps. Zinc oxide (ZnO), an n-type semiconductor, has been used to show the generality of selected steps. Step (i), preparation of high quality inorganic substrates to select inorganic-binding polypeptides, was accomplished using electrochemical microfabrication to grow and pattern Cu2O and ZnO. Raman spectroscopy and x-ray photoelectron spectroscopy were used to verify phase purity and compositional stability of these surfaces during polypeptide screening. Step (ii), accomplished in collaboration with personnel in Prof Baneyx' lab at the University of Washington, involved incubating the inorganic substrates with the FliTrx(TM) random peptide library to identify cysteine-constrained dodecapeptides that bind the targeted inorganic. Insertion of a Cu2O-binding dodecapeptide into the DNA-binding protein TraI endowed the engineered TraI with strong affinity for Cu2O (Kd ≈ 10 -8 M). Finally, step (iii) involved nonequilibrium synthesis and organization of Cu2O nanoparticles, taking advantage of the inorganic and DNA recognition properties of the engineered TraI. The high affinity of the engineered TraI for Cu2O over other related copper compounds led to the formation of Cu2O nanoparticles from a cuprous chloride complex (Cu2Cln1-n, n = 2 or 3) electrolyte under conditions where the mineral atacamite (CuCl(OH) 3) is thermodynamically preferred. The nonequilibrium Cu 2O nanoparticles consisted of 2--3 nm Cu2O cores and functional protein shells that enabled predictable meso-scale assembly on DNA templates. In short, we have rationally designed a protein-based scheme for forming and organizing inorganic materials that Nature has not previous worked with.

Effects of TNF-alpha on Endothelial Cell Collective Migration

NASA Astrophysics Data System (ADS)

Chen, Desu; Wu, Di; Helim Aranda-Espinoza, Jose; Losert, Wolfgang

2013-03-01

Tumor necrosis factor (TNF-alpha) is a small cell-signaling protein usually released by monocytes and macrophages during an inflammatory response. Previous work had shown the effects of TNF-alpha on single cell morphology, migration, and biomechanical properties. However, the effect on collective migrations remains unexplored. In this work, we have created scratches on monolayers of human umbilical endothelial cells (HUVECs) treated with 25ng/mL TNF-alpha on glass substrates. The wound healing like processes were imaged with phase contrast microscopy. Quantitative analysis of the collective migration of cells treated with TNF-alpha indicates that these cells maintain their persistent motion and alignment better than untreated cells. In addition, the collective migration was characterized by measuring the amount of non-affine deformations of the wound healing monolayer. We found a lower mean non-affinity and narrower distribution of non-affinities upon TNF-alpha stimulation. These results suggest that TNF-alpha introduces a higher degree of organized cell collective migration.

The substrate specificity of Metarhizium anisopliae and Bos taurus carboxypeptidases A: Insights into their use as tools for the removal of affinity tags

PubMed Central

Austin, Brian P.; Tözsér, József; Bagossi, Péter; Tropea, Joseph E.; Waugh, David S.

2012-01-01

Carboxypeptidases may serve as tools for removal for C-terminal affinity tags. In the present study, we describe the expression and purification of an A-type carboxypeptidase from the fungal pathogen Metarhizium anisopliae (MeCPA) that has been genetically engineered to facilitate the removal of polyhistidine tags from the C-termini of recombinant proteins. A complete, systematic analysis of the specificity of MeCPA in comparison with that of bovine carboxypeptidase A (BoCPA) was carried out. Our results indicate that the specificity of the two enzymes is similar but not identical. Histidine residues are removed more efficiently by MeCPA. The very inefficient digestion of peptides with C-terminal lysine or arginine residues, along with the complete inability of the enzyme to remove a C-terminal proline suggests a strategy for designing C-terminal affinity tags that can be trimmed by MeCPA (or BoCPA) to produce a digestion product with a homogeneous endpoint. PMID:21073956

The role of Monosaccharide Transport Proteins in carbohydrate assimilation, distribution, metabolism and homeostasis

PubMed Central

Cura, Anthony J.; Carruthers, Anthony

2012-01-01

The facilitated diffusion of glucose, galactose, fructose, urate, myoinositol and dehydroascorbic acid in mammals is catalyzed by a family of 14 monosaccharide transport proteins called GLUTs. These transporters may be divided into 3 classes according to sequence similarity and function/substrate specificity. GLUT1 appears to be highly expressed in glycolytically active cells and has been co-opted in vitamin C auxotrophs to maintain the redox state of the blood through transport of dehydroascorbate. Several GLUTs are definitive glucose/galactose transporters, GLUT2 and GLUT5 are physiologically important fructose transporters, GLUT9 appears to be a urate transporter while GLUT13 (HMIT1) is a proton/myoinositol co-transporter. The physiologic substrates of some GLUTs remain to be established. The GLUTs are expressed in a tissue specific manner where affinity, specificity and capacity for substrate transport are paramount for tissue function. Although great strides have been made in characterizing GLUT-catalyzed monosaccharide transport and mapping GLUT membrane topography and determinants of substrate specificity, a unifying model for GLUT structure and function remains elusive. The GLUTs play a major role in carbohydrate homeostasis and the redistribution of sugar-derived carbons among the various organ systems. This is accomplished through a multiplicity of GLUT-dependent glucose sensing and effector mechanisms that regulate monosaccharide ingestion, absorption, distribution, cellular transport and metabolism and recovery/retention. Glucose transport and metabolism have co-evolved in mammals to support cerebral glucose utilization. PMID:22943001

Isolation and characterization of Dehalobacter sp. strain UNSWDHB capable of chloroform and chlorinated ethane respiration.

PubMed

Wong, Yie K; Holland, Sophie I; Ertan, Haluk; Manefield, Mike; Lee, Matthew

2016-09-01

Dehalobacter sp. strain UNSWDHB can dechlorinate up to 4 mM trichloromethane at a rate of 0.1 mM per day to dichloromethane and 1,1,2-trichloroethane (1 mM, 0.1 mM per day) with the unprecedented product profile of 1,2-dichloroethane and vinyl chloride. 1,1,1-trichloroethane and 1,1-dichloroethane were slowly utilized by strain UNSWDHB and were not completely removed, with minimum threshold concentrations of 0.12 mM and 0.07 mM respectively under growth conditions. Enzyme kinetic experiments confirmed strong substrate affinity for trichloromethane and 1,1,2-trichloroethane (Km  = 30 and 62 µM respectively) and poor substrate affinity for 1,1,1-trichloroethane and 1,1-dichloroethane (Km  = 238 and 837 µM respectively). Comparison of enzyme kinetic and growth data with other trichloromethane respiring organisms (Dehalobacter sp. strain CF and Desulfitobacterium sp. strain PR) suggests an adaptation of strain UNSWDHB to trichloromethane. The trichloromethane RDase (TmrA) expressed by strain UNSWDHB was identified by BN-PAGE and functionally characterized. Amino acid comparison of homologous RDases from all three organisms revealed only six significant amino acid substitutions/deletions, which are likely to be crucial for substrate specificity. Furthermore, strain UNSWDHB was shown to grow without exogenous supply of cobalamin confirming genomic-based predictions of a fully functional cobalamin synthetic pathway. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

PubMed

Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

2015-01-01

A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation. Copyright © 2014 Elsevier B.V. All rights reserved.

A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D-sorbitol

PubMed Central

2009-01-01

Background L-arabitol dehydrogenase (LAD) and xylitol dehydrogenase (XDH) are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological substrate, but also that only XDH has significant activity on D-sorbitol and may therefore be more closely related to D-sorbitol dehydrogenases (SDH). In this study we aimed to identify residues involved in the difference in substrate specificity. Results Phylogenetic analysis demonstrated that LAD, XDH and SDH form 3 distinct groups of the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240) and likely have evolved from a common ancestor. Modelling of LadA and XdhA of the saprobic fungus Aspergillus niger on human SDH identified two residues in LadA (M70 and Y318), that may explain the absence of activity on D-sorbitol. While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Moreover, the affinity for D-sorbitol was increased in this mutant. Conclusion These data demonstrates that Y318 of LadA contributes significantly to the substrate specificity difference between LAD and XDH/SDH. PMID:19674460

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walker, Johnnie A.; Takasuka, Taichi E.; Deng, Kai

Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolyticmore » activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.« less

Structure-based Analysis to Hu-DNA Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swinger,K.; Rice, P.

2007-01-01

HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently publishedmore » Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from 10-14.5 kcal/mol, representing K{sub d} values in the nanomolar to low picomolar range, and a maximum stabilization of at least 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.« less

Species differences in the pharmacokinetics of cefadroxil as determined in wildtype and humanized PepT1 mice.

PubMed

Hu, Yongjun; Smith, David E

2016-05-01

PepT1 (SLC15A1) is a high-capacity low-affinity transporter that is important in the absorption of digested di/tripeptides from dietary protein in the small intestine. PepT1 is also crucial for the intestinal uptake and absorption of therapeutic agents such as the β-lactam aminocephalosporins and antiviral prodrugs. Species differences, however, have been observed in PepT1-mediated intestinal absorption and pharmacokinetics, thereby, making it more difficult to predict systemic drug exposure. In the present study, we evaluated the in situ intestinal permeability of the PepT1 substrate cefadroxil in wildtype and humanized PepT1 (huPepT1) mice, and the in vivo absorption and disposition of drug after escalating oral doses. The in situ perfusions indicated that cefadroxil had a twofold higher affinity (i.e., twofold lower Km) for jejunal PepT1 in huPepT1 mice, lower but substantial permeability in all regions of the small intestine, and low but measureable permeability in the colon as compared to wildtype animals. The in vivo experiments indicated almost superimposable pharmacokinetic profiles between the two genotypes after intravenous bolus dosing of cefadroxil. In contrast, after oral dose escalation, the systemic exposure of cefadroxil was reduced in huPepT1 mice as compared to wildtype animals. Moreover, the AUC and Cmax versus dose relationships were nonlinear for huPepT1 but not wildtype mice, and similar to that observed from human subjects. In conclusion, our findings indicate that huPepT1 mice may provide a valuable tool in the drug discovery process by better predicting the oral pharmacokinetic profiles of PepT1 substrates in humans. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of a nicotinamide/nicotinate mononucleotide adenylyltransferase in Giardia lamblia (GlNMNAT).

PubMed

Forero-Baena, Nicolás; Sánchez-Lancheros, Diana; Buitrago, July Constanza; Bustos, Victor; Ramírez-Hernández, María Helena

2015-01-01

Giardia lamblia is an intestinal protozoan parasite that causes giardiasis, a disease of high prevalence in Latin America, Asia and Africa. Giardiasis leads to poor absorption of nutrients, severe electrolyte loss and growth retardation. In addition to its clinical importance, this parasite is of special biological interest due to its basal evolutionary position and simplified metabolism, which has not been studied thoroughly. One of the most important and conserved metabolic pathways is the biosynthesis of nicotinamide adenine dinucleotide (NAD). This molecule is widely known as a coenzyme in multiple redox reactions and as a substrate in cellular processes such as synthesis of Ca 2+ mobilizing agents, DNA repair and gene expression regulation. There are two pathways for NAD biosynthesis, which converge at the step catalyzed by nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18). Using bioinformatics tools, we found two NMNAT sequences in Giardia lamblia ( glnmnat-a and glnmnat-b ). We first verified the identity of the sequences in silico . Subsequently, glnmnat-a was cloned into an expression vector. The recombinant protein (His-GlNMNAT) was purified by nickel-affinity binding and was used in direct in vitro enzyme assays assessed by C18-HPLC, verifying adenylyltransferase activity with both nicotinamide (NMN) and nicotinic acid (NAMN) mononucleotides. Optimal reaction pH and temperature were 7.3 and 26 °C. Michaelis-Menten kinetics were observed for NMN and ATP, but saturation was not accomplished with NAMN, implying low affinity yet detectable activity with this substrate. Double-reciprocal plots showed no cooperativity for this enzyme. This represents an advance in the study of NAD metabolism in Giardia spp.

A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase from Rahnella aquatilis with Significantly Reduced Glyphosate Sensitivity

PubMed Central

Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Chen, Chen; Jin, Xiao-Fen; Yao, Quan-Hong

2012-01-01

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroAR.aquatilis, was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroAE.coli), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroAR.aquatilis were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroAE.coli. To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious. PMID:22870190

Identification of human leukemia antigen A*0201-restricted epitopes derived from epidermal growth factor pathway substrate number 8.

PubMed

Tang, Baishan; Zhou, Weijun; Du, Jingwen; He, Yanjie; Li, Yuhua

2015-08-01

T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demonstrated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design.

Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

DOE PAGES

Walker, Johnnie A.; Takasuka, Taichi E.; Deng, Kai; ...

2015-12-21

Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolyticmore » activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.« less

Structural Basis of Glyphosate Resistance Resulting from the Double Mutation Thr97 → Ile and Pro101 → Ser in 5-Enolpyruvylshikimate-3-phosphate Synthase from Escherichia coli*S⃞

PubMed Central

Funke, Todd; Yang, Yan; Han, Huijong; Healy-Fried, Martha; Olesen, Sanne; Becker, Andreas; Schönbrunn, Ernst

2009-01-01

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (Ki = 2.4 mm) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (Km = 0.1 mm). The crystal structure at 1.7-Å resolution revealed that the dual mutation causes a shift of residue Gly96 toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile97 points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr97 and Pro101 induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS. PMID:19211556

Structural basis of glyphosate resistance resulting from the double mutation Thr97 -> Ile and Pro101 -> Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli.

PubMed

Funke, Todd; Yang, Yan; Han, Huijong; Healy-Fried, Martha; Olesen, Sanne; Becker, Andreas; Schönbrunn, Ernst

2009-04-10

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (K(i) = 2.4 mm) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (K(m) = 0.1 mm). The crystal structure at 1.7-A resolution revealed that the dual mutation causes a shift of residue Gly(96) toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile(97) points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr(97) and Pro(101) induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS.

The Role of Human Cytochrome P450 Enzymes in the Formation of 2-Hydroxymetronidazole: CYP2A6 is the High Affinity (Low Km) Catalyst

PubMed Central

Cohen-Wolkowiez, Michael; Sampson, Mario R.; Kearns, Gregory L.

2013-01-01

Despite metronidazole’s widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a “therapeutic concentration” of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo. PMID:23813797

Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.

PubMed

Dean, Richard A; Butler, Georgina S; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R; Courty, José; Overall, Christopher M

2007-12-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.

Isolation and characterization of mutated alcohol oxidases from the yeast Hansenula polymorpha with decreased affinity toward substrates and their use as selective elements of an amperometric biosensor

PubMed Central

Dmytruk, Kostyantyn V; Smutok, Oleh V; Ryabova, Olena B; Gayda, Galyna Z; Sibirny, Volodymyr A; Schuhmann, Wolfgang; Gonchar, Mykhailo V; Sibirny, Andriy A

2007-01-01

Background Accurate, rapid, and economic on-line analysis of ethanol is very desirable. However, available biosensors achieve saturation at very low ethanol concentrations and thus demand the time and labour consuming procedure of sample dilution. Results Hansenula polymorpha (Pichia angusta) mutant strains resistant to allyl alcohol in methanol medium were selected. Such strains possessed decreased affinity of alcohol oxidase (AOX) towards methanol: the KM values for AOX of wild type and mutant strains CA2 and CA4 are shown to be 0.62, 2.48 and 1.10 mM, respectively, whereas Vmax values are increased or remain unaffected. The mutant AOX alleles from H. polymorpha mutants CA2 and CA4 were isolated and sequenced. Several point mutations in the AOX gene, mostly different between the two mutant alleles, have been identified. Mutant AOX forms were isolated and purified, and some of their biochemical properties were studied. An amperometric biosensor based on the mutated form of AOX from the strain CA2 was constructed and revealed an extended linear response to the target analytes, ethanol and formaldehyde, as compared to the sensor based on the native AOX. Conclusion The described selection methodology opens up the possibility of isolating modified forms of AOX with further decreased affinity toward substrates without reduction of the maximal velocity of reaction. It can help in creation of improved ethanol biosensors with a prolonged linear response towards ethanol in real samples of wines, beers or fermentation liquids. PMID:17567895

Identification of Candidate Angiogenic Inhibitors Processed by Matrix Metalloproteinase 2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis▿ †

PubMed Central

Dean, Richard A.; Butler, Georgina S.; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R.; Courty, José; Overall, Christopher M.

2007-01-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. PMID:17908800

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet.

PubMed

Yamada, Nana; Sakakibara, Shota; Tsutsumi, Koichi; Waditee, Rungaroon; Tanaka, Yoshito; Takabe, Teruhiro

2011-09-15

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells. Copyright © 2011 Elsevier GmbH. All rights reserved.

Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18

PubMed Central

Hu, Qi; Botuyan, Maria Victoria; Cui, Gaofeng; Zhao, Debiao

2017-01-01

Summary The protein 53BP1 plays a central regulatory role in DNA double-strand break repair. 53BP1 relocates to chromatin by recognizing RNF168-mediated mono-ubiquitylation of histone H2A Lys15 in the nucleosome core particle dimethylated at histone H4 Lys20 (NCP-ubme). 53BP1 relocation is terminated by ubiquitin ligases RNF169 and RAD18 via unknown mechanisms. Using NMR spectroscopy and biochemistry, we show that RNF169 bridges ubiquitin and histone surfaces, stabilizing a pre-existing ubiquitin orientation in NCP-ubme to form a high-affinity complex. This conformational selection mechanism contrasts with the low-affinity binding mode of 53BP1 and ensures 53BP1 displacement by RNF169 from NCP-ubme. We also show that RAD18 binds tightly to NCP-ubme through a ubiquitin-binding domain that contacts ubiquitin and nucleosome surfaces accessed by 53BP1. Our work uncovers diverse ubiquitin recognition mechanisms in the nucleosome, explaining how RNF168, RNF169 and RAD18 regulate 53BP1 chromatin recruitment and how specificity can be achieved in the recognition of a ubiquitin-modified substrate. PMID:28506460

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dedic, Emil; Seweryn, Paulina; Jonstrup, Anette Thyssen

Highlights: • We show that S. cerevisiae Rrp6p and Rrp47p stabilise each other in vitro. • We determine molecular envelopes of the Rrp6p–Rrp47p complex by SAXS. • Rrp47p binds at the top of the Rrp6p exonuclease domain. • Rrp47p modulates the activity of Rrp6p on a variety of RNA substrates. • Rrp47p does not affect RNA affinity by Rrp6p. - Abstract: The RNase D-type 3′–5′ exonuclease Rrp6p from Saccharomyces cerevisiae is a nuclear-specific cofactor of the RNA exosome and associates in vivo with Rrp47p (Lrp1p). Here, we show using biochemistry and small-angle X-ray scattering (SAXS) that Rrp6p and Rrp47p associatemore » into a stable, heterodimeric complex with an elongated shape consistent with binding of Rrp47p to the nuclease domain and opposite of the HRDC domain of Rrp6p. Rrp47p reduces the exonucleolytic activity of Rrp6p on both single-stranded and structured RNA substrates without significantly altering the affinity towards RNA or the ability of Rrp6p to degrade RNA secondary structure.« less

Membrane protease degradomics: proteomic identification and quantification of cell surface protease substrates.

PubMed

Butler, Georgina S; Dean, Richard A; Smith, Derek; Overall, Christopher M

2009-01-01

The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.

The use of nanomaterials for mass spectrometry can be uplifting for analyte detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, J.; Lipson, R. H.

2014-03-31

Surface-Assisted Laser Desorption Ionization (SALDI) involves desorbing and ionizing analyte molecules from a nanoporous substrate by laser irradiation for detection in a mass spectrometer. In this work experiments were designed to better understand the mechanisms governing desorption and ionization for Desorption Ionization On Silicon (DIOS), a variant of SALDI which uses porous silicon (pSi) as a substrate. Experiments are also reported for other nanoporous semiconducting materials (WO{sub 3}, TiO{sub 2}) which exhibit very similar behaviors; specifically, that both protonated analyte ions and analyte radical cations can be generated with relative intensities that depend on the position of the incident lasermore » focus relative to substrate surface. While thermal desorption appears to be important, preliminary evidence suggests that the ionization mechanism leading to protonated analytes involves in part electrons and holes formed when photoexciting the substrate above its electronic band gap, and the presence of defect states within the band gap. Radical cation formation appears to be driven in part by electron transfer due to the large electron affinity of each substrate used in this work.« less

Biphasic Kinetic Behavior of Nitrate Reductase from Heterocystous, Nitrogen-Fixing Cyanobacteria 1

PubMed Central

Martin-Nieto, José; Flores, Enrique; Herrero, Antonia

1992-01-01

Nitrate reductase activity from filamentous, heterocyst-forming cyanobacteria showed a biphasic kinetic behavior with respect to nitrate as the variable substrate. Two kinetic components were detected, the first showing a higher affinity for nitrate (Km, 0.05-0.25 mm) and a lower catalytic activity and the second showing a lower affinity for nitrate (Km, 5-25 mm) and a higher (3- to 5-fold) catalytic activity. In contrast, among unicellular cyanobacteria, most representatives studied exhibited a monophasic, Michaelis-Menten kinetic pattern for nitrate reductase activity. Biphasic kinetics remained unchanged with the use of different assay conditions (i.e. cell disruption or permeabilization, two different electron donors) or throughout partial purification of the enzyme. PMID:16652939

Concentration of (+/-)-propranolol in isolated, perfused lungs of rat.

PubMed Central

Dollery, C T; Junod, A F

1976-01-01

1 The metabolism and the accumulation of (+/-)-propranolol have been studied in isolated lungs of the rat, perfused with an artificial medium. 2 Little or no metabolism took place during the perfusion periods (up to 10 minutes). 3 Accumulation was observed with high tissue/medium ratios for substrate concentrations of 0.2 muM to 1 mM; there was evidence for saturability, but no real plateau could be seen. The presence of two binding sites with different affinities was established. 4 Cold greatly inhibited the accumulation process at low substrate concentrations, but had no effect at 1 mM propranolol. 5 Inhibition of accumulation was measured in the presence of imipramine, desmethylimipramine, nortryptiline, chlorpromazine and of Na+-free medium. Cocaine, 5-hydroxytryptamine and noradrenaline had no effect. Lidocaine enhanced the accumulation process. Release of previously bound propranolol was accelerated in the presence of propranolol and imipramine, unaffected by a Na+-free medium and decreased by cold and by lidocaine. 6 Experiments on lung tissue slices yielded qualitatively similar results to those obtained with perfused lungs. Ouabain and KCN had no or little effect on propranolol accumulation. PMID:1276542

Application of Self-Assembled Monolayers to the Electroless Metallization of High Aspect Ratio Vias for Microelectronics

NASA Astrophysics Data System (ADS)

Bernasconi, R.; Molazemhosseini, A.; Cervati, M.; Armini, S.; Magagnin, L.

2016-10-01

All-wet electroless metallization of through-silicon vias (TSVs) with a width of 5 μm and a 1:10 aspect ratio was carried out. Immersion in a n-(2-aminoethyl) 3-aminopropyl-trimethoxysilane (AEAPTMS) self-assembled monolayer (SAM) was used to enhance the adhesion between the metal film and substrate. Contact angle variation and atomic force microscopy were used to verify the formation of a SAM layer. A PdCl2 solution was later used to activate the silanized substrates, exploiting the affinity of the -NH3 functional group of AEAPTMS to palladium. A nickel-phosphorus-boron electroless bath was employed to deposit the first barrier layer onto silicon. The NiPB growth rate was evaluated on flat silicon wafers, while the structure of the coating obtained was investigated via glow discharge optical emission spectroscopy. Cross-sectional scanning electron microscope observations were carried out on metallized TSVs to characterize the NiPB seed, the Cu seed layer deposited with a second electroless step, and the Cu superfilling obtained with a commercial solution. Complete filling of TSV was achieved.

Crystal structures of substrate-free and retinoic acid-bound cyanobacterial cytochrome P450 CYP120A1.

PubMed

Kühnel, Karin; Ke, Na; Cryle, Max J; Sligar, Stephen G; Schuler, Mary A; Schlichting, Ilme

2008-06-24

The crystal structures of substrate-free and all-trans-retinoic acid-bound CYP120A1 from Synechocystis sp. PCC 6803 were determined at 2.4 and 2.1 A resolution, respectively, representing the first structural characterization of a cyanobacterial P450. Features of CYP120A1 not observed in other P450 structures include an aromatic ladder flanking the channel leading to the active site and a triple-glycine motif within SRS5. Using spectroscopic methods, CYP120A1 is shown to bind 13-cis-retinoic acid, 9-cis-retinoic acid, and retinal with high affinity and dissociation constants of less than 1 microM. Metabolism of retinoic acid by CYP120A1 suggests that CYP120A1 hydroxylates a variety of retinoid derivatives in vivo. On the basis of the retinoic acid-bound CYP120A1 crystal structure, we propose that either carbon 2 or the methyl groups (C16 or C17) of the beta-ionone ring are modified by CYP120A1.

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halavaty, Andrei S.; Rich, Rebecca L.; Chen, Chao

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL ( SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NADmore » +) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD +, NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Finally, five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.« less

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus

DOE PAGES

Halavaty, Andrei S.; Rich, Rebecca L.; Chen, Chao; ...

2015-04-25

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL ( SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NADmore » +) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD +, NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Finally, five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.« less

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

PubMed

Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

2005-08-01

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damage.

PubMed

Wada, Fumitaka; Hasegawa, Hiroki; Nakamura, Akio; Sugimura, Yoshiaki; Kawai, Yoshiki; Sasaki, Narie; Shibata, Hideki; Maki, Masatoshi; Hitomi, Kiyotaka

2007-06-01

Transglutaminases are Ca(2+)-dependent enzymes that post-translationally modify proteins by crosslinking or polyamination at specific polypeptide-bound glutamine residues. Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases. We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage. With use of a biotin-labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity-purified from the damaged slime mold. The purified proteins were Physarum actin, a 40 kDa Ca(2+)-binding protein with four EF-hand motifs (CBP40), and a novel 33 kDa protein highly homologous to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria. Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS/PAGE. Of the three proteins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence. These results suggested that transglutimase reactions function in the response to mechanical injury.

An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

PubMed

Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

2015-05-01

An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity. Copyright © 2015 Elsevier Inc. All rights reserved.

Mechanism of UCH-L5 Activation and Inhibition by DEUBAD Domains in RPN13 and INO80G

PubMed Central

Sahtoe, Danny D.; van Dijk, Willem J.; El Oualid, Farid; Ekkebus, Reggy; Ovaa, Huib; Sixma, Titia K.

2015-01-01

Summary Deubiquitinating enzymes (DUBs) control vital processes in eukaryotes by hydrolyzing ubiquitin adducts. Their activities are tightly regulated, but the mechanisms remain elusive. In particular, the DUB UCH-L5 can be either activated or inhibited by conserved regulatory proteins RPN13 and INO80G, respectively. Here we show how the DEUBAD domain in RPN13 activates UCH-L5 by positioning its C-terminal ULD domain and crossover loop to promote substrate binding and catalysis. The related DEUBAD domain in INO80G inhibits UCH-L5 by exploiting similar structural elements in UCH-L5 to promote a radically different conformation, and employs molecular mimicry to block ubiquitin docking. In this process, large conformational changes create small but highly specific interfaces that mediate activity modulation of UCH-L5 by altering the affinity for substrates. Our results establish how related domains can exploit enzyme conformational plasticity to allosterically regulate DUB activity. These allosteric sites may present novel insights for pharmaceutical intervention in DUB activity. PMID:25702870

Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana L.) fruit.

PubMed

Bravo, Karent; Osorio, Edison

2016-04-15

Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was isolated and biochemically characterized. The enzyme was extracted and purified using acetone and aqueous two-phase systems. The data indicated that PPO had the highest substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid was the most suitable substrate (Km=0.56±0.07 mM and Vmax=53.15±2.03 UPPO mL(-1) min(-1)). The optimal pH values were 5.5 for catechol and 4-methylcatechol and 5.0 for chlorogenic acid. Optimal temperatures were 40°C for catechol, 25°C for 4-methylcatechol and 20°C for chlorogenic acid. In inhibition tests, the most potent inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This study shows possible treatments that can be implemented during the processing of Cape gooseberry fruits to prevent browning. Copyright © 2015 Elsevier Ltd. All rights reserved.

An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.

PubMed

Sueda, Shinji; Shinboku, Yuki; Kusaba, Takeshi

2013-01-01

Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb(3+) complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.

IgG1 memory B cells keep the memory of IgE responses.

PubMed

He, Jin-Shu; Subramaniam, Sharrada; Narang, Vipin; Srinivasan, Kandhadayar; Saunders, Sean P; Carbajo, Daniel; Wen-Shan, Tsao; Hidayah Hamadee, Nur; Lum, Josephine; Lee, Andrea; Chen, Jinmiao; Poidinger, Michael; Zolezzi, Francesca; Lafaille, Juan J; Curotto de Lafaille, Maria A

2017-09-21

The unique differentiation of IgE cells suggests unconventional mechanisms of IgE memory. IgE germinal centre cells are transient, most IgE cells are plasma cells, and high affinity IgE is produced by the switching of IgG1 cells to IgE. Here we investigate the function of subsets of IgG1 memory B cells in IgE production and find that two subsets of IgG1 memory B cells, CD80 + CD73 + and CD80 - CD73 - , contribute distinctively to the repertoires of high affinity pathogenic IgE and low affinity non-pathogenic IgE. Furthermore, repertoire analysis indicates that high affinity IgE and IgG1 plasma cells differentiate from rare CD80 + CD73 + high affinity memory clones without undergoing further mutagenesis. By identifying the cellular origin of high affinity IgE and the clonal selection of high affinity memory B cells into the plasma cell fate, our findings provide fundamental insights into the pathogenesis of allergies, and on the mechanisms of antibody production in memory B cell responses.IgE is an important mediator of protective immunity as well as allergic reaction, but how high affinity IgE antibodies are produced in memory responses is not clear. Here the authors show that IgE can be generated via class-switch recombination in IgG1 memory B cells without additional somatic hypermutation.

Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif

PubMed Central

Song, Ruiwen; Li, Jing; Zhang, Jin; Wang, Lu; Tong, Li; Wang, Ping; Yang, Huan; Wei, Qun; Cai, Huaibin; Luo, Jing

2018-01-01

Calcineurin (CN) is involved in many physiological processes and interacts with multiple substrates. Most of the substrates contain similar motifs recognized by CN. Recent studies revealed a new CN substrate, transcription factor EB (TFEB), which is involved in autophagy. We showed that a 15-mer QSYLENPTSYHLQQS peptide from TFEB (TFEB-YLENP) bound to CN. When the TFEB-YLENP peptide was changed to YLAVP, its affinity for CN increased and it had stronger CN inhibitory activity. Molecular dynamics simulations revealed that the TFEB-YLENP peptide has the same docking sites in CN as the 15-mer DQYLAVPQHPYQWAK motif of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1-YLAVP). Moreover expression of the NFATc1-YLAVP peptide suppressed the TFEB activation in starved Hela cells. Our studies first identified a CN binding site in TFEB and compared the inhibitory capability of various peptides derived from CN substrates. The data uncovered a diversity in recognition sequences that underlies the CN signaling within the cell. Studies of CN-substrate interactions should lay the groundwork for developing selective CN peptide inhibitors that target CN-substrate interaction in vitro experiments. PMID:28890387

Hydrophilic TiO2 porous spheres anchored on hydrophobic polypropylene membrane for wettability induced high photodegrading activities.

PubMed

Niu, Fang; Zhang, Le-Sheng; Chen, Chao-Qiu; Li, Wei; Li, Lin; Song, Wei-Guo; Jiang, Lei

2010-08-01

TiO(2) porous nanospheres on polypropylene (PP) films (TiO(2)/PP composite) are produced at ambient temperature. Particle/pore size match up is the key anchoring point to overcome the low affinity between hydrophilic materials and hydrophobic materials. With the hydrophilic TiO(2) catalyst evenly dispersed on a hydrophobic surface, the aqueous solution will selectively skip the substrate and wet the catalysts. Such a wettability-induced smart system maximizes the degrading activity of the TiO(2) catalyst. In photodegrading reactions, the resulting TiO(2)/PP composite film exhibits a 10 times higher activity in flow-type setup than the same TiO(2) catalyst in a traditional batch-type setup.

Functionalized gold nanostars for label-free detection of PKA phosphorylation using surface-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

He, Shuai; Kah, James C. Y.

2017-04-01

Protein phosphorylation controls fundamental biological processes. Dysregulation of protein kinase is associated with a series of human diseases including cancer. Protein kinase A (PKA) activity has been reported to serve as a potential prognostic marker for cancer. To this end, we developed a non-radioactive, rapid, cheap and robust scheme based on surface-enhanced Raman spectroscopy (SERS) for label-free detection of PKA phosphorylation using gold nanostars (AuNS) functionalized with BSA-kemptide. While bovine serum albumin (BSA) proteins stabilized the AuNS, kemptide, which is a high affinity substrate peptide specific for PKA, were phosphorylated in vitro to generate Raman signals that were identified by performing principal component analysis (PCA) on the acquired SERS spectra.

Spectroscopic analysis and the excellent reusability of sphere-capped ferrocene in the oxidation of glucose oxidase.

PubMed

Antepli, Esin; Sarı, Nurşen

2016-08-01

Sphere-capped ferrocene nanospheres with Schiff base spacers have been prepared using a template, and used as carriers to immobilize glucose oxidase (GOx). GOx immobilized on spheres with one C-spacer (APS-Fc) exhibited high binding affinity to the substrate, which was attributed to appropriate position for the GOx conformation. When glucose oxidase was immobilized with spacers of different lengths, it was found that storage stability decreased with increasing the length of the spacer. It has been found that nanospheres, including capped ferrocene, exhibit good performance as the immobilized supporters of GOx. (APS-EtFc-GOx) retain more than 10% of the initial activity after forty-two successive cycles, which is a remarkable result.

Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sine, Steven M.; Huang, Sun; Li, Shu-Xing

2013-09-01

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less

[Interaction of human factor X with thromboplastin].

PubMed

Kiselev, S V; Zubairov, D M; Timarbaev, V N

2003-01-01

The binding of 125I-labeled human factor X to native and papaine-treated tissue tromboplastin in the presence of CaCl2 or EDTA was studied. The Scatchard analysis suggests the existence of high (Kd=l,8 x10(-9) M) and low affinity binding sites on the thromboplastin surface. The removal of Ca2+ reduced affinity of factor X to the high affinity sites. This was accompanied by some increase of their number. Proteolysis by papaine decreased affinity of high affinity sites and caused the increase of their number in the presence of Ca2+. In the absence of Ca2+ the affinity remained unchanged, but the number of sites decreased. At low concentrations of factor X positive cooperativity for high affinity binding sites was observed. It did not depend on the presence of Ca2+. The results indirectly confirm the role of hydrophobic interactons in Ca2+ dependent binding of factor X to thromboplastin and the fact that heterogeneity of this binding is determined by mesophase structure of the thromboplastin phospholipids.

Enhanced Requirement for TNFR2 in Graft Rejection Mediated by Low Affinity Memory CD8+ T Cells During Heterologous Immunity

PubMed Central

Krummey, Scott M.; Chen, Ching-Wen; Guasch, Sara A.; Liu, Danya; Wagener, Maylene; Larsen, Christian P; Ford, Mandy L.

2016-01-01

The affinity of a T cell receptor (TCR) binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic antigen and mediate graft rejection. We found that low affinity primed memory CD8+ T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared to high affinity primed memory T cells. Low affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2hi proliferating cells. Low affinity primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low but not high affinity primed animals. These data establish a functional connection between TNF signaling and TCR priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation. PMID:27481849

Selecting the correct cellular model for assessing of the biological response of collagen-based biomaterials.

PubMed

Davidenko, Natalia; Hamaia, Samir; Bax, Daniel V; Malcor, Jean-Daniel; Schuster, Carlos F; Gullberg, Donald; Farndale, Richard W; Best, Serena M; Cameron, Ruth E

2018-01-01

Accurate evaluation of the biological performance of biomaterials requires the correct assessment of their native-like cell ligation properties. However, cell attachment studies often overlook the details of the substrate-cell binding mechanisms, be they integrin-mediated or non-specific, and ignore the class- and species-specificities of the cell adhesion receptor involved. In this work we have used different collagen (Col) substrates (fibrillar collagens I, II and III and network-forming Col IV), containing different affinity cell-recognition motifs, to establish the influence of the receptor identity and species-specificity on collagen-cell interactive properties. Receptor expression was varied by using cells of different origin, or transfecting collagen-binding integrins into integrin-null cells. These include mouse C2C12 myoblasts transfected with human α1, α2, α10 or α11; human fibrosarcoma HT1080 cells which constitutively express only human α2β1, and rat glioma Rugli cells, with only rat α1β1. Using these lines, the nature of integrin binding sites was studied in order to delineate the bioactivity of different collagen substrates. Integrin ligation was studied on collagen coatings alongside synthetic (GFOGER/GLOGEN) and Toolkit (Col II-28/Col III-7) triple-helical peptides to evaluate (1) their affinity towards different integrins and (2) to confirm the activity of the inserted integrin in the transfected cells. Thin films of dermal and tendon Col I were used to evaluate the influence of the carbodiimide (EDC)-based treatment on the cellular response on Col of different origin. The results showed that the binding properties of transfected C2C12 cells to collagens depend on the identity of inserted integrin. Similar ligation characteristics were observed using α1+ and α10+ cells, but these were distinct from the similar binding features of α2+ and α11+ cells. Recombinant human and rat-α1 I domain binding to collagens and peptides correlated with the cell adhesion results, showing receptor class- and species-specificities. The understanding of the physiologically relevant cell anchorage characteristics of bio-constructs may assist in the selection of (1) the optimum collagen source for cellular supports and (2) the correct cellular model for their biological assessment. This, in turn, may allow reliable prediction of the biological performance of bio-scaffolds in vivo for specific TE applications. Integrins play a vital role in cellular responses to environmental cues during early-stage cell-substrate interaction. We describe physiologically relevant cell anchorage to collagen substrates that present different affinity cell-recognition motifs, to provide experimental tools to assist in understanding integrin binding. Using different cell types and recombinant integrin α1-I-domains, we found that cellular response was highly dependent on collagen type, origin and EDC-crosslinking status, as well as on the integrin class and species of origin. This comprehensive study establishes selectivity amongst the four collagen-binding integrins and species-specific properties that together may influence choice of cell type and receptor in different experimental settings. This work offers key guidance in selecting of the correct cellular model for the biological testing of collagen-based biomaterials. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Information encoded in non-native states drives substrate-chaperone pairing.

PubMed

Mapa, Koyeli; Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik

2012-09-05

Many proteins refold in vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, in vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated in vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones. Copyright © 2012 Elsevier Ltd. All rights reserved.

An allosteric conduit facilitates dynamic multisite substrate recognition by the SCFCdc4 ubiquitin ligase

NASA Astrophysics Data System (ADS)

Csizmok, Veronika; Orlicky, Stephen; Cheng, Jing; Song, Jianhui; Bah, Alaji; Delgoshaie, Neda; Lin, Hong; Mittag, Tanja; Sicheri, Frank; Chan, Hue Sun; Tyers, Mike; Forman-Kay, Julie D.

2017-01-01

The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.

Improved xylose uptake in Saccharomyces cerevisiae due to directed evolution of galactose permease Gal2 for sugar co-consumption.

PubMed

Reznicek, O; Facey, S J; de Waal, P P; Teunissen, A W R H; de Bont, J A M; Nijland, J G; Driessen, A J M; Hauer, B

2015-07-01

Saccharomyces cerevisiae does not express any xylose-specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed. Three rounds of error-prone PCR were used to generate mutants with improved xylose-transport characteristics. After developing a fast and reliable high-throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild-type Gal2 out of 41 200 single yeast cells. Gal2 variant 2·1 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3·1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose. Random mutagenesis of the S. cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co-consumption. Random mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co-consumption of new substrates. Using a high-throughput screening system based on flow-through cytometry, various mutants were identified with improved xylose-transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass. © 2015 The Society for Applied Microbiology.

Characterization of loxoprofen transport in Caco-2 cells: the involvement of a proton-dependent transport system in the intestinal transport of loxoprofen.

PubMed

Narumi, Katsuya; Kobayashi, Masaki; Kondo, Ayuko; Furugen, Ayako; Yamada, Takehiro; Takahashi, Natsuko; Iseki, Ken

2016-11-01

Loxoprofen, a propionate non-steroidal anti-inflammatory drug (NSAID), is used widely in East Asian countries. However, little is known about the transport mechanisms contributing to its intestinal absorption. The objectives of this study were to characterize the intestinal transport of loxoprofen using the human intestinal Caco-2 cell model. The transport of loxoprofen was investigated in cellular uptake studies. The uptake of loxoprofen into Caco-2 cells was pH- and concentration-dependent, and was described by a Michaelis-Menten equation with passive diffusion (K m : 4.8 mm, V max : 142 nmol/mg protein/30 s, and K d : 2.2 μl/mg protein/30 s). Moreover, the uptake of loxoprofen was inhibited by a typical monocarboxylate transporter (MCT) inhibitor as well as by various monocarboxylates. The uptake of [ 14 C] l-lactic acid, a typical MCT substrate, in Caco-2 cells was saturable with relatively high affinity for MCT. Because loxoprofen inhibited the uptake of [ 14 C] l-lactic acid in a noncompetitive manner, it was unlikely that loxoprofen uptake was mediated by high-affinity MCT(s). Our results suggest that transport of loxoprofen in Caco-2 cells is, at least in part, mediated by a proton-dependent transport system. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase (HePTP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Critton, D.; Tortajada, A; Stetson, G

2008-01-01

Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPsmore » as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.« less

Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

PubMed

Batra, Jyotica; Szabó, András; Caulfield, Thomas R; Soares, Alexei S; Sahin-Tóth, Miklós; Radisky, Evette S

2013-04-05

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

D-Amino acid oxidase bio-functionalized platforms: Toward an enhanced enzymatic bio-activity

NASA Astrophysics Data System (ADS)

Herrera, Elisa; Valdez Taubas, Javier; Giacomelli, Carla E.

2015-11-01

The purpose of this work is to study the adsorption process and surface bio-activity of His-tagged D-amino acid oxidase (DAAO) from Rhodotorula gracilis (His6-RgDAAO) as the first step for the development of an electrochemical bio-functionalized platform. With such a purpose this work comprises: (a) the His6-RgDAAO bio-activity in solution determined by amperometry, (b) the adsorption mechanism of His6-RgDAAO on bare gold and carboxylated modified substrates in the absence (substrate/COO-) and presence of Ni(II) (substrate/COO- + Ni(II)) determined by reflectometry, and (c) the bio-activity of the His6-RgDAAO bio-functionalized platforms determined by amperometry. Comparing the adsorption behavior and bio-activity of His6-RgDAAO on these different solid substrates allows understanding the contribution of the diverse interactions responsible for the platform performance. His6-RgDAAO enzymatic performance in solution is highly improved when compared to the previously used pig kidney (pk) DAAO. His6-RgDAAO exhibits an amperometrically detectable bio-activity at concentrations as low as those expected on a bio-functional platform; hence, it is a viable bio-recognition element of D-amino acids to be coupled to electrochemical platforms. Moreover, His6-RgDAAO bio-functionalized platforms exhibit a higher surface activity than pkDAAO physically adsorbed on gold. The platform built on Ni(II) modified substrates present enhanced bio-activity because the surface complexes histidine-Ni(II) provide with site-oriented, native-like enzymes. The adsorption mechanism responsible of the excellent performance of the bio-functionalized platform takes place in two steps involving electrostatic and bio-affinity interactions whose prevalence depends on the degree of surface coverage.

Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

PubMed Central

Hu, L.; Borleske, B. L.; Colman, R. F.

1997-01-01

Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes. PMID:9007975

Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

PubMed

Hu, L; Borleske, B L; Colman, R F

1997-01-01

Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes.

A Single Amino Acid Substitution in the Active Site of Escherichia coli Aspartate Transcarbamoylase Prevents the Allosteric Transition

PubMed Central

Stieglitz, Kimberly A.; Pastra-Landis, Styliani C.; Xia, Jiarong; Tsuruta, Hiro; Kantrowitz, Evan R.

2005-01-01

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7Å resolution. Time-resolved small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low activity low affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase. PMID:15890205

Immobilized metal affinity cryogel-based high-throughput platform for screening bioprocess and chromatographic parameters of His6-GTPase.

PubMed

Sarkar, Joyita; Kumar, Ashok

2017-04-01

Among various tools of product monitoring, chromatography is of vital importance as it also extends to the purification of product. Immobilized metal affinity cryogel (Cu(II)-iminodiacetic acid- and Ni(II)-nitrilotriacetic acid-polyacrylamide) minicolumns (diameter 8 mm, height 4 mm, void volume 250 μl) were inserted in open-ended 96-well plate and different chromatographic parameters and bioprocess conditions were analysed. The platform was first validated with lysozyme. Optimum binding of lysozyme (∼90%) was achieved when 50 μg of protein in 20 mM Tris, pH 8.0 was applied to the minicolumns with maximum recovery (∼90%) upon elution with 300 mM imidazole. Thereafter, the platform was screened for chromatographic conditions of His 6 -GTPase. Since cryogels have large pore size, they can easily process non-clarified samples containing debris and particulate matters. The bound enzymes on the gel retain its activity and therefore can be assayed on-column by adding substrate and then displacing the product. Highest binding of His 6 -GTPase was achieved when 50 μl of non-clarified cell lysate was applied to the cryogel and subsequently washed with 50 mM Tris, 150 mM NaCl, 5 mM MgCl 2 , 10 mM imidazole, pH 8.0 with dynamic and static binding capacities of ∼1.5 and 3 activity units. Maximum recovery was obtained upon elution with 300 mM imidazole with a purification fold of ∼10; the purity was also analysed by SDS-PAGE. The platform showed reproducible results which were validated by Bland-Altman plot. The minicolumn was also scaled up for chromatographic capture and recovery of His 6 -GTPase. The bioprocess conditions were monitored which displayed that optimum production of His 6 -GTPase was attained by induction with 200 μM isopropyl-β-D-thiogalactoside at 25 °C for 12 h. It was concluded that immobilized metal affinity cryogel-based platform can be successfully used as a high-throughput platform for screening of bioprocess and chromatographic parameters. Graphical abstract Capture and on-column analysis of bound enzyme from non-clarified cell lysate on immobilized metal affinity cryogel minicolumn-based high-throughput platform.

Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

PubMed

Glisic, Branka; Mihaljevic, Ivan; Popovic, Marta; Zaja, Roko; Loncar, Jovica; Fent, Karl; Kovacevic, Radmila; Smital, Tvrtko

2015-01-01

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28 mM (Gstp2) to 3.69 mM (Gstm3), while affinity toward MCB was in the range of 5 μM (Gstt1a) to 250 μM (Gstp1). Affinity toward GSH varied from 0.27 mM (Gstz1) to 4.45 mM (Gstt1a). Turnover number for CDNB varied from 5.25s(-1) (Gstt1a) to 112s(-1) (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the first comprehensive analysis of GST superfamily in zebrafish, presents new insight into distinct functions of individual Gsts, and offers methodological protocols that can be used for further verification of interaction of environmental contaminants with fish Gsts. Copyright © 2014 Elsevier B.V. All rights reserved.

Substrate-induced stable enzyme-inhibitor complex formation allows tight binding of novel 2-aminopyrimidin-4(3H)-ones to drug-resistant HIV-1 reverse transcriptase mutants.

PubMed

Samuele, Alberta; Facchini, Marcella; Rotili, Dante; Mai, Antonello; Artico, Marino; Armand-Ugón, Mercedes; Esté, José A; Maga, Giovanni

2008-09-01

We recently reported the synthesis and biological evaluation of a novel series of 5-alkyl-2-(N,N-disubstituted)amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F(2)-N,N-DABOs). These compounds are highly active against both wild-type HIV-1 and the K103N, Y181C, and Y188L mutant strains. Herein we present novel 6-(2-chloro-6-fluorophenylalkyl)-N,N-DABO (2-Cl-6-F-N,N-DABO) derivatives and investigate the molecular basis for their high-affinity binding to HIV-1 reverse transcriptase (RT). Our results show that the new compounds display higher association rates than the difluoro derivatives toward wild-type HIV-1 RT or drug-resistant RT mutant forms. We also show that they preferentially associate to either the free enzyme or the enzyme-nucleic acid binary complex, and that this binding is stabilized upon formation of the ternary complex between HIV-1 RT and both the nucleic acid and nucleotide substrates. Interestingly, one compound showed dissociation rates from the ternary complex with RT mutants K103N and Y181I 10-20-fold slower than from the corresponding complex with wild-type RT.

Characterization of Cinnamoyl Esterases from Different Lactobacilli and Bifidobacteria.

PubMed

Fritsch, Caroline; Jänsch, André; Ehrmann, Matthias A; Toelstede, Simone; Vogel, Rudi F

2017-02-01

A high variety of plants that are used for food production contain esterified hydroxycinnamic acids. As their free forms display several benefits, like an enhanced absorption in human intestinal tract, anti-oxidative and anti-carcinogenic effects, an improved protein solubility and reduced discoloration, the microbial ability to cleave the ester bond is highly desired. In order to examine potential fermentation strains for this purpose, six different lactic acid bacteria and one bifidobacterial strain were screened for their ability to degrade esterified hydroxycinnamic acids because these strains are commonly used for fermentation of plant-based foods. Moreover, their cinnamoyl esterase activity was examined by molecular biological analyses. The enzymes were heterologously expressed in Escherichia coli, purified and biochemically characterized. The purified esterases with a molecular mass around 27-29 kDa had their optimum predominantly between pH 7 and 8 at 20-30 °C. Bifidobacterium animalis subsp. lactis, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus fermentum displayed activities against a broad substrate range (methyl caffeate, methyl trans-p-coumarate, chlorogenic acid as well as partially ethyl ferulate). Concerning substrate affinity, reaction velocity, thermal and pH stability, Lactobacillus gasseri showed the overall best performance. The herein studied lactic acid- and bifidobacteria are promising for the production of fermented plant-based foods with an increased quality and nutritional value.

Emulsifying Activity and Stability of a Non-Toxic Bioemulsifier Synthesized by Microbacterium sp. MC3B-10

PubMed Central

Camacho-Chab, Juan Carlos; Guézennec, Jean; Chan-Bacab, Manuel Jesús; Ríos-Leal, Elvira; Sinquin, Corinne; Muñiz-Salazar, Raquel; De la Rosa-García, Susana del C.; Reyes-Estebanez, Manuela; Ortega-Morales, Benjamín Otto

2013-01-01

A previously reported bacterial bioemulsifier, here termed microbactan, was further analyzed to characterize its lipid component, molecular weight, ionic character and toxicity, along with its bioemulsifying potential for hydrophobic substrates at a range of temperatures, salinities and pH values. Analyses showed that microbactan is a high molecular weight (700 kDa), non-ionic molecule. Gas chromatography of the lipid fraction revealed the presence of palmitic, stearic, and oleic acids; thus microbactan may be considered a glycolipoprotein. Microbactan emulsified aromatic hydrocarbons and oils to various extents; the highest emulsification index was recorded against motor oil (96%). The stability of the microbactan-motor oil emulsion model reached its highest level (94%) at 50 °C, pH 10 and 3.5% NaCl content. It was not toxic to Artemia salina nauplii. Microbactan is, therefore, a non-toxic and non-ionic bioemulsifier of high molecular weight with affinity for a range of oily substrates. Comparative phylogenetic assessment of the 16S rDNA gene of Microbacterium sp. MC3B-10 with genes derived from other marine Microbacterium species suggested that this genus is well represented in coastal zones. The chemical nature and stability of the bioemulsifier suggest its potential application in bioremediation of marine environments and in cosmetics. PMID:24065097

Computational design of a Diels-Alderase from a thermophilic esterase: the importance of dynamics

NASA Astrophysics Data System (ADS)

Linder, Mats; Johansson, Adam Johannes; Olsson, Tjelvar S. G.; Liebeschuetz, John; Brinck, Tore

2012-09-01

A novel computational Diels-Alderase design, based on a relatively rare form of carboxylesterase from Geobacillus stearothermophilus, is presented and theoretically evaluated. The structure was found by mining the PDB for a suitable oxyanion hole-containing structure, followed by a combinatorial approach to find suitable substrates and rational mutations. Four lead designs were selected and thoroughly modeled to obtain realistic estimates of substrate binding and prearrangement. Molecular dynamics simulations and DFT calculations were used to optimize and estimate binding affinity and activation energies. A large quantum chemical model was used to capture the salient interactions in the crucial transition state (TS). Our quantitative estimation of kinetic parameters was validated against four experimentally characterized Diels-Alderases with good results. The final designs in this work are predicted to have rate enhancements of ≈103-106 and high predicted proficiencies. This work emphasizes the importance of considering protein dynamics in the design approach, and provides a quantitative estimate of the how the TS stabilization observed in most de novo and redesigned enzymes is decreased compared to a minimal, `ideal' model. The presented design is highly interesting for further optimization and applications since it is based on a thermophilic enzyme ( T opt = 70 °C).

DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

PubMed

Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

2018-01-01

Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases.

PubMed

Soya, Naoto; Shoemaker, Glen K; Palcic, Monica M; Klassen, John S

2009-11-01

The first comparative thermodynamic study of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24 degrees C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn(2+), both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 10(4) M(-1) was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (approximately 1 x 10(4) M(-1)), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn(2+), both GTA and GTB recognize their respective trisaccharide products but with a low affinity approximately 10(3) M(-1); the presence of UDP and Mn(2+) has no effect on A trisaccharide binding but precludes B-trisaccharide binding.

An RNA motif that binds ATP

NASA Technical Reports Server (NTRS)

Sassanfar, M.; Szostak, J. W.

1993-01-01

RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

Rapid dehalogenation of pesticides and organics at the interface of reduced graphene oxide-silver nanocomposite.

PubMed

Koushik, Dibyashree; Sen Gupta, Soujit; Maliyekkal, Shihabudheen M; Pradeep, T

2016-05-05

This paper reports dehalogenation of various organohalides, especially aliphatic halocarbons and pesticides at reduced graphene oxide-silver nanocomposite (RGO@Ag). Several pesticides as well as chlorinated and fluorinated alkyl halides were chosen for this purpose. The composite and the products of degradation were characterized thoroughly by means of various microscopic and spectroscopic techniques. A sequential two-step mechanism involving dehalogenation of the target pollutants by silver nanoparticles followed by adsorption of the degraded compounds onto RGO was revealed. The composite showed unusual adsorption capacity, as high as 1534 mg/g, which facilitated the complete removal of the pollutants. Irrespective of the pollutants tested, a pseudo-second-order rate equation best described the adsorption kinetics. The affinity of the composite manifested chemical differences. The high adsorption capacity and re-usability makes the composite an excellent substrate for purification of water. Copyright © 2016 Elsevier B.V. All rights reserved.

Transport of angiotensin-converting enzyme inhibitors by H+/peptide transporters revisited.

PubMed

Knütter, Ilka; Wollesky, Claudia; Kottra, Gabor; Hahn, Martin G; Fischer, Wiebke; Zebisch, Katja; Neubert, Reinhard H H; Daniel, Hannelore; Brandsch, Matthias

2008-11-01

Angiotensin-converting enzyme (ACE) inhibitors are often regarded as substrates for the H+/peptide transporters (PEPT)1 and PEPT2. Even though the conclusions drawn from published data are quite inconsistent, in most review articles PEPT1 is claimed to mediate the intestinal absorption of ACE inhibitors and thus to determine their oral availability. We systematically investigated the interaction of a series of ACE inhibitors with PEPT1 and PEPT2. First, we studied the effect of 14 ACE inhibitors including new drugs on the uptake of the dipeptide [14C]glycylsarcosine into human intestinal Caco-2 cells constitutively expressing PEPT1 and rat renal SKPT cells expressing PEPT2. In a second approach, the interaction of ACE inhibitors with heterologously expressed human PEPT1 and PEPT2 was determined. In both assay systems, zofenopril and fosinopril were found to have very high affinity for binding to peptide transporters. Medium to low affinity for transporter interaction was found for benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, moexipril, quinaprilat, and perindopril. For enalapril, lisinopril, and captopril, very weak affinity or lack of interaction was found. Transport currents of PEPT1 and PEPT2 expressed in Xenopus laevis oocytes were recorded by the two-electrode voltage-clamp technique. Statistically significant, but very low currents were only observed for lisinopril, enalapril, quinapril, and benazepril at PEPT1 and for spirapril at PEPT2. For the other ACE inhibitors, electrogenic transport activity was extremely low or not measurable at all. The present results suggest that peptide transporters do not control intestinal absorption and renal reabsorption of ACE inhibitors.

A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

PubMed

Wall, Jonathan S; Williams, Angela; Richey, Tina; Stuckey, Alan; Huang, Ying; Wooliver, Craig; Macy, Sallie; Heidel, Eric; Gupta, Neil; Lee, Angela; Rader, Brianna; Martin, Emily B; Kennel, Stephen J

2013-01-01

Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

2017-08-01

The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

In Vitro and Clinical Evaluations of the Drug-Drug Interaction Potential of a Metabotropic Glutamate 2/3 Receptor Agonist Prodrug with Intestinal Peptide Transporter 1

PubMed Central

Long, Amanda J.; Annes, William F.; Witcher, Jennifer W.; Knadler, Mary Pat; Ayan-Oshodi, Mosun A.; Mitchell, Malcolm I.; Leese, Phillip; Hillgren, Kathleen M.

2017-01-01

Despite peptide transporter 1 (PEPT1) being responsible for the bioavailability for a variety of drugs, there has been little study of its potential involvement in drug-drug interactions. Pomaglumetad methionil, a metabotropic glutamate 2/3 receptor agonist prodrug, utilizes PEPT1 to enhance absorption and bioavailability. In vitro studies were conducted to guide the decision to conduct a clinical drug interaction study and to inform the clinical study design. In vitro investigations determined the prodrug (LY2140023 monohydrate) is a substrate of PEPT1 with Km value of approximately 30 µM, whereas the active moiety (LY404039) is not a PEPT1 substrate. In addition, among the eight known PEPT1 substrates evaluated in vitro, valacyclovir was the most potent inhibitor (IC50 = 0.46 mM) of PEPT1-mediated uptake of the prodrug. Therefore, a clinical drug interaction study was conducted to evaluate the potential interaction between the prodrug and valacyclovir in healthy subjects. No effect of coadministration was observed on the pharmacokinetics of the prodrug, valacyclovir, or either of their active moieties. Although in vitro studies showed potential for the prodrug and valacyclovir interaction via PEPT1, an in vivo study showed no interaction between these two drugs. PEPT1 does not appear to easily saturate because of its high capacity and expression in the intestine. Thus, a clinical interaction at PEPT1 is unlikely even with a compound with high affinity for the transporter. PMID:27895114

Ultra-hard amorphous AlMgB14 films RF sputtered onto curved substrates

NASA Astrophysics Data System (ADS)

Grishin, A. M.; Putrolaynen, V. V.; Yuzvyuk, M. H.

2017-03-01

Recently, hard AlMgB14 (BAM) coatings were deposited for the first time by RF magnetron sputtering using a single stoichiometric ceramic target. High target sputtering power and sufficiently short target-to-substrate distance were found to be critical processing conditions. They enabled fabrication of stoichiometric in-depth compositionally homogeneous films with the peak values of nanohardness 88 GPa and Young’s modulus 517 GPa at the penetration depth of 26 nm and, respectively, 35 GPa and 275 GPa at 200 nm depth in 2 µm thick film (Grishin et al 2014 JETP Lett. 100 680). The narrow range of sufficiently short target-to-substrate distance makes impossible to coat non flat specimens. To achieve ultimate BAM films’ characteristics onto curved surfaces we developed two-step sputtering process. The first thin layer is deposited as a template at low RF power that facilitates a layered Frank van der Merwe mode growth of smooth film occurs. The next layer is grown at high RF target sputtering power. The affinity of subsequent flow of sputtered atoms to already evenly condensed template fosters the development of smooth film surface. As an example, we made BAM coating onto hemispherical 5 mm in diameter ball made from a hard tool steel and used as a head of a special gauge. Very smooth (6.6 nm RMS surface roughness) and hard AlMgB14 films fabricated onto commercial ball-shaped items enhance hardness of tool steel specimens by a factor of four.

Differential Temperature Dependence of Tobacco Etch Virus and Rhinovirus 3C Proteases

PubMed Central

Raran-Kurussi, Sreejith; Tözsér, József; Cherry, Scott; Tropea, Joseph E.; Waugh, David S.

2014-01-01

Because of their stringent sequence specificity, the 3C-like proteases from tobacco etch virus (TEV3) and human rhinovirus are often used for the removal of affinity tags. The latter enzyme is rumored to have greater catalytic activity at 4°C, the temperature at which fusion protein substrates are usually digested. Here, we report that experiments with fusion protein and peptide substrates confirm this conjecture. Whereas the catalytic efficiency of rhinovirus 3C protease is approximately the same at its optimum temperature (30°C) and at 4°C, TEV protease is 10-fold less active at the latter temperature, due primarily to a reduction in kcat. PMID:23395976

Surface conformations of an anti-ricin aptamer and its affinity for ricin determined by atomic force microscopy and surface plasmon resonance.

PubMed

Wang, B; Lou, Z; Park, B; Kwon, Y; Zhang, H; Xu, B

2015-01-07

We used atomic force microscopy (AFM) and surface plasmon resonance (SPR) to study the surface conformations of an anti-ricin aptamer and its specific binding affinity for ricin molecules. The effect of surface modification of the Au(111) substrate on the aptamer affinity was also estimated. The AFM topography images had a resolution high enough to distinguish different aptamer conformations. The specific binding site on the aptamer molecule was clearly located by the AFM recognition images. The aptamer on a Au(111) surface modified with carboxymethylated-dextran (CD) showed both similarities to and differences from the one without CD modification. The influence of CD modification was evaluated using AFM images of various aptamer conformations on the Au(111) surface. The affinity between ricin and the anti-ricin aptamer was estimated using the off-rate values measured using AFM and SPR. The SPR measurements of the ricin sample were conducted in the range from 83.3 pM to 8.33 nM, and the limit of detection was estimated as 25 pM (1.5 ng mL(-1)). The off-rate values of the ricin-aptamer interactions were estimated using both single-molecule dynamic force spectroscopy (DFS) and SPR as (7.3 ± 0.4) × 10(-4) s(-1) and (1.82 ± 0.067) × 10(-2) s(-1), respectively. The results show that single-molecule measurements can obtain different reaction parameters from bulk solution measurements. In AFM single-molecule measurements, the various conformations of the aptamer immobilized on the gold surface determined the availability of each specific binding site to the ricin molecules. The SPR bulk solution measurements averaged the signals from specific and non-specific interactions. AFM images and DFS measurements provide more specific information on the interactions of individual aptamer and ricin molecules.

[Role of hemoglobin affinity to oxygen in adaptation to hypoxemia].

PubMed

Kwasiborski, Przemysław Jerzy; Kowalczyk, Paweł; Zieliński, Jakub; Przybylski, Jacek; Cwetsch, Andrzej

2010-04-01

One of the basic mechanisms of adapting to hypoxemia is a decrease in the affinity of hemoglobin for oxygen. This process occurs mainly due to the increased synthesis of 2,3-diphosphoglycerate (2,3-DPG) in the erythrocytes, as well as through the Bohr effect. Hemoglobin with decreased affinity for oxygen increases the oxygenation of tissues, because it gives up oxygen more easily during microcirculation. In foetal circulation, however, at a partial oxygen pressure (pO2) of 25 mmHg in the umbilical vein, the oxygen carrier is type F hemoglobin which has a high oxygen affinity. The commonly accepted role for hemoglobin F is limited to facilitating diffusion through the placenta. Is fetal life the only moment when haemoglobin F is useful? THE AIM OF STUDY was to create a mathematical model, which would answer the question at what conditions an increase, rather than a decrease, in haemoglobin oxygen affinity is of benefit to the body. Using the kinetics of dissociation of oxygen from hemoglobin described by the Hill equation as the basis for further discussion, we created a mathematical model describing the pO2 value in the microcirculatory system and its dependence on arterial blood pO2. The calculations were performed for hemoglobin with low oxygen affinity (adult type) and high-affinity hemoglobin (fetal type). The modelling took into account both physiological and pathological ranges of acid-base equilibrium and tissue oxygen extraction parameters. It was shown that for the physiological range of acid-base equilibrium and the resting level of tissue oxygen extraction parameters, with an arterial blood pO2 of 26.8 mmHg, the higher-affinity hemoglobin becomes the more effective oxygen carrier. It was also demonstrated that the arterial blood pO2, below which the high-affinity hemoglobin becomes the more effective carrier, is dependent on blood pH and the difference between the arterial and venous oxygen saturation levels. Simulations performed for the pathological states showed that acidosis and increased tissue oxygen demand lead to a broadened arterial blood pO2 range, in which the high-affinity hemoglobin is more efficient. Contrary to the widely held view that the only response to hypoxemia is a decrease in haemoglobin oxygen affinity, it was shown that under extreme hypoxemic conditions, an increased haemoglobin oxygen affinity improves the oxygenation of tissues. It was also shown that the dominance of hemoglobin with a high oxygen affinity rapidly exceeds hemoglobin with low oxygen affinity in the case of acidosis with its accompanying high tissue oxygen extraction. In cases of extreme disruptions of the acid-base equilibrium, the dominance of high-oxygen-affinity hemoglobin spans over the entire possible range of pO2 in arterial blood.

Rifampin phosphotransferase is an unusual antibiotic resistance kinase

PubMed Central

Stogios, Peter J.; Cox, Georgina; Spanogiannopoulos, Peter; Pillon, Monica C.; Waglechner, Nicholas; Skarina, Tatiana; Koteva, Kalinka; Guarné, Alba; Savchenko, Alexei; Wright, Gerard D.

2016-01-01

Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds. PMID:27103605

Catalytic growth of carbon nanofibers on Cr nanoparticles on a carbon substrate: adsorbents for organic dyes in water

NASA Astrophysics Data System (ADS)

de Oliveira, Luiz Carlos Alves; da Silva, Adilson Cândido; Machado, Alan Rodrigues Teixeira; Diniz, Renata; Pereira, Márcio César

2013-05-01

We have produced carbon nanofibers (CNFs) using leather waste that had been tanned with a chromium bath, and when dried contained Cr2O3. Suitable reduction processing produced a carbon substrate with supported nanoparticles of chromium metal. Powder X-ray diffraction showed that the Cr2O3 is reduced on the carbon surface to produce CrC and metal Cr, which is the effective catalyst for the CNFs growth. The CNF arrays were confirmed by TEM images. Raman data revealed that the synthesized CNFs have a poor-quality graphite structure which favors their use in adsorption processes. These CNFs presented higher affinity to adsorb anionic dyes, whereas the cationic dyes are better adsorbed on the carbon substrate. The low-cost and availability of the carbon precursor makes their potential use to produce CNFs of interest.

Affinity proteomics to study endogenous protein complexes: Pointers, pitfalls, preferences and perspectives

PubMed Central

LaCava, John; Molloy, Kelly R.; Taylor, Martin S.; Domanski, Michal; Chait, Brian T.; Rout, Michael P.

2015-01-01

Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls. PMID:25757543

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose

PubMed Central

Edwards, J. Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle nee; French, Alfred D.; Condon, Brian D.

2018-01-01

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis–Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding. PMID:29534033

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose.

PubMed

Edwards, J Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle Nee; French, Alfred D; Condon, Brian D

2018-03-13

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis-Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity ( K m ) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased K m observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency ( k cat / K m ), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding.

Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

PubMed Central

Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

2013-01-01

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

Adenosylcobinamide methyl phosphate as a pseudocoenzyme for diol dehydrase.

PubMed

Ishida, A; Toraya, T

1993-02-16

Adenosylcobinamide methyl phosphate, a novel analog of adenosylcobalamin lacking the nucleotide loop moiety, was synthesized. It did not show detectable coenzymic activity but behaved as a strong competitive inhibitor against AdoCbl with relatively high affinity (Ki = 2.5 microM). When apoenzyme was incubated at 37 degrees C with this analog in the presence of substrate, the Co-C bond of the analog was almost completely and irreversibly cleaved within 10 min, forming an enzyme-bound Co(II)-containing species. The cleavage was not observed in the absence of substrate. The Co-C bond cleavage in the presence of substrate was not catalytic but stoichiometric, implying that the Co-C bond of the analog undergoes activation when the analog binds to the active site of the enzyme. 5'-Deoxyadenosine was the only product derived from the adenosyl group of the analog upon the Co-C bond cleavage. Apoenzyme did not undergo modification during this process. Therefore, it seems likely that adenosylcobinamide methyl phosphate acts as a pseudocoenzyme or a potent suicide coenzyme. Since adenosylcobinamide neither functions as coenzyme nor binds tightly to apoenzyme, it can be concluded that the phosphodiester moiety of the nucleotide loop of adenosylcobalamin is essential for tight binding to apoenzyme and therefore for subsequent activation of the Co-C bond and catalysis. It is also evident that the nucleotide loop is obligatory for the normal progress of catalytic cycle.

Basic Residues R260 and K357 Affect the Conformational Dynamics of the Major Facilitator Superfamily Multidrug Transporter LmrP

PubMed Central

Wang, Wei; van Veen, Hendrik W.

2012-01-01

Secondary-active multidrug transporters can confer resistance on cells to pharmaceuticals by mediating their extrusion away from intracellular targets via substrate/H+(Na+) antiport. While the interactions of catalytic carboxylates in these transporters with coupling ions and substrates (drugs) have been studied in some detail, the functional importance of basic residues has received much less attention. The only two basic residues R260 and K357 in transmembrane helices in the Major Facilitator Superfamily transporter LmrP from Lactococcus lactis are present on the outer surface of the protein, where they are exposed to the phospholipid head group region of the outer leaflet (R260) and inner leaflet (K357) of the cytoplasmic membrane. Although our observations on the proton-motive force dependence and kinetics of substrate transport, and substrate-dependent proton transport demonstrate that K357A and R260A mutants are affected in ethidium-proton and benzalkonium-proton antiport compared to wildtype LmrP, our findings suggest that R260 and K357 are not directly involved in the binding of substrates or the translocation of protons. Secondary-active multidrug transporters are thought to operate by a mechanism in which binding sites for substrates are alternately exposed to each face of the membrane. Disulfide crosslinking experiments were performed with a double cysteine mutant of LmrP that reports the substrate-stimulated transition from the outward-facing state to the inward-facing state with high substrate-binding affinity. In the experiments, the R260A and K357A mutations were found to influence the dynamics of these major protein conformations in the transport cycle, potentially by removing the interactions of R260 and K357 with phospholipids and/or other residues in LmrP. The R260A and K357A mutations therefore modify the maximum rate at which the transport cycle can operate and, as the transitions between conformational states are differently affected by components of the proton-motive force, the mutations also influence the energetics of transport. PMID:22761697

The chitin-binding domain of a GH-18 chitinase from Vibrio harveyi is crucial for chitin-chitinase interactions.

PubMed

Suginta, Wipa; Sirimontree, Paknisa; Sritho, Natchanok; Ohnuma, Takayuki; Fukamizo, Tamo

2016-12-01

Vibrio harveyi chitinase A (VhChiA) is a GH-18 glycosyl hydrolase with a structure containing three distinct domains: i) the N-terminal chitin-binding domain; ii) the (α/β) 8 TIM barrel catalytic domain; and iii) the α+β insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBD Vh ChiA . The recombinant ChBD Vh ChiA was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBD Vh ChiA contained β-sheets as major structural components and fluorescence spectroscopy showed that the protein domain was folded correctly, and suitable for functional characterization. Chitin binding assays showed that ChBD Vh ChiA bound to both α- and β-chitins, with the greatest affinity for β-colloidal chitin, but barely bound to polymeric chitosan. These results identified the tandem N-acetamido functionality on chitin chains as the specific sites of enzyme-substrate interactions. The binding affinity of the isolated domain was significantly lower than that of intact VhChiA, suggesting that the catalytic domain works synergistically with the chitin-binding domain to guide the polymeric substrate into the substrate binding cleft. These data confirm the physiological role of the chitin-binding domain of the marine bacterial GH-18 chitinase A in chitin-chitinase interactions. Copyright © 2016 Elsevier B.V. All rights reserved.

A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

PubMed Central

Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G.

2014-01-01

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM−1 s−1, 7.63 mM−1 s−1, 3.83 mM−1 s−1 and 3.75 mM−1 s−1, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. PMID:25548041

Thermally-induced transition of lamellae orientation in block-copolymer films on ‘neutral’ nanoparticle-coated substrates

DOE PAGES

Yager, Kevin G.; Forrey, Christopher; Singh, Gurpreet; ...

2015-06-01

Block-copolymer orientation in thin films is controlled by the complex balance between interfacial free energies, including the inter-block segregation strength, the surface tensions of the blocks, and the relative substrate interactions. While block-copolymer lamellae orient horizontally when there is any preferential affinity of one block for the substrate, we recently described how nanoparticle-roughened substrates can be used to modify substrate interactions. We demonstrate how such ‘neutral’ substrates can be combined with control of annealing temperature to generate vertical lamellae orientations throughout a sample, at all thicknesses. We observe an orientational transition from vertical to horizontal lamellae upon heating, as confirmedmore » using a combination of atomic force microscopy (AFM), neutron reflectometry (NR) and rotational small-angle neutron scattering (RSANS). Using molecular dynamics (MD) simulations, we identify substrate-localized distortions to the lamellar morphology as the physical basis of the novel behavior. In particular, under strong segregation conditions, bending of horizontal lamellae induce a large energetic cost. At higher temperatures, the energetic cost of conformal deformations of lamellae over the rough substrate is reduced, returning lamellae to the typical horizontal orientation. Thus, we find that both surface interactions and temperature play a crucial role in dictating block-copolymer lamellae orientation. As a result, our combined experimental and simulation findings suggest that controlling substrate roughness should provide a useful and robust platform for controlling block-copolymer orientation in applications of these materials.« less

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis.

PubMed

Papadakos, Grigorios A; Nastri, Horacio; Riggs, Paul; Dupureur, Cynthia M

2007-05-01

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.

Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

PubMed

Schimpl, Flávia Camila; Kiyota, Eduardo; Mayer, Juliana Lischka Sampaio; Gonçalves, José Francisco de Carvalho; da Silva, José Ferreira; Mazzafera, Paulo

2014-09-01

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase(s) in human platelets.

PubMed Central

Bakken, A M; Farstad, M

1992-01-01

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferases (EC 2.3.1.23) have been studied in human platelet lysates by using endogenously formed [14C]acyl-CoA from [14C]fatty acid, ATP and CoA in the presence of 1-acyl-lysophosphatidyl-choline (lysoPC), -ethanolamine (lysoPE), -serine (lysoPS) or -inositol (lysoPI). Linoleic acid as fatty acid substrate had the highest affinity to acyl-CoA:1-acyl-lysophospholipid acyltransferase with lysoPC as variable substrate, followed by eicosapentaenoic acid (EPA) and arachidonic acid (AA). The activity at optimal conditions was 7.4, 7.3 and 7.2 nmol/min per 10(9) platelets with lysoPC as substrate, with linoleic acid, AA and EPA respectively. EPA and AA were incorporated into all lyso-forms. Linoleic acid was also incorporated into lysoPE at a high rate, but less into lysoPS and lysoPI. DHA was incorporated into lysoPC and lysoPE, but only slightly into lysoPI and lysoPS. Whereas incorporation of all fatty acids tested was maximal for lysoPC and lysoPI at 200 and 80 microM respectively, maximal incorporation needed over 500 microM for lysoPE and lysoPS. The optimal concentration for [14C]fatty acid substrates was in the range 15-150 microM for all lysophospholipids. Competition experiments with equimolar concentrations of either lysoPC and lysoPI or lysoPE resulted in formation of [14C]PC almost as if lysoPI or lysoPE were not added to the assay medium. PMID:1471991

HIGH-AFFINITY T CELL RECEPTOR DIFFERENTIATES COGNATE PEPTIDE-MHC AND ALTERED PEPTIDE LIGANDS WITH DISTINCT KINETICS AND THERMODYNAMICS

PubMed Central

Persaud, Stephen P.; Donermeyer, David L.; Weber, K. Scott; Kranz, David M.; Allen, Paul M.

2010-01-01

Interactions between the T cell receptor and cognate peptide-MHC are crucial initiating events in the adaptive immune response. These binding events are highly specific yet occur with micromolar affinity. Even weaker interactions between TCR and self-pMHC complexes play critical regulatory roles in T cell development, maintenance and coagonist activity. Due to their low affinity, the kinetics and thermodynamics of such weak interactions are difficult to study. In this work, we used M15, a high-affinity TCR engineered from the 3.L2 TCR system, to study the binding properties, thermodynamics, and specificity of two altered peptide ligands (APLs). Our affinity measurements of the high-affinity TCR support the view that the wild type TCR binds these APLs in the millimolar affinity range, and hence very low affinities can still elicit biological functions. Finally, single methylene differences among the APLs gave rise to strikingly different binding thermodynamics. These minor changes in the pMHC antigen were associated with significant and unpredictable changes in both the entropy and enthalpy of the reaction. As the identical TCR was analyzed with several structurally similar ligands, the distinct thermodynamic binding profiles provide a mechanistic perspective on how exquisite antigen specificity is achieved by the T cell receptor. PMID:20334923

Let's get specific: the relationship between specificity and affinity.

PubMed

Eaton, B E; Gold, L; Zichi, D A

1995-10-01

The factors that lead to high-affinity binding are a good fit between the surfaces of the two molecules in their ground state and charge complementarity. Exactly the same factors give high specificity for a target. We argue that selection for high-affinity binding automatically leads to highly specific binding. This principle can be used to simplify screening approaches aimed at generating useful drugs.

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine.

PubMed

Köhrle, J; Rasmussen, U B; Rokos, H; Leonard, J L; Hesch, R D

1990-04-15

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man*

PubMed Central

Macauley, Matthew S.; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M.; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C.

2015-01-01

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. PMID:26507663

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man.

PubMed

Macauley, Matthew S; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C

2015-12-11

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2-6Galβ1-4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential effects of ethanol and other inducers of drug metabolism on the two forms of hamster liver microsomal aniline hydroxylase.

PubMed

McCoy, G D

1980-03-01

The aniline hydroxylase activity of microsomes isolated from hamster liver can be differentiated kinetically into high affinity (low K(m), form I) and low affinity (high K(m), form II) forms. Microsomes isolated from uninduced animals contain slightly more form I activity. The activity of the low affinity form (form II) is preferentially enhanced by Aroclor or 3-methylcholanthrene treatment, while phenobarbital treatment increases the activity of both forms. Chronic ethanol consumption results in enhancement of only the high affinity form (form I).

Effect of atomic layer deposition temperature on current conduction in Al{sub 2}O{sub 3} films formed using H{sub 2}O oxidant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hiraiwa, Atsushi, E-mail: hiraiwa@aoni.waseda.jp, E-mail: qs4a-hriw@asahi-net.or.jp; Matsumura, Daisuke; Kawarada, Hiroshi, E-mail: kawarada@waseda.jp

To develop high-performance, high-reliability gate insulation and surface passivation technologies for wide-bandgap semiconductor devices, the effect of atomic layer deposition (ALD) temperature on current conduction in Al{sub 2}O{sub 3} films is investigated based on the recently proposed space-charge-controlled field emission model. Leakage current measurement shows that Al{sub 2}O{sub 3} metal-insulator-semiconductor capacitors formed on the Si substrates underperform thermally grown SiO{sub 2} capacitors at the same average field. However, using equivalent oxide field as a more practical measure, the Al{sub 2}O{sub 3} capacitors are found to outperform the SiO{sub 2} capacitors in the cases where the capacitors are negatively biased andmore » the gate material is adequately selected to reduce virtual dipoles at the gate/Al{sub 2}O{sub 3} interface. The Al{sub 2}O{sub 3} electron affinity increases with the increasing ALD temperature, but the gate-side virtual dipoles are not affected. Therefore, the leakage current of negatively biased Al{sub 2}O{sub 3} capacitors is approximately independent of the ALD temperature because of the compensation of the opposite effects of increased electron affinity and permittivity in Al{sub 2}O{sub 3}. By contrast, the substrate-side sheet of charge increases with increasing ALD temperature above 210 °C and hence enhances the current of positively biased Al{sub 2}O{sub 3} capacitors more significantly at high temperatures. Additionally, an anomalous oscillatory shift of the current-voltage characteristics with ALD temperature was observed in positively biased capacitors formed by low-temperature (≤210 °C) ALD. This shift is caused by dipoles at the Al{sub 2}O{sub 3}/underlying SiO{sub 2} interface. Although they have a minimal positive-bias leakage current, the low-temperature-grown Al{sub 2}O{sub 3} films cause the so-called blisters problem when heated above 400 °C. Therefore, because of the absence of blistering, a 450 °C ALD process is presently the most promising technology for growing high-reliability Al{sub 2}O{sub 3} films.« less

Chapter 8: Selective Stoichiometric and Catalytic Reactivity in the Confines of a Chiral Supramolecular Assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

University of California, Berkeley; Lawrence Berkeley National Laboratory; Raymond, Kenneth

2007-09-27

Nature uses enzymes to activate otherwise unreactive compounds in remarkable ways. For example, DNases are capable of hydrolyzing phosphate diester bonds in DNA within seconds,[1-3]--a reaction with an estimated half-life of 200 million years without an enzyme.[4] The fundamental features of enzyme catalysis have been much discussed over the last sixty years in an effort to explain the dramatic rate increases and high selectivities of enzymes. As early as 1946, Linus Pauling suggested that enzymes must preferentially recognize and stabilize the transition state over the ground state of a substrate.[5] Despite the intense study of enzymatic selectivity and ability tomore » catalyze chemical reactions, the entire nature of enzyme-based catalysis is still poorly understood. For example, Houk and co-workers recently reported a survey of binding affinities in a wide variety of enzyme-ligand, enzyme-transition-state, and synthetic host-guest complexes and found that the average binding affinities were insufficient to generate many of the rate accelerations observed in biological systems.[6] Therefore, transition-state stabilization cannot be the sole contributor to the high reactivity and selectivity of enzymes, but rather, other forces must contribute to the activation of substrate molecules. Inspired by the efficiency and selectivity of Nature, synthetic chemists have admired the ability of enzymes to activate otherwise unreactive molecules in the confines of an active site. Although much less complex than the evolved active sites of enzymes, synthetic host molecules have been developed that can carry out complex reactions with their cavities. While progress has been made toward highly efficient and selective reactivity inside of synthetic hosts, the lofty goal of duplicating enzymes specificity remains.[7-9] Pioneered by Lehn, Cram, Pedersen, and Breslow, supramolecular chemistry has evolved well beyond the crown ethers and cryptands originally studied.[10-12] Despite the increased complexity of synthetic host molecules, most assembly conditions utilize self-assembly to form complex highly-symmetric structures from relatively simple subunits. For supramolecular assemblies able to encapsulate guest molecules, the chemical environment in each assembly--defined by the size, shape, charge, and functional group availability--greatly influences the guest-binding characteristics.[6, 13-17]« less

Molecular self-assembly for biological investigations and nanoscale lithography

NASA Astrophysics Data System (ADS)

Cheunkar, Sarawut

Small, diffusible molecules when recognized by their binding partners, such as proteins and antibodies, trigger enzymatic activity, cell communication, and immune response. Progress in analytical methods enabling detection, characterization, and visualization of biological dynamics at the molecular level will advance our exploration of complex biological systems. In this dissertation, analytical platforms were fabricated to capture membrane-associated receptors, which are essential proteins in cell signaling pathways. The neurotransmitter serotonin and its biological precursor were immobilized on gold substrates coated with self-assembled monolayers (SAMs) of oligo(ethylene glycol)alkanethiols and their reactive derivatives. The SAM-coated substrates present the biologically selective affinity of immobilized molecules to target native membrane-associated receptors. These substrates were also tested for biospecificity using antibodies. In addition, small-molecule-functionalized platforms, expressing neurotransmitter pharmacophores, were employed to examine kinetic interactions between G-protein-coupled receptors and their associated neurotransmitters. The binding interactions were monitored using a quartz crystal microbalance equipped with liquid-flow injection. The interaction kinetics of G-protein-coupled serotonin 1A receptor and 5-hydroxytyptophan-functionalized surfaces were studied in a real-time, label-free environment. Key binding parameters, such as equilibrium dissociation constants, binding rate constants, and dissociative half-life, were extracted. These parameters are critical for understanding and comparing biomolecular interactions in modern biomedical research. By integrating self-assembly, surface functionalization, and nanofabrication, small-molecule microarrays were created for high-throughput screening. A hybrid soft-lithography, called microcontact insertion printing, was used to pattern small molecules at the dilute scales necessary for highly selective biorecognition. By carefully tuning the polar surface energy of polymeric stamps, problems associated with patterning hydrophilic tether molecules inserted into hydrophilic preformed SAMs are surmounted. The patterned substrates presenting neurotransmitter precursors selectively capture membrane-associated receptors. These advances provide new avenues for fabricating small-molecule arrays. Furthermore, a novel strategy based on a conventional microcontact printing, called chemical lift-off lithography, was invented to overcome the micrometer-scale resolution limits of molecular ink diffusion in soft lithography. Self-assembled monolayers of hydroxyl-terminated alkanethiols, preformed on gold substrates, were selectively removed by oxygen-plasma-treated polymeric stamps in a subtractive stamping process with high pattern fidelity. The covalent interactions formed at the stamp-substrate interface are believed to be responsible for removing not only alkanethiol molecules but also a monolayer of gold atoms from the substrates. A variety of high-resolution patterned features were fabricated, and stamps were cleaned and reused many times without feature deterioration. The remaining SAMs acted as resists for etching exposed gold features. Monolayer backfilling into lifted-off areas enabled patterned protein capture, and 40-nanometer chemical patterns were achieved.

Purification and characterization of Phaseolus vulgaris alpha-D-galactosidase isozymes.

PubMed

Dhar, M; Mitra, M; Hata, J; Butnariu, O; Smith, D

1994-11-01

A highly purified preparation of alpha-D-galactosidase [E.C. 3.2.1.22] isozymes was obtained from Phaseolus vulgaris (pinto bean) seeds by extraction, salt precipitation, ion exchange, and affinity chromatography. The final preparation was homogeneous by SDS-PAGE but revealed isozymes of relative mass of 38.3 and 39.6 kDa. The N-terminal sequence for both isozymes was identical, LANGLAKT (one letter code for amino acids). Relative native molecular mass was estimated at 149.3 kDa by Sephacryl S-200 chromatography. Activity was unaffected by ionic strength at high enzyme concentrations, and was specific for alpha-D-galactoside conjugates. No protease or hemagglutinin activity was detected, and activity was stable at 4 degrees C. Studies with soluble oligosaccharides demonstrated high activity against the selected straight and branched-chain substrates. The enzyme was active against terminal alpha 1-3 galactosyl residues on human and rabbit erythrocyte membranes. Because of its activity against membrane glycoconjugates, these isozymes may have potential utility for modifying membrane epitopes on native erythrocytes.

Absolute cross section for loss of supercoiled topology induced by 10 eV electrons in highly uniform /DNA/1,3-diaminopropane films deposited on highly ordered pyrolitic graphite

PubMed Central

Boulanouar, Omar; Fromm, Michel; Bass, Andrew D.; Cloutier, Pierre; Sanche, Léon

2013-01-01

It was recently shown that the affinity of doubly charged, 1–3 diaminopropane (Dap2+) for DNA permits the growth on highly ordered pyrolitic graphite (HOPG) substrates, of plasmid DNA films, of known uniform thickness [O. Boulanouar, A. Khatyr, G. Herlem, F. Palmino, L. Sanche, and M. Fromm, J. Phys. Chem. C 115, 21291–21298 (2011)]. Post-irradiation analysis by electrophoresis of such targets confirms that electron impact at 10 eV produces a maximum in the yield of single strand breaks that can be associated with the formation of a DNA− transient anion. Using a well-adapted deterministic survival model for the variation of electron damage with fluence and film thickness, we have determined an absolute cross section for strand-break damage by 10 eV electrons and inelastic scattering attenuation length in DNA-Dap complex films. PMID:23927289

Structure of the G225P/G226P mutant of mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) ternary complex: implications for the binding of inhibitor and substrate.

PubMed

Dhagat, Urmi; Endo, Satoshi; Mamiya, Hiroaki; Hara, Akira; El-Kabbani, Ossama

2009-03-01

3(17)alpha-Hydroxysteroid dehydrogenase (AKR1C21) is a unique member of the aldo-keto reductase (AKR) superfamily owing to its ability to reduce 17-ketosteroids to 17alpha-hydroxysteroids, as opposed to other members of the AKR family, which can only produce 17beta-hydroxysteroids. In this paper, the crystal structure of a double mutant (G225P/G226P) of AKR1C21 in complex with the coenzyme NADP(+) and the inhibitor hexoestrol refined at 2.1 A resolution is presented. Kinetic analysis and molecular-modelling studies of 17alpha- and 17beta-hydroxysteroid substrates in the active site of AKR1C21 suggested that Gly225 and Gly226 play an important role in determining the substrate stereospecificity of the enzyme. Additionally, the G225P/G226P mutation of the enzyme reduced the affinity (K(m)) for both 3alpha- and 17alpha-hydroxysteroid substrates by up to 160-fold, indicating that these residues are critical for the binding of substrates.

Purification and properties of aryl acylamidase from Pseudomonas fluorescens ATCC 39004.

PubMed

Hammond, P M; Price, C P; Scawen, M D

1983-05-16

Aryl acylamidase has been purified from a strain of Pseudomonas fluorescens ATCC 39004, selected from soil on the basis of its ability to utilise acylanilide compounds as a sole source of carbon. The enzyme was purified to homogeneity by a combination of ion-exchange, hydrophobic and gel-permeation chromatography. A relative molecular mass of about 52 500 was estimated by gel filtration. The native enzyme was shown to be a monomeric protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The enzyme was maximally active at a pH of 8.6 and at a temperature of 45 degrees C. The enzyme shows Michaelis-Menten kinetics; Km values for nitroacetanilide (69 microM) and hydroxyacetanilide (6.1 microM) were low, indicating that the enzyme has a very high affinity for both substrates.

Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study†

PubMed Central

Peng, Dungeng; Satterlee, James D.; Ma, Li-Hua; Dallas, Jerry L.; Smith, Kevin M.; Zhang, Xuhong; Sato, Michihiko; La Mar, Gerd N.

2011-01-01

Heme oxygenase, HO, from the pathogenic bacterium N. meningitidis, NmHO, which secures host iron, shares many properties with mammalian HOs, but also exhibits some key differences. The crystal structure appears more compact and the crystal-undetected C-terminus interacts with substrate in solution. The unique nature of substrate-protein, specifically pyrrole-I/II-helix-2, peripheral interactions in NmHO are probed by 2D 1H NMR to reveal unique structural features controlling substrate orientation. The thermodynamics of substrate orientational isomerism are mapped for substrates with individual vinyl → methyl → hydrogen substitutions and with enzyme C-terminal deletions. NmHO exhibits significantly stronger orientational preference, reflecting much stronger and selective pyrrole-I/II interactions with the protein matrix, than in mammalian HOs. Thus, replacing bulky vinyls with hydrogens results in a 180° rotation of substrate about the α,γ-meso axis in the active site. A "collapse" of the substrate pocket as substrate size decreases is reflected in movement of helix-2 toward the substrate as indicated by significant and selective increased NOESY cross peak intensity, increase in steric Fe-CN tilt reflected in the orientation of the major magnetic axis, and decrease in steric constraints controlling the rate of aromatic ring reorientation. The active site of NmHO appears "stressed" for native protohemin and its "collapse" upon replacing vinyls by hydrogen leads to a factor ~102 increase in substrate affinity. Interaction of the C-terminus with the active site destabilizes the crystallographic protohemin orientation by ~0.7 kcal/mol, which is consistent with optimizing the His207-Asp27 H-bond. Implications of the active site "stress" for product release are discussed. PMID:21870860

Purine uptake in Plasmodium: transport versus metabolism.

PubMed

Kirk, Kiaran; Howitt, Susan M; Bröer, Stefan; Saliba, Kevin J; Downie, Megan J

2009-06-01

In a recent paper, Quashie et al. have proposed that purine uptake into the intraerythrocytic malaria parasite involves four different plasma membrane transporters - two high affinity and two low affinity. They equate one of the two high-affinity transporters with PfNT1, a transporter reported previously to be a low-affinity system. Here, we offer an alternative interpretation of their data, suggesting that the conclusions drawn by Quashie et al. take insufficient account of metabolism.

Genomic Instability and Breast Cancer

DTIC Science & Technology

2011-01-01

interaction between CCDC98 and BRCA1 (Kim et al., 2007; Liu et al., 2007; Wang et al., 2007). BRCC36 expressed and purified from insect cells was...Figure 7. (A) An in vitro DUB assay was conducted using K63 ubiquitin chains as substrate and insect cell-expressed BRCC36, the BRCC36/KIAA0157...Tandom Affinity Purification (TAP), Irradiation , Immuno- staining, and Immunoprecipitation—All of these procedures were performed as described

Mass spectrometry-based methods for detection and differentiation of botulinum neurotoxins

DOEpatents

Schmidt, Jurgen G [Los Alamos, NM; Boyer, Anne E [Atlanta, GA; Kalb, Suzanne R [Atlanta, GA; Moura, Hercules [Tucker, GA; Barr, John R [Suwannee, GA; Woolfitt, Adrian R [Atlanta, GA

2009-11-03

The present invention is directed to a method for detecting the presence of clostridial neurotoxins in a sample by mixing a sample with a peptide that can serve as a substrate for proteolytic activity of a clostridial neurotoxin; and measuring for proteolytic activity of a clostridial neurotoxin by a mass spectroscopy technique. In one embodiment, the peptide can have an affinity tag attached at two or more sites.

Host-Guest Complexes with Protein-Ligand-Like Affinities: Computational Analysis and Design

PubMed Central

Moghaddam, Sarvin; Inoue, Yoshihisa

2009-01-01

It has recently been discovered that guests combining a nonpolar core with cationic substituents bind cucurbit[7]uril (CB[7]) in water with ultra-high affinities. The present study uses the Mining Minima algorithm to study the physics of these extraordinary associations and to computationally test a new series of CB[7] ligands designed to bind with similarly high affinity. The calculations reproduce key experimental observations regarding the affinities of ferrocene-based guests with CB[7] and β-cyclodextrin and provide a coherent view of the roles of electrostatics and configurational entropy as determinants of affinity in these systems. The newly designed series of compounds is based on a bicyclo[2.2.2]octane core, which is similar in size and polarity to the ferrocene core of the existing series. Mining Minima predicts that these new compounds will, like the ferrocenes, bind CB[7] with extremely high affinities. PMID:19133781

Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

PubMed Central

Nika, Heinz; Nieves, Edward; Hawke, David H.; Angeletti, Ruth Hogue

2013-01-01

A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O-GlcNAc)-modified counterparts. Solid-phase enzymatic dephosphorylation proved to be a viable tool to condition O-GlcNAcylated peptide in mixtures with phosphopeptides for selective affinity purification. Acetylation, as an integral step of the sample-preparation method, precluded reduction in recovery of the thiolation substrate caused by intrapeptide lysine-dehydroalanine cross-link formation. The solid-phase analytical platform provides robustness and simplicity of operation using equipment readily available in most biological laboratories and is expected to accommodate additional chemistries to expand the scope of solid-phase serial derivatization for protein structural characterization. PMID:23997662

Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase, with Potential for Biotechnological Application

PubMed Central

Souza, Amanda Araújo; Leitão, Vanessa Oliveira; Ramada, Marcelo Henrique; Mehdad, Azadeh; Georg, Raphaela de Castro; Ulhôa, Cirano José; de Freitas, Sonia Maria

2016-01-01

Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65°C, respectively, and is stable at 55°C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments. PMID:26938873

Adsorption behavior of methacryloyloxydecyl dihydrogen phosphate on an apatite surface at neutral pH.

PubMed

Bista, Baba; Nakashima, Syozi; Nikaido, Toru; Sadr, Alireza; Takagaki, Tomohiro; Romero, Maria J R H; Sato, Takaaki; Tagami, Junji

2016-04-01

This study aimed to quantify the adsorption affinity of neutralized 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP-N) toward hydroxyapatite (HA) and dicalcium phosphate dihydrate (DCPD) at pH 7.0 by employing the Langmuir isotherm model. Furthermore, the effects of inorganic phosphate (Pi) and fluoride (F(-) ) ions on the adsorption of 10-MDP-N onto HA and DCPD were examined. Fixed amounts of HA and DCPD powders were suspended in different concentrations of 10-MDP-N solutions and were incubated for 18 h. Equilibrated concentrations of 10-MDP-N were measured by spectrophotometry and the adsorption affinity was estimated using the Langmuir model. Moreover, the adsorption was examined by zeta-potential analysis. The results indicated that significant Langmuir correlation was noted in both substrates, along with an increasing negative zeta-potential; however, in DCPD the correlation was less strong. The addition of 1.0 mM Pi slightly delayed the adsorption of 10-MDP-N onto both substrates, whereas 3.0 mM Pi drastically delayed adsorption onto HA but completely inhibited adsorption onto DCPD. Up to 50 ppm, F(-) enhanced the adsorption onto HA, and the adsorption plateaued at higher concentrations of F(-) , whereas no obvious influence of F(-) on the adsorption onto DCPD was noted. © 2016 Eur J Oral Sci.

Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase, with Potential for Biotechnological Application.

PubMed

Souza, Amanda Araújo; Leitão, Vanessa Oliveira; Ramada, Marcelo Henrique; Mehdad, Azadeh; Georg, Raphaela de Castro; Ulhôa, Cirano José; de Freitas, Sonia Maria

2016-01-01

Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65 °C, respectively, and is stable at 55 °C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0 was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments.

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*

PubMed Central

Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

2015-01-01

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

Affinity alkylation of the active site of C/sub 21/ steroid side-chain cleavage cytochrome P-450 from neonatal porcine testis: a unique cysteine residue alkylated by 17-(bromoacetoxy)progesterone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Onoda, M.; Haniu, M.; Yanagibashi, K.

1987-01-27

The affinity alkylating progesterone analogue 17-(bromoacetoxy)progesterone has been used to label the active site of a microsomal cytochrome P-450 enzyme from neonatal pig testis. The enzyme causes removal of the C/sub 20/ and C/sub 21/ side chains from the substrates progesterone and pregnenolone by catalyzing both 17-hydroxylase and C/sub 17,20/-lyase reactions, which produce the corresponding C/sub 1//sup 9/ steroidal precursors of testosterone. The progesterone analogue causes simultaneous inactivation of the two catalytic activities of the enzyme by a first-order kinetic process that obeys saturation kinetics. Progesterone and 17-hydroxyprogesterone each protect the enzyme against inactivation. The progesterone analogue is a competitivemore » inhibitor of the enzyme with K/sub i/ values of 8.4 ..mu..M and 7.8 ..mu..M for progesterone and 17-hydroxyprogesterone, respectively. The enzyme inactivation and kinetic data are consistent with a theory proposing that the analogue and the two substrates compete for the same active site. The radioactive analogue 17-((/sup 14/C)bromoacetoxy)progesterone causes inactivation of the enzyme with incorporation of 1.5-2.2 mol of the analogue per mole of inactivated enzyme. When this experiment is carried out in the presence of a substrate, then 0.9-1.2 mol of radioactive analogue is incorporated per mole of inactivated enzyme. The data suggest that the analogue can bind to two different sites, one of which is related to the catalytic site. Radiolabeled enzyme samples, from reactions of the /sup 14/C-labeled analogue with the enzyme alone or with enzyme in the presence of a substrate, were subjected to amino acid analysis and also in tryptic digestion and peptide mapping.« less

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

PubMed Central

Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

2013-01-01

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

Conformational state interactions provide clues to the pharmacochaperone potential of serotonin transporter partial substrates

PubMed Central

Bhat, Shreyas; Hasenhuetl, Peter S.; Kasture, Ameya; El-Kasaby, Ali; Baumann, Michael H.; Blough, Bruce E.; Sucic, Sonja; Sandtner, Walter; Freissmuth, Michael

2017-01-01

Point mutations in SLC6 transporters cause misfolding, which can be remedied by pharmacochaperones. The serotonin transporter (SERT/SLC6A4) has a rich pharmacology including inhibitors, releasers (amphetamines, which promote the exchange mode), and more recently, discovered partial substrates. We hypothesized that partial substrates trapped the transporter in one or several states of the transport cycle. This conformational trapping may also be conducive to folding. We selected naphthylpropane-2-amines of the phenethylamine library (PAL) including the partial substrate PAL1045 and its congeners PAL287 and PAL1046. We analyzed their impact on the transport cycle of SERT by biochemical approaches and by electrophysiological recordings; substrate-induced peak currents and steady-state currents monitored the translocation of substrate and co-substrate Na+ across the lipid bilayer and the transport cycle, respectively. These experiments showed that PAL1045 and its congeners bound with different affinities (ranging from nm to μm) to various conformational intermediates of SERT during the transport cycle. Consistent with the working hypothesis, PAL1045 was the most efficacious compound in restoring surface expression and transport activity to the folding-deficient mutant SERT-601PG602-AA. These experiments provide a proof-of-principle for a rational search for pharmacochaperones, which may be useful to restore function to clinically relevant folding-deficient transporter mutants. PMID:28842491

High-affinity K+ uptake in pepper plants.

PubMed

Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

2005-06-01

High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

High l-Trp affinity of indoleamine 2,3-dioxygenase 1 is attributed to two residues located in the distal heme pocket.

PubMed

Yuasa, Hajime J

2016-10-01

Indoleamine 2, 3-dioxygenase (IDO) catalyzes the oxidative cleavage of the pyrrole ring of l-Trp to generate N-formyl-kynurenine. Two IDO genes, IDO1 and IDO2, are found in vertebrates. Mammalian IDO1s are high-affinity, l-Trp-degrading enzymes, whereas IDO2s generally have a relatively low affinity. It has been suggested that the distal-Ser (corresponding to Ser167 of human IDO1) was crucial for improvement in the affinity for l-Trp but this idea was insufficient to explain the high affinity shown by mammalian IDO1. In this study, the amino acid sequences of vertebrate ancestral IDO1 and ancestral IDO2 were inferred, and bacterially expressed ancestral IDOs were characterized. Although the amino acid sequences of the enzymes shared high identity (86%) with each other, they showed distinct enzymatic properties. In analyses of a series of ancestral IDO1/IDO2 chimeric enzymes and their variants, the distal-Tyr (corresponding to Tyr126 of human IDO1) was detected as another and was probably the most crucial residue for high l-Trp affinity. The two amino acid substitutions (distal-Ser to Thr and distal-Tyr to His) drastically decreased the l-Trp affinity and catalytic efficiency of IDO1s. Conversely, two substitutions (distal-Thr to Ser and distal-His to Tyr) were sufficient to bestow IDO1-like high affinity on ancestral and chicken IDO2. © 2016 Federation of European Biochemical Societies.