Sample records for kangur tiiu koff

  1. Effects of target binding kinetics on in vivo drug efficacy: koff , kon and rebinding.

    PubMed

    Vauquelin, Georges

    2016-08-01

    Optimal drug therapy often requires continuing high levels of target occupancy. Besides the traditional pharmacokinetic contribution, target binding kinetics is increasingly considered to play an important role as well. While most attention has been focused on the dissociation rate of the complex, recent reports expressed doubt about the unreserved translatability of this pharmacodynamic property into clinical efficacy. 'Micro'-pharmacokinetic mechanisms like drug rebinding and partitioning into the cell membrane may constitute a potential fix. Simulations were based on solving differential equations. Based on a selected range of association and dissociation rate constants, kon and koff , and rebinding potencies of the drugs as variables, their effects on the temporal in vivo occupancy profile of their targets, after one or multiple repetitive dosings, have here been simulated. Most strikingly, the simulations show that, when rebinding is also taken into account, increasing kon may produce closely the same outcome as decreasing koff when dosing is performed in accordance with the therapeutically most relevant constant [Lmax ]/KD ratio paradigm. Also, under certain conditions, rebinding may produce closely the same outcome as invoking slow diffusion of the drug between the plasma compartment and a target-containing 'effect' compartment. Although the present simulations should only be regarded as a 'proof of principle', these findings may help pharmacologists and medicinal chemists to devise ex vivo and in vitro binding kinetic assays that are more relevant and translatable to in vivo settings. © 2016 The British Pharmacological Society.

  2. Effects of target binding kinetics on in vivo drug efficacy: koff, kon and rebinding

    PubMed Central

    2016-01-01

    Abstract Background and Purpose Optimal drug therapy often requires continuing high levels of target occupancy. Besides the traditional pharmacokinetic contribution, target binding kinetics is increasingly considered to play an important role as well. While most attention has been focused on the dissociation rate of the complex, recent reports expressed doubt about the unreserved translatability of this pharmacodynamic property into clinical efficacy. ‘Micro’‐pharmacokinetic mechanisms like drug rebinding and partitioning into the cell membrane may constitute a potential fix. Experimental Approach Simulations were based on solving differential equations. Key Results Based on a selected range of association and dissociation rate constants, kon and koff, and rebinding potencies of the drugs as variables, their effects on the temporal in vivo occupancy profile of their targets, after one or multiple repetitive dosings, have here been simulated. Conclusions and Implications Most strikingly, the simulations show that, when rebinding is also taken into account, increasing kon may produce closely the same outcome as decreasing koff when dosing is performed in accordance with the therapeutically most relevant constant [Lmax]/K D ratio paradigm. Also, under certain conditions, rebinding may produce closely the same outcome as invoking slow diffusion of the drug between the plasma compartment and a target‐containing ‘effect’ compartment. Although the present simulations should only be regarded as a ‘proof of principle’, these findings may help pharmacologists and medicinal chemists to devise ex vivo and in vitro binding kinetic assays that are more relevant and translatable to in vivo settings. PMID:27129075

  3. Non-Markovian effects in the first-passage dynamics of obstructed tracer particle diffusion in one-dimensional systems

    NASA Astrophysics Data System (ADS)

    Forsling, Robin; Sanders, Lloyd P.; Ambjörnsson, Tobias; Lizana, Ludvig

    2014-09-01

    The standard setup for single-file diffusion is diffusing particles in one dimension which cannot overtake each other, where the dynamics of a tracer (tagged) particle is of main interest. In this article, we generalize this system and investigate first-passage properties of a tracer particle when flanked by identical crowder particles which may, besides diffuse, unbind (rebind) from (to) the one-dimensional lattice with rates koff (kon). The tracer particle is restricted to diffuse with rate kD on the lattice and the density of crowders is constant (on average). The unbinding rate koff is our key parameter and it allows us to systematically study the non-trivial transition between the completely Markovian case (koff ≫ kD) to the non-Markovian case (koff ≪ kD) governed by strong memory effects. This has relevance for several quasi one-dimensional systems. One example is gene regulation where regulatory proteins are searching for specific binding sites on a crowded DNA. We quantify the first-passage time distribution, f (t) (t is time), numerically using the Gillespie algorithm, and estimate f (t) analytically. In terms of koff (keeping kD fixed), we study the transition between the two known regimes: (i) when koff ≫ kD the particles may effectively pass each other and we recover the single particle result f (t) ˜ t-3/2, with a reduced diffusion constant; (ii) when koff ≪ kD unbinding is rare and we obtain the single-file result f (t) ˜ t-7/4. The intermediate region displays rich dynamics where both the characteristic f (t) - peak and the long-time power-law slope are sensitive to koff.

  4. Transient kinetic studies of pH-dependent hydrolyses by exo-type carboxypeptidase P on a 27-MHz quartz crystal microbalance.

    PubMed

    Furusawa, Hiroyuki; Takano, Hiroki; Okahata, Yoshio

    2008-02-15

    pH-Dependent kinetic parameters (k(on), k(off), and k(cat)) of protein (myoglobin) hydrolyses catalyzed by exo-enzyme (carboxypeptidase P, CPP) were obtained by using a protein-immobilized quartz crystal microbalance (QCM) in acidic aqueous solutions. The formation of the enzyme-substrate (ES) complex (k(on)), the decay of the ES complex (k(off)), and the formation of the product (k(cat)) could be analyzed by transient kinetics as mass changes on the QCM plate. The Kd (k(off)/k(on)) value was different from the Michaelis constant Km calculated from (k(off) + k(cat))/k(on) due to k(cat) > k(off). The rate-determining step was the binding step (k(on), and the catalytic rate k(cat) was faster than other k(on) and k(off) values. In the range of pH 2.5-5.0, values of k(on) gradually increased with decreasing pH showing a maximum at pH 3.7, values of k(off) were independent of pH, and k(cat) increased gradually with decreasing pH. As a result, the apparent rate constant (k(cat)/Km) showed a maximum at pH 3.7 and gradually increased with decreasing pH. The optimum pH at 3.7 of k(on) is explained by the optimum binding ability of CPP to the COOH terminus of the substrate with hydrogen bonds. The increase of k(cat) at the lower pH correlated with the decrease of alpha-helix contents of the myoglobin substrate on the QCM.

  5. Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases*

    PubMed Central

    Kurašin, Mihhail; Kuusk, Silja; Kuusk, Piret; Sørlie, Morten; Väljamäe, Priit

    2015-01-01

    Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (PIntr) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a 14C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site −3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils. PMID:26468285

  6. Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases.

    PubMed

    Kurašin, Mihhail; Kuusk, Silja; Kuusk, Piret; Sørlie, Morten; Väljamäe, Priit

    2015-11-27

    Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (P(Intr)) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a (14)C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site -3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices

    PubMed Central

    Dupuis, Nicholas F.; Holmstrom, Erik D.; Nesbitt, David J.

    2013-01-01

    In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′on = 2.1(1) × 106 M−1 s−1) and 6 bp (k′on = 5.0(1) × 106 M−1 s−1) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s−1) to 6 bp (koff = 14 s−1) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, konincreases by >40-fold (kon = 0.10(1) s−1 to 4.0(4) s−1 between [NaCl] = 25 mM and 1 M), whereas in contrast, koffdecreases by fourfold (0.72(3) s−1 to 0.17(7) s−1) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation. PMID:23931323

  8. Free energy landscape for the binding process of Huperzine A to acetylcholinesterase

    PubMed Central

    Bai, Fang; Xu, Yechun; Chen, Jing; Liu, Qiufeng; Gu, Junfeng; Wang, Xicheng; Ma, Jianpeng; Li, Honglin; Onuchic, José N.; Jiang, Hualiang

    2013-01-01

    Drug-target residence time (t = 1/koff, where koff is the dissociation rate constant) has become an important index in discovering better- or best-in-class drugs. However, little effort has been dedicated to developing computational methods that can accurately predict this kinetic parameter or related parameters, koff and activation free energy of dissociation (). In this paper, energy landscape theory that has been developed to understand protein folding and function is extended to develop a generally applicable computational framework that is able to construct a complete ligand-target binding free energy landscape. This enables both the binding affinity and the binding kinetics to be accurately estimated. We applied this method to simulate the binding event of the anti-Alzheimer’s disease drug (−)−Huperzine A to its target acetylcholinesterase (AChE). The computational results are in excellent agreement with our concurrent experimental measurements. All of the predicted values of binding free energy and activation free energies of association and dissociation deviate from the experimental data only by less than 1 kcal/mol. The method also provides atomic resolution information for the (−)−Huperzine A binding pathway, which may be useful in designing more potent AChE inhibitors. We expect this methodology to be widely applicable to drug discovery and development. PMID:23440190

  9. Free energy landscape for the binding process of Huperzine A to acetylcholinesterase.

    PubMed

    Bai, Fang; Xu, Yechun; Chen, Jing; Liu, Qiufeng; Gu, Junfeng; Wang, Xicheng; Ma, Jianpeng; Li, Honglin; Onuchic, José N; Jiang, Hualiang

    2013-03-12

    Drug-target residence time (t = 1/k(off), where k(off) is the dissociation rate constant) has become an important index in discovering better- or best-in-class drugs. However, little effort has been dedicated to developing computational methods that can accurately predict this kinetic parameter or related parameters, k(off) and activation free energy of dissociation (ΔG(off)≠). In this paper, energy landscape theory that has been developed to understand protein folding and function is extended to develop a generally applicable computational framework that is able to construct a complete ligand-target binding free energy landscape. This enables both the binding affinity and the binding kinetics to be accurately estimated. We applied this method to simulate the binding event of the anti-Alzheimer's disease drug (-)-Huperzine A to its target acetylcholinesterase (AChE). The computational results are in excellent agreement with our concurrent experimental measurements. All of the predicted values of binding free energy and activation free energies of association and dissociation deviate from the experimental data only by less than 1 kcal/mol. The method also provides atomic resolution information for the (-)-Huperzine A binding pathway, which may be useful in designing more potent AChE inhibitors. We expect this methodology to be widely applicable to drug discovery and development.

  10. Pollen-inferred quantitative reconstructions of Holocene land-cover in NW Europe for the evaluation of past climate-vegetation feedbacks - The Swedish LANDCLIM project and the NordForsk LANDCLIM network

    NASA Astrophysics Data System (ADS)

    Gaillard, Marie-Jose; Sugita, Shinya; Rundgren, Mats; Smith, Benjamin; Mazier, Florence; Trondman, Anna-Kari; Fyfe, Ralph; Kokfelt, Ulla; Nielsen, Anne-Birgitte; Strandberg, Gustav

    2010-05-01

    of ca. 1o x 1o. The REVEALS estimates of the past cover of PFTs will be 1) compared with the outputs of the LPJ-GUESS (10 PFTs), a widely-used dynamic vegetation model and 2) used as an alternative to the LPJ-GUESS-simulated vegetation (3 PFTs) to run for the past the regional climate model RCA3 developed at the Rossby Centre, Norrköping, Sweden. The study will evaluate and further refine these models (RCA3 and LPJ-GUESS) using a data-model comparison approach that incorporates new syntheses of palaeoclimatic data as well. It will lead to new assessments of the possible effect of various factors on climate, such as deforestations and afforestations, and changes in vegetation composition and spatial patterns of land cover/land use. Refined climate models and empirical land-cover reconstructions will shed new light on controversial hypotheses of past climate change and human impacts, such as the "Ruddiman hypothesis". First maps of REVEALS estimates of plant functional types (PFTs) are now available for Sweden, Norway, Finland, Denmark, Estonia, Poland, Germany, The Czech Republic, Switzerland and Britain (see Mazier et al. C1.21 and Trondman et al. C1.22). Correlation tests show that the REVEALS estimates are robust in terms of ranking of the PFTs' abundance (see Mazier et al, C1.21). The LANDCLIM project and network are a contribution to the IGBP-PAGES-Focus 4 PHAROS programme on human impact on environmental changes in the past. The following LANDCLIM members are acknowledged for providing pollen records, for help with pollen databases, and for providing results to the project: Mihkel Kangur and Tiiu Koff (Univ. Tallinn, Tallinn); Erik Kjellström (SMHI, Norrköping), Anna Broström, Lena Barnekow and Thomas Persson (GeoBiosphere Science Centre, Lund University); Anneli Poska (Physical Geography and Ecosystems Analysis, Lund University); Thomas Giesecke (Albrecht-von-Haller-Institute for Plant Sciences, University of Göttingen), Anne Bjune and John Birks (Dept. of

  11. Urethral advancement in hypospadias with a distal division of the corpus spongiosum: outcome in 158 cases.

    PubMed

    Thiry, S; Gorduza, D; Mouriquand, P

    2014-06-01

    Outcome of urethral mobilization and advancement (Koff procedure) in hypospadias with a distal division of the corpus spongiosum and redo cases with distal urethral failure. From January 1999 to November 2012, 158 children with a distal hypospadias (115 primary cases and 43 redo cases) underwent surgical repair using the Koff technique with a median age at surgery of 21 months (range, 12-217 months). Mean follow-up was 19 months (median, 14 months). Thirty patients (19%) presented with a complication (13.9% in primary cases and 32.5% in redo surgery) mostly at the beginning of our experience. Meatal stenosis was the most common one (3.5% in primary case, 6% overall). Ventral curvature (>10°), which is considered as a possible long-term iatrogenic complication of the Koff procedure, was not found in patients with fully grown penis except in one redo patient who had, retrospectively, an inadequate indication for this type of repair. Of 158 patients, 33 reached the age of puberty (>14 years old) with a mean follow-up of 34 months, only one presented with a significant ventral curvature. Urethral mobilization and advancement is a reasonable alternative for anterior hypospadias and distal fistula repair in selected cases. It has two major advantages compared to other techniques: it avoids any urethroplasty with non-urethral tissue and eliminates dysplastic tissues located beyond the division of the corpus spongiosum, which may not grow at the same pace as the rest of the penis. Significant iatrogenic curvature in fully grown penis is not supported by this series. Copyright © 2013 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

  12. Stress: Specific Life Events in the Teaching Profession.

    ERIC Educational Resources Information Center

    Martray, Carl R.; Adams, Ronald D.

    This study examined the greatest stressors in teaching situations that affect teachers, and how these events vary for groups of elementary, middle, and secondary school teachers. The list of possibly stressful situations was taken from the Teaching Events Stress Inventory (TESI), developed by Cichon and Koff in 1978. Data were collected from…

  13. THE HEME BINDING PROPERTIES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

    PubMed Central

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H.; Stuehr, Dennis J.

    2012-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for cellular heme insertion into inducible nitric oxide synthase (Chakravarti et al, PNAS 2010, 107(42):18004-9), we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (1 heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418 and 537 nm, and when reduced to ferrous gave maxima at 424, 527 and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were kon =17,800 M−1s−1 and koff1 = 7.0 × 10−3 s−1; koff2 = 3.3 × 10−4 s−1 respectively, giving approximate affinities of 19–390 nM. Ferrous heme bound more poorly to GAPDH and dissociated with a koff = 4.2 × 10−3 s−1. Magnetic circular dichroism (MCD), resonance Raman (rR) and EPR spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in ferric complex was not displaced by CN− or N3− but in ferrous complex was displaceable by CO at a rate of 1.75 s−1 (for [CO]>0.2 mM). Studies with heme analogs revealed selectivity toward the coordinating metal and porphyrin ring structure. GAPDH-heme was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-amino levulinic acid. Our finding of heme binding to GAPDH expands the protein’s potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH is consistent with it performing heme sensing or heme chaperone-like functions in cells. PMID:22957700

  14. Improving estimation of kinetic parameters in dynamic force spectroscopy using cluster analysis

    NASA Astrophysics Data System (ADS)

    Yen, Chi-Fu; Sivasankar, Sanjeevi

    2018-03-01

    Dynamic Force Spectroscopy (DFS) is a widely used technique to characterize the dissociation kinetics and interaction energy landscape of receptor-ligand complexes with single-molecule resolution. In an Atomic Force Microscope (AFM)-based DFS experiment, receptor-ligand complexes, sandwiched between an AFM tip and substrate, are ruptured at different stress rates by varying the speed at which the AFM-tip and substrate are pulled away from each other. The rupture events are grouped according to their pulling speeds, and the mean force and loading rate of each group are calculated. These data are subsequently fit to established models, and energy landscape parameters such as the intrinsic off-rate (koff) and the width of the potential energy barrier (xβ) are extracted. However, due to large uncertainties in determining mean forces and loading rates of the groups, errors in the estimated koff and xβ can be substantial. Here, we demonstrate that the accuracy of fitted parameters in a DFS experiment can be dramatically improved by sorting rupture events into groups using cluster analysis instead of sorting them according to their pulling speeds. We test different clustering algorithms including Gaussian mixture, logistic regression, and K-means clustering, under conditions that closely mimic DFS experiments. Using Monte Carlo simulations, we benchmark the performance of these clustering algorithms over a wide range of koff and xβ, under different levels of thermal noise, and as a function of both the number of unbinding events and the number of pulling speeds. Our results demonstrate that cluster analysis, particularly K-means clustering, is very effective in improving the accuracy of parameter estimation, particularly when the number of unbinding events are limited and not well separated into distinct groups. Cluster analysis is easy to implement, and our performance benchmarks serve as a guide in choosing an appropriate method for DFS data analysis.

  15. Quantitative determination of conformational, dynamic, and kinetic parameters of a ligand-protein/DNA complex from a complete relaxation and conformational exchange matrix analysis of intermolecular transferred NOESY.

    PubMed

    Moseley, H N; Lee, W; Arrowsmith, C H; Krishna, N R

    1997-05-06

    We report a quantitative analysis of the 13C-edited intermolecular transferred NOESY (inter-TrNOESY) spectrum of the trp-repressor/operator complex (trp-rep/op) with [ul-13C/15N]-L-tryptophan corepressor using a computer program implementing complete relaxation and conformational exchange matrix (CORCEMA) methodology [Moseley et al. (1995) J. Magn. Reson. 108B, 243-261]. Using complete mixing time curves of three inter-TrNOESY peaks between the tryptophan and the Trp-rep/op, this self-consistent analysis determined the correlation time of the bound species (tauB = 13.5 ns) and the exchange off-rate (k(off) = 3.6 s(-1)) of the corepressor. In addition, the analysis estimated the correlation time of the free species (tauF approximately 0.15 ns). Also, we demonstrate the sensitivity of these inter-TrNOESY peaks to several factors including the k(off) and orientation of the tryptophan corepressor within the binding site. The analysis indicates that the crystal structure orientation for the corepressor is compatible with the solution NMR data.

  16. [Crypto-hypospadias: a new therapeutic approach].

    PubMed

    Ottolenghi, A; Belligoli, A

    1984-01-01

    The AA present 26 cases of crypto-hypospadias traited by different methods. Particularly they describe a new technique, suitable to avoid urethroplasty; it consists in a wide mobilization of the urethra as Koff (modified) and a plasty of the tunica albuginea on the dorsal aspect of the shaft as Nesbit (modified). The first results are much satisfactories.

  17. Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos

    PubMed Central

    Liu, Tianbin; Dou, Hongwei; Xiang, Xi; Li, Yong; Pang, Xinzhi; Zhang, Yijie; Chen, Yu; Luan, Jing; Xu, Ying; Yang, Zhenzhen; Yang, Wenxian; Liu, Huan; Li, Feida; Wang, Hui; Yang, Huanming; Bolund, Lars; Vajta, Gabor

    2015-01-01

    Abstract Data analysis in somatic cell nuclear transfer (SCNT) research is usually limited to several hundreds or thousands of reconstructed embryos. Here, we report mass results obtained with an established and consistent porcine SCNT system (handmade cloning [HMC]). During the experimental period, 228,230 reconstructed embryos and 82,969 blastocysts were produced. After being transferred into 656 recipients, 1070 piglets were obtained. First, the effects of different types of donor cells, including fetal fibroblasts (FFs), adult fibroblasts (AFs), adult preadipocytes (APs), and adult blood mesenchymal (BM) cells, were investigated on the further in vitro and in vivo development. Compared to adult donor cells (AFs, APs, BM cells, respectively), FF cells resulted in a lower blastocyst/reconstructed embryo rate (30.38% vs. 37.94%, 34.65%, and 34.87%, respectively), but a higher overall efficiency on the number of piglets born alive per total blastocysts transferred (1.50% vs. 0.86%, 1.03%, and 0.91%, respectively) and a lower rate of developmental abnormalities (10.87% vs. 56.57%, 24.39%, and 51.85%, respectively). Second, recloning was performed with cloned adult fibroblasts (CAFs) and cloned fetal fibroblasts (CFFs). When CAFs were used as the nuclear donor, fewer developmental abnormalities and higher overall efficiency were observed compared to AFs (56.57% vs. 28.13% and 0.86% vs. 1.59%, respectively). However, CFFs had an opposite effect on these parameters when compared with CAFs (94.12% vs. 10.87% and 0.31% vs. 1.50%, respectively). Third, effects of genetic modification on the efficiency of SCNT were investigated with transgenic fetal fibroblasts (TFFs) and gene knockout fetal fibroblasts (KOFFs). Genetic modification of FFs increased developmental abnormalities (38.96% and 25.24% vs. 10.87% for KOFFs, TFFs, and FFs, respectively). KOFFs resulted in lower overall efficiency compared to TFFs and FFs (0.68% vs. 1.62% and 1.50%, respectively). In conclusion

  18. Heme binding properties of glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H; Stuehr, Dennis J

    2012-10-30

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004-18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: k(on) = 17800 M(-1) s(-1), k(off1) = 7.0 × 10(-3) s(-1), and k(off2) = 3.3 × 10(-4) s(-1) (giving approximate affinities of 19-390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a k(off) of 4.2 × 10(-3) s(-1). Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN(-) or N(3)(-) but in the ferrous complex could be displaced by CO at a rate of 1.75 s(-1) (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH-heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-aminolevulinic acid. Our finding of heme binding to GAPDH expands the protein's potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells.

  19. Utilizing a Water-Soluble Cryptophane with Fast Xenon Exchange Rates for Picomolar Sensitivity NMR Measurements

    PubMed Central

    Bai, Yubin; Hill, P. Aru; Dmochowski, Ivan J.

    2012-01-01

    Hyperpolarized 129Xe chemical exchange saturation transfer (129Xe Hyper-CEST) NMR is a powerful technique for the ultrasensitive, indirect detection of Xe host molecules (e.g., cryptophane-A). Irradiation at the appropriate Xe-cryptophane resonant radio frequency results in relaxation of the bound hyperpolarized 129Xe and rapid accumulation of depolarized 129Xe in bulk solution. The cryptophane effectively ‘catalyzes’ this process by providing a unique molecular environment for spin depolarization to occur, while allowing xenon exchange with the bulk solution during the hyperpolarized lifetime (T1 ≈ 1 min). Following this scheme, a triacetic acid cryptophane-A derivative (TAAC) was indirectly detected at 1.4 picomolar concentration at 320 K in aqueous solution, which is the record for a single-unit xenon host. To investigate this sensitivity enhancement, the xenon binding kinetics of TAAC in water was studied by NMR exchange lifetime measurement. At 297 K, kon ≈ 1.5 × 106 M−1s−1 and koff = 45 s−1, which represent the fastest Xe association and dissociation rates measured for a high-affinity, water-soluble xenon host molecule near rt. NMR linewidth measurements provided similar exchange rates at rt, which we assign to solvent-Xe exchange in TAAC. At 320 K, koff was estimated to be 1.1 × 103 s−1. In Hyper-CEST NMR experiments, the rate of 129Xe depolarization achieved by 14 pM TAAC in the presence of RF pulses was calculated to be 0.17 µM·s−1. On a per cryptophane basis, this equates to 1.2 × 104 129Xe atoms s−1 (or 4.6 × 104 Xe atoms s−1, all Xe isotopes), which is more than an order of magnitude faster than koff, the directly measurable Xe-TAAC exchange rate. This compels us to consider multiple Xe exchange processes for cryptophane-mediated bulk 129Xe depolarization, which provide at least 107-fold sensitivity enhancements over directly detected hyperpolarized 129Xe NMR signals. PMID:23106513

  20. Engineering and characterization of fluorogenic glycine riboswitches

    PubMed Central

    Ketterer, Simon; Gladis, Lukas; Kozica, Adnan; Meier, Matthias

    2016-01-01

    A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch library. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570 μM. Furthermore, the kinetic glycine binding (kon), and dissociation (koff) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. kon and koff were in the order of 10−3s−1 and 10−2s−1, respectively. Conclusively, we demonstrate that systematic screening of synthetic and natural linked RNA parts with microfluidic chip technology is an effective approach to rapidly generate fluorogenic metabolite riboswitches with a broad range of biophysical response properties. PMID:27220466

  1. Förster resonance energy transfer competitive displacement assay for human soluble epoxide hydrolase

    PubMed Central

    Lee, Kin Sing Stephen; Morisseau, Christophe; Yang, Jun; Wang, Peng; Hwang, Sung Hee; Hammock, Bruce D.

    2013-01-01

    The soluble epoxide hydrolase (sEH), responsible for the hydrolysis of various fatty acid epoxides to their corresponding 1,2-diols, is becoming an attractive pharmaceutical target. These fatty acid epoxides, particularly epoxyeicosatrienoic acids (EETs), play an important role in human homeostatic and inflammation processes. Therefore, inhibition of human sEH, which stabilizes EETs in vivo, brings several beneficial effects to human health. Although there are several catalytic assays available to determine the potency of sEH inhibitors, measuring the in vitro inhibition constant (Ki) for these inhibitors using catalytic assay is laborious. In addition, koff, which has been recently suggested to correlate better with the in vivo potency of inhibitors, has never been measured for sEH inhibitors. To better measure the potency of sEH inhibitors, a reporting ligand, 1-(adamantan-1-yl)-3-(1-(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetyl) piperidin-4-yl)urea (ACPU), was designed and synthesized. With ACPU, we have developed a Förster resonance energy transfer (FRET)-based competitive displacement assay using intrinsic tryptophan fluorescence from sEH. In addition, the resulting assay allows us to measure the Ki values of very potent compounds to the picomolar level and to obtain relative koff values of the inhibitors. This assay provides additional data to evaluate the potency of sEH inhibitors. PMID:23219719

  2. Engineering and characterization of fluorogenic glycine riboswitches.

    PubMed

    Ketterer, Simon; Gladis, Lukas; Kozica, Adnan; Meier, Matthias

    2016-07-08

    A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch library. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570 μM. Furthermore, the kinetic glycine binding (k(on)), and dissociation (k(off)) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. k(on) and k(off) were in the order of 10(-3)s(-1) and 10(-2)s(-1), respectively. Conclusively, we demonstrate that systematic screening of synthetic and natural linked RNA parts with microfluidic chip technology is an effective approach to rapidly generate fluorogenic metabolite riboswitches with a broad range of biophysical response properties. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle.

    PubMed

    Skinner, Gary M; Baumann, Christoph G; Quinn, Diana M; Molloy, Justin E; Hoggett, James G

    2004-01-30

    A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape.

  4. von Willebrand factor (VWF) propeptide binding to VWF D'D3 domain attenuates platelet activation and adhesion.

    PubMed

    Madabhushi, Sri R; Shang, Chengwei; Dayananda, Kannayakanahalli M; Rittenhouse-Olson, Kate; Murphy, Mary; Ryan, Thomas E; Montgomery, Robert R; Neelamegham, Sriram

    2012-05-17

    Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.

  5. Modeling neutralization kinetics of HIV by broadly neutralizing monoclonal antibodies in genital secretions coating the cervicovaginal mucosa.

    PubMed

    McKinley, Scott A; Chen, Alex; Shi, Feng; Wang, Simi; Mucha, Peter J; Forest, M Gregory; Lai, Samuel K

    2014-01-01

    Eliciting broadly neutralizing antibodies (bnAb) in cervicovaginal mucus (CVM) represents a promising "first line of defense" strategy to reduce vaginal HIV transmission. However, it remains unclear what levels of bnAb must be present in CVM to effectively reduce infection. We approached this complex question by modeling the dynamic tally of bnAb coverage on HIV. This analysis introduces a critical, timescale-dependent competition: to protect, bnAb must accumulate at sufficient stoichiometry to neutralize HIV faster than virions penetrate CVM and reach target cells. We developed a model that incorporates concentrations and diffusivities of HIV and bnAb in semen and CVM, kinetic rates for binding (kon) and unbinding (koff) of select bnAb, and physiologically relevant thicknesses of CVM and semen layers. Comprehensive model simulations lead to robust conclusions about neutralization kinetics in CVM. First, due to the limited time virions in semen need to penetrate CVM, substantially greater bnAb concentrations than in vitro estimates must be present in CVM to neutralize HIV. Second, the model predicts that bnAb with more rapid kon, almost independent of koff, should offer greater neutralization potency in vivo. These findings suggest the fastest arriving virions at target cells present the greatest likelihood of infection. It also implies the marked improvements in in vitro neutralization potency of many recently discovered bnAb may not translate to comparable reduction in the bnAb dose needed to confer protection against initial vaginal infections. Our modeling framework offers a valuable tool to gaining quantitative insights into the dynamics of mucosal immunity against HIV and other infectious diseases.

  6. Antigen Potency and Maximal Efficacy Reveal a Mechanism of Efficient T Cell Activation

    PubMed Central

    Wheeler, Richard J.; Zhang, Hao; Cordoba, Shaun-Paul; Peng, Yan-Chun; Chen, Ji-Li; Cerundolo, Vincenzo; Dong, Tao; Coombs, Daniel; van der Merwe, P. Anton

    2014-01-01

    T cell activation, a critical event in adaptive immune responses, follows productive interactions between T cell receptors (TCRs) and antigens, in the form of peptide-bound major histocompatibility complexes (pMHCs) on the surfaces of antigen-presenting-cells. Upon activation, T cells can lyse infected cells, secrete cytokines, such as interferon-γ (IFN-γ), and perform other effector functions with various efficiencies that directly depend on the binding parameters of the TCR-pMHC complex. The mechanism that relates binding parameters to the efficiency of activation of the T cell remains controversial; some studies suggest that the dissociation constant (KD) determines the response (the “affinity model”), whereas others suggest that the off-rate (koff) is critical (the “productive hit rate model”). Here, we used mathematical modeling to show that antigen potency, as determined by the EC50, the functional correlate that is used to support KD-based models, could not be used to discriminate between the affinity and productive hit rate models. Our theoretical work showed that both models predicted a correlation between antigen potency and KD, but only the productive hit rate model predicted a correlation between maximal efficacy (Emax) and koff. We confirmed the predictions made by the productive hit rate model in experiments with cytotoxic T cell clones and a panel of pMHC variants. Therefore, we suggest that the activity of an antigen is determined by both its potency and maximal efficacy. We discuss the implications of our findings to the practical evaluation of T cell activation, for example in adoptive immunotherapies, and relate our work to the pharmacological theory of dose-response. PMID:21653229

  7. Control of the Ability of Profilin to Bind and Facilitate Nucleotide Exchange from G-actin*

    PubMed Central

    Wen, Kuo-Kuang; McKane, Melissa; Houtman, Jon C. D.; Rubenstein, Peter A.

    2008-01-01

    A major factor in profilin regulation of actin cytoskeletal dynamics is its facilitation of G-actin nucleotide exchange. However, the mechanism of this facilitation is unknown. We studied the interaction of yeast (YPF) and human profilin 1 (HPF1) with yeast and mammalian skeletal muscle actins. Homologous pairs (YPF and yeast actin, HPF1 and muscle actin) bound more tightly to one another than heterologous pairs. However, with saturating profilin, HPF1 caused a faster etheno-ATP exchange with both yeast and muscle actins than did YPF. Based on the -fold change in ATP exchange rate/Kd, however, the homologous pairs are more efficient than the heterologous pairs. Thus, strength of binding of profilin to actin and nucleotide exchange rate are not tightly coupled. Actin/HPF interactions were entropically driven, whereas YPF interactions were enthalpically driven. Hybrid yeast actins containing subdomain 1 (sub1) or subdomain 1 and 2 (sub12) muscle actin residues bound more weakly to YPF than did yeast actin (Kd = 2 μm versus 0.6 μm). These hybrids bound even more weakly to HPF than did yeast actin (Kd = 5 μm versus 3.2 μm). sub1/YPF interactions were entropically driven, whereas the sub12/YPF binding was enthalpically driven. Compared with WT yeast actin, YPF binding to sub1 occurred with a 5 times faster koff and a 2 times faster kon. sub12 bound with a 3 times faster koff and a 1.5 times slower kon. Profilin controls the energetics of its interaction with nonhybrid actin, but interactions between actin subdomains 1 and 2 affect the topography of the profilin binding site. PMID:18223293

  8. Multi-Ligand-Binding Flavoprotein Dodecin as a Key Element for Reversible Surface Modification in Nano-biotechnology.

    PubMed

    Gutiérrez Sánchez, Cristina; Su, Qiang; Schönherr, Holger; Grininger, Martin; Nöll, Gilbert

    2015-01-01

    In this paper the multiple (re)programming of protein-DNA nanostructures comprising generation, deletion, and reprogramming on the same flavin-DNA-modified surface is introduced. This work is based on a systematic study of the binding affinity of the multi-ligand-binding flavoprotein dodecin on flavin-terminated DNA monolayers by surface plasmon resonance and quartz crystal microbalance with dissipation (QCM-D) measurements, surface plasmon fluorescence spectroscopy (SPFS), and dynamic AFM force spectroscopy. Depending on the flavin surface coverage, a single apododecin is captured by one or more surface-immobilized flavins. The corresponding complex binding and unbinding rate constants kon(QCM) = 7.7 × 10(3) M(-1)·s(-1) and koff(QCM) = 4.5 × 10(-3) s(-1) (Kd(QCM) = 580 nM) were determined by QCM and were found to be in agreement with values for koff determined by SPFS and force spectroscopy. Even though a single apododecin-flavin bond is relatively weak, stable dodecin monolayers were formed on flavin-DNA-modified surfaces at high flavin surface coverage due to multivalent interactions between apododecin bearing six binding pockets and the surface-bound flavin-DNA ligands. If bi- or multivalent flavin ligands are adsorbed on dodecin monolayers, stable sandwich-type surface-DNA-flavin-apododecin-flavin ligand arrays are obtained. Nevertheless, the apododecin flavin complex is easily and quantitatively disassembled by flavin reduction. Binding and release of apododecin are reversible processes, which can be carried out alternatingly several times to release one type of ligand by an external redox trigger and subsequently replace it with a different ligand. Hence the versatile concept of reprogrammable functional biointerfaces with the multi-ligand-binding flavoprotein dodecin is demonstrated.

  9. A specific transition state for S-peptide combining with folded S-protein and then refolding

    PubMed Central

    Goldberg, Jonathan M.; Baldwin, Robert L.

    1999-01-01

    We measured the folding and unfolding kinetics of mutants for a simple protein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form ribonuclease S analogues: initially, S-peptide is disordered whereas S-protein is folded. The fluorescent probe provides a convenient spectroscopic probe for the reaction. The association rate constant, kon, and the dissociation rate constant, koff, were both determined for two sets of mutants. The dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows kon and koff to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding and refolding. The first set of mutants tests the role of the α-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the α-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log kon vs. log Kd for this series of mutants is linear over a very wide range, with a slope of −0.3, indicating that almost all of the molecules fold via a transition state involving the helix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, which are known to be important for binding S-peptide to S-protein and which also contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transition state. The results provide a remarkably clear description of a folding transition state. PMID:10051587

  10. Image Restoration and Analysis of Influenza Virions Binding to Membrane Receptors Reveal Adhesion-Strengthening Kinetics

    PubMed Central

    Lee, Donald W.; Hsu, Hung-Lun; Bacon, Kaitlyn B.; Daniel, Susan

    2016-01-01

    With the development of single-particle tracking (SPT) microscopy and host membrane mimics called supported lipid bilayers (SLBs), stochastic virus-membrane binding interactions can be studied in depth while maintaining control over host receptor type and concentration. However, several experimental design challenges and quantitative image analysis limitations prevent the widespread use of this approach. One main challenge of SPT studies is the low signal-to-noise ratio of SPT videos, which is sometimes inevitable due to small particle sizes, low quantum yield of fluorescent dyes, and photobleaching. These situations could render current particle tracking software to yield biased binding kinetic data caused by intermittent tracking error. Hence, we developed an effective image restoration algorithm for SPT applications called STAWASP that reveals particles with a signal-to-noise ratio of 2.2 while preserving particle features. We tested our improvements to the SPT binding assay experiment and imaging procedures by monitoring X31 influenza virus binding to α2,3 sialic acid glycolipids. Our interests lie in how slight changes to the peripheral oligosaccharide structures can affect the binding rate and residence times of viruses. We were able to detect viruses binding weakly to a glycolipid called GM3, which was undetected via assays such as surface plasmon resonance. The binding rate was around 28 folds higher when the virus bound to a different glycolipid called GD1a, which has a sialic acid group extending further away from the bilayer surface than GM3. The improved imaging allowed us to obtain binding residence time distributions that reflect an adhesion-strengthening mechanism via multivalent bonds. We empirically fitted these distributions using a time-dependent unbinding rate parameter, koff, which diverges from standard treatment of koff as a constant. We further explain how to convert these models to fit ensemble-averaged binding data obtained by assays such

  11. Flight of a cytidine deaminase complex with an imperfect transition state analogue inhibitor: mass spectrometric evidence for the presence of a trapped water molecule.

    PubMed

    Schroeder, Gottfried K; Zhou, Li; Snider, Mark J; Chen, Xian; Wolfenden, Richard

    2012-08-14

    Cytidine deaminase (CDA) binds the inhibitor zebularine as its 3,4-hydrate (K(d) ~ 10(-12) M), capturing all but ~5.6 kcal/mol of the free energy of binding expected of an ideal transition state analogue (K(tx) ~ 10(-16) M). On the basis of its entropic origin, that shortfall was tentatively ascribed to the trapping of a water molecule in the enzyme-inhibitor complex, as had been observed earlier for product uridine [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364-11371]. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of CDA nebularized in the presence of saturating 5-fluorozebularine reveals peaks corresponding to the masses of E(2)Zn(2)W(2) (dimeric Zn-CDA with two water molecules), E(2)Zn(2)W(2)Fz, and E(2)Zn(2)W(2)Fz(2), where Fz represents the 3,4-hydrate of 5-fluorozebularine. In the absence of an inhibitor, E(2)Zn(2) is the only dimeric species detected, with no additional water molecules. Experiments conducted in H(2)(18)O indicate that the added mass W represents a trapped water molecule rather than an isobaric ammonium ion. This appears to represent the first identification of an enzyme-bound water molecule at a subunit interface (active site) using FTICR-MS. The presence of a 5-fluoro group appears to retard the decomposition of the inhibitory complex kinetically in the vapor phase, as no additional dimeric complexes (other than E(2)Zn(2)) are observed when zebularine is used in place of 5-fluorozebularine. Substrate competition assays show that in solution zebularine is released from CDA (k(off) > 0.14 s(-1)) much more rapidly than is 5-fluorozebularine (k(off) = 0.014 s(-1)), despite the greater thermodynamic stability of the zebularine complex.

  12. Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation

    PubMed Central

    Casillas-Ituarte, Nadia N.; Cruz, Carlos H. B.; Lins, Roberto D.; DiBartola, Alex C.; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F. T.; Sierra-Hernández, M. Roxana; Lower, Steven K.

    2017-01-01

    The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2–0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections. PMID:28400484

  13. High-affinity dopamine D2/D3 PET radioligands 18F-fallypride and 11C-FLB457: A comparison of kinetics in extrastriatal regions using a multiple-injection protocol

    PubMed Central

    Vandehey, Nicholas T; Moirano, Jeffrey M; Converse, Alexander K; Holden, James E; Mukherjee, Jogesh; Murali, Dhanabalan; Nickles, R Jerry; Davidson, Richard J; Schneider, Mary L; Christian, Bradley T

    2010-01-01

    18F-Fallypride and 11C-FLB457 are commonly used PET radioligands for imaging extrastriatal dopamine D2/D3 receptors, but differences in their in vivo kinetics may affect the sensitivity for measuring subtle changes in receptor binding. Focusing on regions of low binding, a direct comparison of the kinetics of 18F-fallypride and 11C-FLB457 was made using a MI protocol. Injection protocols were designed to estimate K1, k2, fNDkon, Bmax, and koff in the midbrain and cortical regions of the rhesus monkey. 11C-FLB457 cleared from the arterial plasma faster and yielded a ND space distribution volume (K1/k2) that is three times higher than 18F-fallypride, primarily due to a slower k2 (FAL:FLB; k2=0.54 min−1:0.18 min−1). The dissociation rate constant, koff, was slower for 11C-FLB457, resulting in a lower KDapp than 18F-fallypride (FAL:FLB; 0.39 nM:0.13 nM). Specific D2/D3 binding could be detected in the cerebellum for 11C-FLB457 but not 18F-fallypride. Both radioligands can be used to image extrastriatal D2/D3 receptors, with 11C-FLB457 providing greater sensitivity to subtle changes in low-receptor-density cortical regions and 18F-fallypride being more sensitive to endogenous dopamine displacement in medium-to-high-receptor-density regions. In the presence of specific D2/D3 binding in the cerebellum, reference region analysis methods will give a greater bias in BPND with 11C-FLB457 than with 18F-fallypride. PMID:20040928

  14. Pollen-inferred quantitative reconstructions of Holocene land-cover in NW Europe for the evaluation of past climate-vegetation feedbacks - methods and first maps of the cover of plant functional types at 6000, 3000, 600, 200 and 0 BP.

    NASA Astrophysics Data System (ADS)

    Trondman, Anna-Kari; Gaillard, Marie-José; Sugita, Shinya; Mazier, Florence; Fyfe, Ralph; Nielsen, Anne-Birgitte; Leydet, Michelle; Members, Landclim

    2010-05-01

    ) number of pollen taxa, 3) PPEs dataset, and 4) number of dates per record used to establish the chronology (≥3 or ≥5) was tested (see Mazier et al. CL 1.21). Following the results of these tests, the first maps are based on REVEALS runs using pollen records from both lakes and bogs with ≥3 dates, 24 taxa (entomophilous taxa excluded), and the mean of all PPEs available in the study area. The maps are produced for 10 PFTs (LPJ-GUESS) and 3 PFTs (RCA3) at a spatial resolution of 1o x 1o for five selected time windows of the Holocene with contrasting human-induced land-cover (0-100 cal BP, 100-350 cal BP, 350-700 cal BP, 2700-3200 cal BP and 5700-6200 cal BP). The maps of PFTs show significant changes in the degree of human-induced vegetation openness through the Holocene over most of the study area. There are large discrepancies between these first quantitative land-cover maps and earlier maps based on pollen data and other methods such as biomization and the modern analogue approach. * The following LANDCLIM members are acknowledged for providing pollen records and for help with pollen databases: Teija Alenius (Espoo), Heather Almquist-Jacobson (Montana, USA), Lena Barnekow and Thomas Persson (Lund), Jonas Bergman (Stockholm), Anne Bjune and John Birks (Bergen), Thomas Giesecke (Göttingen), Rixt de Jong (Bern), Mihkel Kangur and Tiiuu Koff (Tallinn), Malgorzata Latalowa (Gdansk), Ann-Marie Robertsson (Stockholm), Ulf Segerström and Henrik von Stedingk (Umeå), Heikki Seppä (Helsinki). Sugita, S. 2007. The Holocene, 17, 229-241.

  15. Measuring the equation of state for a 2D colloidal membrane: A microfluidic approach to buffer exchange

    NASA Astrophysics Data System (ADS)

    Balchunas, Andrew; Cabanas, Rafael; Fraden, Seth; Dogic, Zvonimir

    Previous work has shown that monodisperse rod-like colloidal particles, such as a filamentous bacteriophage, self assemble into a 2D monolayer smectic in the presence of a non-adsorbing depleting polymer. These structures have the same functional form of bending rigidity and lateral compressibility as conventional lipid bi-layers, so we name the monolayer smectic a colloidal membrane. We have developed a microfluidic device such that the osmotic pressure acting on a colloidal membrane may be controlled via a full in situ buffer exchange. Rod density within individual colloidal membranes was measured as a function of osmotic pressure and a first order phase transition, from 2D fluid to 2D solid, was observed. kon and koff rates of rod to membrane binding were measured by lowering the osmotic pressure until membrane evaporation occurred.

  16. Tubulin Dimer Reversible Dissociation

    PubMed Central

    Schuck, Peter; Sackett, Dan L.

    2016-01-01

    Tubulins are evolutionarily conserved proteins that reversibly polymerize and direct intracellular traffic. Of the tubulin family only αβ-tubulin forms stable dimers. We investigated the monomer-dimer equilibrium of rat brain αβ-tubulin using analytical ultracentrifugation and fluorescence anisotropy, observing tubulin in virtually fully monomeric and dimeric states. Monomeric tubulin was stable for a few hours and exchanged into preformed dimers, demonstrating reversibility of dimer dissociation. Global analysis combining sedimentation velocity and fluorescence anisotropy yielded Kd = 84 (54–123) nm. Dimer dissociation kinetics were measured by analyzing the shape of the sedimentation boundary and by the relaxation of fluorescence anisotropy following rapid dilution of labeled tubulin, yielding koff in the range 10−3–10−2 s−1. Thus, tubulin dimers reversibly dissociate with moderately fast kinetics. Monomer-monomer association is much less sensitive than dimer-dimer association to solution changes (GTP/GDP, urea, and trimethylamine oxide). PMID:26934918

  17. Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.

    PubMed

    Brewer, Laurence R; Friddle, Raymond; Noy, Aleksandr; Baldwin, Enoch; Martin, Shelley S; Corzett, Michele; Balhorn, Rod; Baskin, Ronald J

    2003-10-01

    Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

  18. Clues for discovering a new biological function of Vitreoscilla hemoglobin in organisms: potential sulfide receptor and storage.

    PubMed

    Wang, Dandan; Liu, Li; Wang, Hui; Xu, Haoran; Chen, Lei; Ma, Li; Li, Zhengqiang

    2016-04-01

    The interaction between H2 S and Vitreoscilla hemoglobin (VHb) has been studied by UV-Vis and Resonance Raman spectroscopes to confirm the binding between the ligand and the protein. Kinetic constants, kon = 1.2 × 10(5) m(-1) ·s(-1) and koff = 2.5 × 10(-4) ·s(-1) , have been determined and compared with those for mammalian hemoglobins. Density Functional Theory study supports the binding of H2 S by modeling the configurations of HOMO dispersions. We hypothesized that VHb is involved in H2 S reception and storage. Different from Lucina pectinata HbI, a typical H2 S-binding hemoglobin, VHb, exhibits unusual properties on H2 S reactivity such as steric constraints playing an important role in modulating H2 S entry. A distinct mechanism of VHb interaction with H2 S is supported by studies of variant forms of VHb. © 2016 Federation of European Biochemical Societies.

  19. Studies on the contributions of steric and polarity effects to the H2S-binding properties of Vitreoscilla hemoglobin

    NASA Astrophysics Data System (ADS)

    Wang, Dandan; Wang, Hui; Li, Haichao; Liu, Li; Li, Zhengqiang

    2017-01-01

    We have reported recently that Vitreoscilla hemoglobin (VHb) is a potential H2S receptor and storage molecule in bacterial metabolism. In this study, molecular cloning and site-directed mutagenesis were employed to investigate the structural basis for H2S binding. Association and dissociation rate constants (kon and koff) were determined using stopped-flow rapid-scanning spectrophotometry and compared with those for wild type VHb. Several unanticipated factors were found to govern H2S binding properties, due to the distinct structure of VHb. The results presented in this paper show that: i) bulkier residues at positions E7 and E11 decrease H2S binding accessibility, while the residue located at position B10 blocks bound H2S from escaping. ii) hydroxyl sidechains within the distal heme pocket reduce H2S reactivity to VHb; iii) Pro(E8) is involved in moving the E7-E10 loop region to trigger opening of the distal heme pocket to facilitate H2S binding.

  20. Long residence times revealed by Aurora A kinase-targeting fluorescent probes derived from inhibitors MLN8237 and VX-689.

    PubMed

    Lavogina, Darja; Enkvist, Erki; Viht, Kaido; Uri, Asko

    2014-02-10

    We report the development of three fluorescent probes for protein kinase Aurora A that are derived from the well-known inhibitors MLN8237 and VX-689 (MK-5108). Two of these probes target the ATP site of Aurora A, and one targets simultaneously the ATP and substrate sites of the kinase. The probes were tested in an assay with fluorescence polarisation/anisotropy readout, and we demonstrated slow association kinetics and long residence time of the probes (kon 10(5)-10(7) M(-1) s(-1), koff 10(-3)-10(-4) s(-1); residence time 500-3000 s). The presence of the Aurora A activator TPX2 caused a significant reduction in the on-rate and increase in the off-rate of fluorescent probes targeting ATP site. These observations were supported by Aurora A inhibition assays with MLN8237 and VX-689. Overall, our results emphasise the importance of rational design of experiments with these compounds and correct interpretation of the obtained data. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Single-molecule force measurement via optical tweezers reveals different kinetic features of two BRaf mutants responsible for cardio-facial-cutaneous (CFC) syndrome

    PubMed Central

    Wen, Cheng; Ye, Anpei

    2013-01-01

    BRaf (B- Rapid Accelerated Fibrosarcoma) protein is an important serine/threonine-protein kinase. Two domains on BRaf can independently bind its upstream kinase, Ras (Rat Sarcoma) protein. These are the Ras binding domain (RBD) and cysteine-rich-domain (CRD). Herein we use customized optical tweezers to compare the Ras binding process in two pathological mutants of BRaf responsible for CFC syndrome, abbreviated BRaf (A246P) and BRaf (Q257R). The two mutants differ in their kinetics of Ras-binding, though both bind Ras with similar increased overall affinity. BRaf (A246P) exhibits a slightly higher Ras/CRD unbinding force and a significantly higher Ras/RBD unbinding force versus the wild type. The contrary phenomenon is observed in the Q257R mutation. Simulations of the unstressed-off rate, koff(0), yield results in accordance with the changes revealed by the mean unbinding force. Our approach can be applied to rapidly assess other mutated proteins to deduce the effects of mutation on their kinetics compared to wild type proteins and to each other. PMID:24409384

  2. Proteins feel more than they see: fine-tuning of binding affinity by properties of the non-interacting surface.

    PubMed

    Kastritis, Panagiotis L; Rodrigues, João P G L M; Folkers, Gert E; Boelens, Rolf; Bonvin, Alexandre M J J

    2014-07-15

    Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct contributions from the interface, the non-interacting surface (NIS) also plays an important role in binding affinity, especially polar and charged residues. Their percentage on the NIS is conserved over orthologous complexes indicating an evolutionary selection pressure. Their effect on binding affinity can be explained by long-range electrostatic contributions and surface-solvent interactions that are known to determine the local frustration of the protein complex surface. Including these in a simple model significantly improves the affinity prediction of protein complexes from structural models. The impact of mutations outside the interacting surface on binding affinity is supported by experimental alanine scanning mutagenesis data. These results enable the development of more sophisticated and integrated biophysical models of binding affinity and open new directions in experimental control and modulation of biomolecular interactions. Copyright © 2014. Published by Elsevier Ltd.

  3. A new approach for the extraction of pollutants from wastewaters handled by the graphic industry.

    PubMed

    Monteiro, C; Ventura, C; Martins, F

    2013-06-15

    It is widely recognized that the Graphic Industry handles toxic products and produces, in its various operations, toxic wastes. These wastes can cause serious environmental damages and can lead to severe health problems. In this work we report an efficient, simple and cheap to run method for the removal of some of the most common pollutants involved in the various stages of the Graphic Industry production, using a Solid-Phase Extraction (SPE) methodology. We have determined equilibrium constants, K(eq), and adsorption (k(up)) and desorption (k(off)) rate constants for the extraction of benzene, xylene, toluene and ethylbenzene (BXTE) from water, using C18 disks. The removal of these compounds was monitored by UV-vis spectroscopy, at room temperature. Average extraction efficiencies were of 60% in a mixture of BXTEs and close to 80% when pollutants were assessed separately. Since the retention mechanism in the C18 disk is essentially governed by hydrophobic interactions between the compounds and the alkyl chains of the disk, we have also shown that these pollutants' lipophilicity plays an important role in the rationalization of their behavior during the extraction process. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Nitrous oxide pollution from aircraft to increase by 2050

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Atreyee

    2012-09-01

    The transportation industry is not only one of the biggest sources of air pollution and a significant player in greenhouse gas-induced global warming, but, as a new study shows, the industry could also be responsible for episodes of ozone (O3 ) pollution, particularly over the United States and northern Europe. Combustion of fuel in cars, shipping vessels, and low-flying aircraft produce nitrogen oxides (NOx), which not only decrease the lifetime of greenhouse gases such as methane but also react with other molecules in the atmosphere to form tropospheric O3, another, more lethal, air pollutant. Hauglustaine and Koff used a global three-dimensional chemistry-climate model to investigate how different components of the transportation industry—cars, ships, and low-flying aircraft—would contribute to NOx pollution over the next few decades under several projected emission scenarios. They found that as road transportation stagnates or even declines due to stricter regulations and congestion, NOx emissions from cars will decrease over time. However, aircraft will increase in number and could contribute between 25% and 48% of NOx emissions, which will be most severe over the United States and Europe—two regions with the highest growth rate in commercial aviation.

  5. A semi-mechanistic model of CP-690,550-induced reduction in neutrophil counts in patients with rheumatoid arthritis.

    PubMed

    Gupta, Pankaj; Friberg, Lena E; Karlsson, Mats O; Krishnaswami, Sriram; French, Jonathan

    2010-06-01

    CP-690,550, a selective inhibitor of the Janus kinase family, is being developed as an oral disease-modifying antirheumatic drug for the treatment of rheumatoid arthritis (RA). A semi-mechanistic model was developed to characterize the time course of drug-induced absolute neutrophil count (ANC) reduction in a phase 2a study. Data from 264 RA patients receiving 6-week treatment (placebo, 5, 15, 30 mg bid) followed by a 6-week off-treatment period were analyzed. The model included a progenitor cell pool, a maturation chain comprising transit compartments, a circulation pool, and a feedback mechanism. The model was adequately described by system parameters (BASE(h), ktr(h), gamma, and k(circ)), disease effect parameters (DIS), and drug effect parameters (k(off) and k(D)). The disease manifested as an increase in baseline ANC and reduced maturation time due to increased demand from the inflammation site. The drug restored the perturbed system parameters to their normal values via an indirect mechanism. ANC reduction due to a direct myelosuppressive drug effect was not supported. The final model successfully described the dose- and time-dependent changes in ANC and predicted the incidence of neutropenia at different doses reasonably well.

  6. Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings

    PubMed Central

    Lowry, Troy W.; Hariri, Hanaa; Prommapan, Plengchart; Kusi-Appiah, Aubrey; Vafai, Nicholas; Bienkiewicz, Ewa A.; Van Winkle, David H.; Stagg, Scott M.

    2016-01-01

    The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid–protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (KD) and kinetics (kon and koff). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached. PMID:26649649

  7. Investigation of the heparin-thrombin interaction by dynamic force spectroscopy.

    PubMed

    Wang, Congzhou; Jin, Yingzi; Desai, Umesh R; Yadavalli, Vamsi K

    2015-06-01

    The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process. In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin-thrombin interaction from an energy viewpoint at the molecular scale. Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters. An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces. These results contribute to understanding the role of specific and nonspecific forces that drive heparin-thrombin interactions under applied force or flow conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Decoding the Role of Water Dynamics in Ligand-Protein Unbinding: CRF1R as a Test Case.

    PubMed

    Bortolato, Andrea; Deflorian, Francesca; Weiss, Dahlia R; Mason, Jonathan S

    2015-09-28

    The residence time of a ligand-protein complex is a crucial aspect in determining biological effect in vivo. Despite its importance, the prediction of ligand koff still remains challenging for modern computational chemistry. We have developed aMetaD, a fast and generally applicable computational protocol to predict ligand-protein unbinding events using a molecular dynamics (MD) method based on adiabatic-bias MD and metadynamics. This physics-based, fully flexible, and pose-dependent ligand scoring function evaluates the maximum energy (RTscore) required to move the ligand from the bound-state energy basin to the next. Unbinding trajectories are automatically analyzed and translated into atomic solvation factor (SF) values representing the water dynamics during the unbinding event. This novel computational protocol was initially tested on two M3 muscarinic receptor and two adenosine A2A receptor antagonists and then evaluated on a test set of 12 CRF1R ligands. The resulting RTscores were used successfully to classify ligands with different residence times. Additionally, the SF analysis was used to detect key differences in the degree of accessibility to water molecules during the predicted ligand unbinding events. The protocol provides actionable working hypotheses that are applicable in a drug discovery program for the rational optimization of ligand binding kinetics.

  9. Kinetics of protein–ligand unbinding: Predicting pathways, rates, and rate-limiting steps

    PubMed Central

    Tiwary, Pratyush; Limongelli, Vittorio; Salvalaglio, Matteo; Parrinello, Michele

    2015-01-01

    The ability to predict the mechanisms and the associated rate constants of protein–ligand unbinding is of great practical importance in drug design. In this work we demonstrate how a recently introduced metadynamics-based approach allows exploration of the unbinding pathways, estimation of the rates, and determination of the rate-limiting steps in the paradigmatic case of the trypsin–benzamidine system. Protein, ligand, and solvent are described with full atomic resolution. Using metadynamics, multiple unbinding trajectories that start with the ligand in the crystallographic binding pose and end with the ligand in the fully solvated state are generated. The unbinding rate koff is computed from the mean residence time of the ligand. Using our previously computed binding affinity we also obtain the binding rate kon. Both rates are in agreement with reported experimental values. We uncover the complex pathways of unbinding trajectories and describe the critical rate-limiting steps with unprecedented detail. Our findings illuminate the role played by the coupling between subtle protein backbone fluctuations and the solvation by water molecules that enter the binding pocket and assist in the breaking of the shielded hydrogen bonds. We expect our approach to be useful in calculating rates for general protein–ligand systems and a valid support for drug design. PMID:25605901

  10. Role of the C-terminal residue of the DNA polymerase of bacteriophage T7.

    PubMed

    Kumar, J K; Tabor, S; Richardson, C C

    2001-09-14

    The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.

  11. The flexibility of a distant loop modulates active site motion and product release in ribonuclease A.

    PubMed

    Doucet, Nicolas; Watt, Eric D; Loria, J Patrick

    2009-08-04

    The role of the flexible loop 1 in protein conformational motion and in the dissociation of enzymatic product from ribonuclease A (RNase A) was investigated by creation of a chimeric enzyme in which a 6-residue loop 1 from the RNase A homologue, eosinophil cationic protein (ECP), replaced the 12-residue loop 1 in RNase A. The chimera (RNase A(ECP)) experiences only local perturbations in NMR backbone chemical shifts compared to WT RNase A. Many of the flexible residues that were previously identified in WT as involved in an important conformational change now experience no NMR-detected millisecond motions in the chimera. Likewise, binding of the product analogue, 3'-CMP, to RNase A(ECP) results in only minor chemical shift changes in the enzyme similar to what is observed for the H48A mutant of RNase A and in contrast to WT enzyme. For both RNase A(ECP) and H48A there is a 10-fold decrease in the product release rate constant, k(off), compared to WT, in agreement with previous studies indicating the importance of flexibility in RNase A in the overall rate-limiting product release step. Together, these NMR and biochemical experiments provide additional insight into the mechanism of millisecond motions in the RNase A catalytic cycle.

  12. Effect of contact time and force on monocyte adhesion to vascular endothelium.

    PubMed Central

    Rinker, K D; Prabhakar, V; Truskey, G A

    2001-01-01

    In this study we examined whether monocytic cell attachment to vascular endothelium was affected by elevating shear stress at a constant shear rate. Contact time, which is inversely related to the shear rate, was fixed and viscosity elevated with dextran to increase the shear stress (and hence the net force on the cell) independently of shear rate. At a fixed contact time, tethering frequencies increased, rolling velocities decreased, and median arrest durations increased with increasing shear stress. Rolling and short arrests (< 0.2 s) were well fit by a single exponential consistent with adhesion via the formation of a single additional bond. The cell dissociation constant, k(off), increased when the shear stress was elevated at constant shear rate. Firmly adherent cells arresting for at least 0.2 s were well fit by a stochastic model involving dissociation from multiple bonds. Therefore, at a fixed contact time and increasing shear stress, bonds formed more frequently for rolling cells resulting in more short arrests, and more bonds formed for firmly arresting cells resulting in longer arrest durations. Possible mechanisms for this increased adhesion include greater monocyte deformation and/or more frequent penetration of microvilli through steric and charge barriers. PMID:11259286

  13. KINETIC CHARACTERIZATION AND MOLECULAR DOCKING OF A NOVEL, POTENT, AND SELECTIVE SLOW-BINDING INHIBITOR OF HUMAN CATHEPSIN L

    PubMed Central

    Shah, Parag P.; Myers, Michael C.; Beavers, Mary Pat; Purvis, Jeremy E.; Jing, Huiyan; Grieser, Heather J.; Sharlow, Elizabeth R.; Napper, Andrew D.; Huryn, Donna M.; Cooperman, Barry S.; Smith, Amos B.; Diamond, Scott L.

    2008-01-01

    A novel small molecule thiocarbazate (PubChem SID 26681509), a potent inhibitor of human cathepsin L (EC 3.4.22.15) with an IC50 of 56 nM, was developed following a 57,821 compound screen of the NIH Molecular Libraries Small Molecule Repository. After a 4 hr preincubation with cathepsin L, this compound became even more potent, demonstrating an IC50 of 1.0 nM. The thiocarbazate was determined to be a slow-binding and slowly reversible competitive inhibitor. Through a transient kinetic analysis for single-step reversibility, inhibition rate constants were kon = 24,000 M-1s-1 and koff = 2.2 × 10-5 s-1 (Ki = 0.89 nM). Molecular docking studies were undertaken using the experimentally-derived X-ray crystal structure of papain/CLIK-148 (1cvz.pdb). These studies revealed critical hydrogen bonding patterns of the thiocarbazate with key active site residues in papain. The thiocarbazate displayed 7- to 151-fold greater selectivity toward cathepsin L than papain and cathepsins B, K, V, and S with no activity against cathepsin G. The inhibitor demonstrated a lack of toxicity in human aortic endothelial cells and zebrafish. Additionally, the thiocarbazate inhibited in vitro propagation of malaria parasite Plasmodium falciparum with an IC50 of 15.4 μM and inhibited Leishmania major with an IC50 of 12.5 μM. PMID:18403718

  14. Erratum: Variational Principles for Stellar Structure

    NASA Astrophysics Data System (ADS)

    Kennedy, Dallas C.; Bludman, Sidney A.

    1998-01-01

    In the paper ``HST and MERLIN Observations of 3C 264--A Laboratory for Jet Physics and Unified Schemes'' by Stefi A. Baum, Christopher P. O'Dea, Gabriele Giovannini, John Biretta, William B. Cotton, Sigrid de Koff, Luigina Feretti, Daniel Golombek, Lucas Lara, Ferdinando D. Macchetto, G. K. Miley, William B. Sparks, Tiziana Venturi, and Serguei S. Komissarov (ApJ, 483, 178 [1997]), there are errors in some of the exponents in equations (18), (20), and (21). The correct equations were used in the analysis, and the results of the paper are not affected. Section 5.4 including equations (18)-(21) with the correct exponents is presented here. 5.4. Brightness Variations Combining the results of previous sections, we obtain the following equations for the proper emissivity and the observed brightness variations (intensity per unit area Iν = εν rj) along a steady relativistic jet: Predominantly parallel field (case of 3C 264): ε˜ν~(Γjvj)-(γ+2)/3r-(5γ+7)/3j ,Iν~(Γjvj)-(γ+2)/3r-(5γ+4)/3jD2+α . (18) (19) Predominantly transverse field: ε˜ν~(Γjvj)-(5γ+7)/6r-(7γ+11)/6j ,Iν~(Γjvj)-(5γ+7)/6r-(7γ+5)/6jD2+α . (20) (21)

  15. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    PubMed Central

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  16. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

    PubMed

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-03-03

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )< 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  17. Probing the effect of the non-active-site mutation Y229W in New Delhi metallo-β-lactamase-1 by site-directed mutagenesis, kinetic studies, and molecular dynamics simulations.

    PubMed

    Chen, Jiao; Chen, Hui; Shi, Yun; Hu, Feng; Lao, Xingzhen; Gao, Xiangdong; Zheng, Heng; Yao, Wenbing

    2013-01-01

    New Delhi metallo-β-lactamase-1 (NDM-1) has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactamases (MβLs), but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD) simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1 ∼ 7 fold increases in k(cat) /K(m) values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon) and product egress (koff). The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1.

  18. Biophysical and physicochemical methods differentiate highly ligand-efficient human D-amino acid oxidase inhibitors.

    PubMed

    Lange, Jos H M; Venhorst, Jennifer; van Dongen, Maria J P; Frankena, Jurjen; Bassissi, Firas; de Bruin, Natasja M W J; den Besten, Cathaline; de Beer, Stephanie B A; Oostenbrink, Chris; Markova, Natalia; Kruse, Chris G

    2011-10-01

    Many early drug research efforts are too reductionist thereby not delivering key parameters such as kinetics and thermodynamics of target-ligand binding. A set of human D-Amino Acid Oxidase (DAAO) inhibitors 1-6 was applied to demonstrate the impact of key biophysical techniques and physicochemical methods in the differentiation of chemical entities that cannot be adequately distinguished on the basis of their normalized potency (ligand efficiency) values. The resulting biophysical and physicochemical data were related to relevant pharmacodynamic and pharmacokinetic properties. Surface Plasmon Resonance data indicated prolonged target-ligand residence times for 5 and 6 as compared to 1-4, based on the observed k(off) values. The Isothermal Titration Calorimetry-derived thermodynamic binding profiles of 1-6 to the DAAO enzyme revealed favorable contributions of both ΔH and ΔS to their ΔG values. Surprisingly, the thermodynamic binding profile of 3 elicited a substantially higher favorable contribution of ΔH to ΔG in comparison with the structurally closely related fused bicyclic acid 4. Molecular dynamics simulations and free energy calculations of 1, 3, and 4 led to novel insights into the thermodynamic properties of the binding process at an atomic level and in the different thermodynamic signatures of 3 and 4. The presented holistic approach is anticipated to facilitate the identification of compounds with best-in-class properties at an early research stage. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  19. Force measurements on the molecular interactions between ligand (RGD) and human platelet α IIbβ 3 receptor system

    NASA Astrophysics Data System (ADS)

    Lee, ImShik; Marchant, Roger E.

    2001-10-01

    The peptide sequence arginine-glycine-aspartate (RGD) found in fibrinogen, von Willebrand factor, fibronectin, and vitronectin, plays a critical role in platelet adhesion and thrombus formation, when bound to the platelet α IIbβ 3 integrin receptor. Using atomic force microscopy (AFM), we have measured the debonding interaction between an RGD peptide-modified AFM probe tip and a human platelet surface from pN to nN levels of force. The peptide sequence, GSSSGRGDSPA, which contains the biologically active RGDSP sequence with a hydrophilic spacer sequence (GSSSG), was covalently coupled to AFM probe tips. Direct measurements on the debonding force for the RGD ligand - α IIbβ 3 platelet receptor system were carried out in Tyrode buffer at room temperature. Our results show three distinct distributions of debonding forces at a loading rate of 12 nN/s, from which we estimate the debonding force for the single ligand-receptor to be ˜93 pN. The results also show evidence for considerable extension in the flexible sample surface during the debonding process, and a linear correlation between the debonding force and the logarithm of the rate of loading. From our analysis, the zero kinetic off-rate Koff(0), the single molecular binding energy Eb, and the transition state xB, assuming rigid binding, were extracted from the data, and estimated to be 22.6 s -1, -2.64×10 -20 J and 0.1 nm, respectively.

  20. Molecular Basis of the Bohr Effect in Arthropod Hemocyanin

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

    Hirota, S.; Kawahara, T; Beltramini, M

    2008-01-01

    Flash photolysis and K-edge x-ray absorption spectroscopy (XAS) were used to investigate the functional and structural effects of pH on the oxygen affinity of three homologous arthropod hemocyanins (Hcs). Flash photolysis measurements showed that the well-characterized pH dependence of oxygen affinity (Bohr effect) is attributable to changes in the oxygen binding rate constant, kon, rather than changes in koff. In parallel, coordination geometry of copper in Hc was evaluated as a function of pH by XAS. It was found that the geometry of copper in the oxygenated protein is unchanged at all pH values investigated, while significant changes were observedmore » for the deoxygenated protein as a function of pH. The interpretation of these changes was based on previously described correlations between spectral lineshape and coordination geometry obtained for model compounds of known structure A pH-dependent change in the geometry of cuprous copper in the active site of deoxyHc, from pseudotetrahedral toward trigonal was assigned from the observed intensity dependence of the 1s ? 4pz transition in x-ray absorption near edge structure (XANES) spectra. The structural alteration correlated well with increase in oxygen affinity at alkaline pH determined in flash photolysis experiments. These results suggest that the oxygen binding rate in deoxyHc depends on the coordination geometry of Cu(I) and suggest a structural origin for the Bohr effect in arthropod Hcs.« less

  1. The fast release of sticky protons: Kinetics of substrate binding and proton release in a multidrug transporter

    PubMed Central

    Adam, Yoav; Tayer, Naama; Rotem, Dvir; Schreiber, Gideon; Schuldiner, Shimon

    2007-01-01

    EmrE is an Escherichia coli H+-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP+, a high-affinity substrate, is 2 × 107 M−1·s−1, a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the koff of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs. PMID:17984053

  2. Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry

    PubMed Central

    Sedlak, Steffen M.; Bauer, Magnus S.; Kluger, Carleen; Schendel, Leonard C.; Milles, Lukas F.; Pippig, Diana A.

    2017-01-01

    The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin’s tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10−6 s-1 range. PMID:29206886

  3. Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry.

    PubMed

    Sedlak, Steffen M; Bauer, Magnus S; Kluger, Carleen; Schendel, Leonard C; Milles, Lukas F; Pippig, Diana A; Gaub, Hermann E

    2017-01-01

    The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin's tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10-6 s-1 range.

  4. Tailoring in vitro evolution for protein affinity or stability

    PubMed Central

    Jermutus, Lutz; Honegger, Annemarie; Schwesinger, Falk; Hanes, Jozef; Plückthun, Andreas

    2001-01-01

    We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure. Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of single-chain Fv antibody fragments (scFvs). To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties. A scFv with an initial affinity of 1.1 nM (koff at 4°C of 10−4 s−1) was improved 30-fold by the use of off-rate selections over a period of several days. As a second example, a generic selection strategy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed protein occurs. We used decreasing redox potentials in the selection step to select for molecules stable in the absence of disulfide bonds. They could be functionally expressed in the reducing cytoplasm, and, when allowed to form disulfides again, their stability had increased to 54 kJ/mol from an initial value of 24 kJ/mol. Sequencing revealed that the evolved mutant proteins had used different strategies of residue changes to adapt to the selection pressure. Therefore, by a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible. PMID:11134506

  5. A heteronuclear zero quantum coherence Nz-exchange experiment that resolves resonance overlap and its application to measure the rates of heme binding to the IsdC protein.

    PubMed

    Robson, Scott A; Peterson, Robert; Bouchard, Louis-S; Villareal, Valerie A; Clubb, Robert T

    2010-07-21

    Chemical exchange phenomena in NMR spectra can be quantitatively interpreted to measure the rates of ligand binding, as well as conformational and chemical rearrangements. In macromolecules, processes that occur slowly on the chemical shift time scale are frequently studied using 2D heteronuclear ZZ or N(z)-exchange spectroscopy. However, to successfully apply this method, peaks arising from each exchanging species must have unique chemical shifts in both dimensions, a condition that is often not satisfied in protein-ligand binding equilibria for (15)N nuclei. To overcome the problem of (15)N chemical shift degeneracy we developed a heteronuclear zero-quantum (and double-quantum) coherence N(z)-exchange experiment that resolves (15)N chemical shift degeneracy in the indirect dimension. We demonstrate the utility of this new experiment by measuring the heme binding kinetics of the IsdC protein from Staphylococcus aureus. Because of peak overlap, we could not reliably analyze binding kinetics using conventional methods. However, our new experiment resulted in six well-resolved systems that yielded interpretable data. We measured a relatively slow k(off) rate of heme from IsdC (<10 s(-1)), which we interpret as necessary so heme loaded IsdC has time to encounter downstream binding partners to which it passes the heme. The utility of using this new exchange experiment can be easily expanded to (13)C nuclei. We expect our heteronuclear zero-quantum coherence N(z)-exchange experiment will expand the usefulness of exchange spectroscopy to slow chemical exchange events that involve ligand binding.

  6. Investigating the binding behaviour of two avidin-based testosterone binders using molecular recognition force spectroscopy.

    PubMed

    Rangl, Martina; Leitner, Michael; Riihimäki, Tiina; Lehtonen, Soili; Hytönen, Vesa P; Gruber, Hermann J; Kulomaa, Markku; Hinterdorfer, Peter; Ebner, Andreas

    2014-02-01

    Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand-receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin-based proteins called sbAvd-1 and sbAvd-2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone-binding protein was immobilized on the surface. Repeated formation and rupture of the ligand-receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex-rupturing force. In this way, we obtained the molecular dissociation rate (k(off)) and energy landscape distances (x(β)) of the four possible complexes: sbAvd-1-biotin, sbAvd-1-testosterone, sbAvd-2-biotin and sbAvd-2-testosterone. It was found that the kinetic off-rates for both proteins and both ligands are similar. In contrast, the x(β) values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone-binding proteins, implying a decreased cross-reactivity of sbAvd-2. Unravelling the binding behaviour of the investigated testosterone-binding proteins is expected to improve their usability for possible sensing applications. Copyright © 2014 John Wiley & Sons, Ltd.

  7. Improving the affinity of an antibody for its antigen via long-range electrostatic interactions.

    PubMed

    Fukunaga, Atsushi; Tsumoto, Kouhei

    2013-12-01

    To address how long-range electrostatic force can affect antibody-antigen binding, we focused on the interactions between human cardiac troponin I and its specific single-chain antibodies (scFvs). We first isolated two scFvs against two linear epitopes with distinct isoelectric points. For the scFv against the acidic epitope (A1scFv), we mutated five residues of framework region 3 of the light chain to Lys or Arg, designated as the K- or R-mutant, respectively. For the scFv against the basic epitope (A2scFv), we mutated four or three residues in framework region 3 of the light or heavy chain to Asp, to generate the VL- and VH-mutant, respectively. Surface plasmon resonance analyses showed that the kon values of all of the mutants were greater than that of wild type, even though framework region 3 does not make direct contact with the epitope. The affinity of the K-mutant was pM range, and that of the R-mutant improved further by more than two orders of magnitude due to a decrease in the dissociation rate constant. For the A2scFv mutants, the affinity of the VL-mutant for its target improved through an increase in the kon value without a decrease in the koff value. The stability slightly decreased in all mutants. These results suggest that introducing electrostatic interaction can improve the affinity of an antibody for its target, even if the mutation reduces stability of the antibody.

  8. Kinetic analysis of central ( sup 11 C)raclopride binding to D2-dopamine receptors studied by PET--a comparison to the equilibrium analysis

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

    Farde, L.; Eriksson, L.; Blomquist, G.

    1989-10-01

    (11C)Raclopride binding to central D2-dopamine receptors in humans has previously been examined by positron emission tomography (PET). Based on the rapid occurrence of binding equilibrium, a saturation analysis has been developed for the determination of receptor density (Bmax) and affinity (Kd). For analysis of PET measurements obtained with other ligands, a kinetic three-compartment model has been used. In the present study, the brain uptake of (11C)raclopride was analyzed further by applying both a kinetic and an equilibrium analysis to data obtained from four PET experiments in each of three healthy subjects. First regional CBV was determined. In the second andmore » third experiment, (11C)-raclopride with high and low specific activity was used. In a fourth experiment, the (11C)raclopride enantiomer (11C)FLB472 was used to examine the concentration of free radioligand and nonspecific binding in brain. Radio-activity in arterial blood was measured using an automated blood sampling system. Bmax and Kd values for (11C)raclopride binding could be determined also with the kinetic analysis. As expected theoretically, those values were similar to those obtained with the equilibrium analysis. In addition, the kinetic analysis allowed separate determination of the association and dissociation rate constants, kon and koff, respectively. Examination of (11C)raclopride and (11C)FLB472 uptake in brain regions devoid of specific D2-dopamine receptor binding indicated a fourth compartment in which uptake was reversible, nonstereoselective, and nonsaturable in the dose range studied.« less

  9. Kinetics, mechanism, and spectroscopy of the reversible binding of nitric oxide to aquated iron(II). An undergraduate text book reaction revisited.

    PubMed

    Wanat, Alicja; Schneppensieper, Thorsten; Stochel, Grazyna; van Eldik, Rudi; Bill, Eckhard; Wieghardt, Karl

    2002-01-14

    A detailed kinetic and mechanistic analysis of the classical "brown-ring" reaction of [Fe(H(2)O)(6)](2+) with NO was performed using stopped-flow and laser flash photolysis techniques at ambient and high pressure. The kinetic parameters for the "on" and "off" reactions at 25 degrees C were found to be k(on) = 1.42 x 10(6) M(-1) s(-1), DeltaH(++)(on) = 37.1 +/- 0.5 kJ mol(-1), DeltaS(++)(on) = -3 +/- 2 J K(-1) mol(-1), DeltaV(++)(on) = +6.1 +/- 0.4 cm(3) mol(-1), and k(off) = 3240 +/- 750 s(-1), DeltaH(++)(off) = 48.4 +/- 1.4 kJ mol(-1), DeltaS(++)(off) = -15 +/- 5 J K(-1) mol(-1), DeltaV(++)(off) = +1.3 +/- 0.2 cm(3) mol(-1). These parameters suggest that both reactions follow an interchange dissociative (I(d)) ligand substitution mechanism, which correlates well with the suggested mechanism for the water exchange reaction on [Fe(H(2)O)(6)](2+). In addition, Mössbauer spectroscopy and EPR measurements were performed on the reaction product [Fe(H(2)O)(5)(NO)](2+). The Mössbauer and EPR parameters closely resemble those of the [FeNO](7) units in any of the other well-characterized nitrosyl complexes. It is concluded that its electronic structure is best described by the presence of high-spin Fe(III) antiferromagnetically coupled to NO(-) (S = 1) yielding the observed spin quartet ground state (S = (3)/(2)), i.e., [Fe(III)(H(2)O)(5)(NO(-))](2+), and not [Fe(I)(H(2)O)(5)(NO(+))](2+) as usually quoted in undergraduate text books.

  10. Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions.

    PubMed

    Khan, Mateen A; Ma, Jia; Walden, William E; Merrick, William C; Theil, Elizabeth C; Goss, Dixie J

    2014-06-01

    Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn(2+) decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn(2+) increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn(2+) eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Development of a Tetrameric Streptavidin Mutein with Reversible Biotin Binding Capability: Engineering a Mobile Loop as an Exit Door for Biotin

    PubMed Central

    O'Sullivan, Valerie J.; Barrette-Ng, Isabelle; Hommema, Eric; Hermanson, Greg T.; Schofield, Mark; Wu, Sau-Ching; Honetschlaeger, Claudia; Ng, Kenneth K.-S.; Wong, Sui-Lam

    2012-01-01

    A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop3–4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop3–4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop7–8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop7–8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (koff of 4.28×10−4 s−1 and Kd of 1.9×10−8 M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible. PMID:22536357

  12. Dissecting the Binding between Glutamine Synthetase and Its Two Natively Unfolded Protein Inhibitors.

    PubMed

    Pantoja-Uceda, David; Neira, José L; Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel; Santoro, Jorge

    2016-06-21

    Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue (IF7) and the 149-residue one (IF17). In this work, we studied both IF7 and IF17 by nuclear magnetic resonance (NMR), and we described their binding to GS by using NMR and biolayer interferometry. We assigned the backbone nuclei of all residues of IF7. Analyses of chemical shifts and the (15)N-{(1)H} NOEs at two field strengths suggest that IF7 region Thr3-Arg13 and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The two-dimensional (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting first with IF7 were Gln6 and Ser27, belonging to those regions that appeared to be ordered in the isolated protein. We also determined the kon values and koff values for the binding of both IF7 and IF17 to GS, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model ([Formula: see text]), (ii) there is a strong electrostatic component in the determined kon, and (iii) the binding is not diffusion-limited.

  13. Development of peptide and protein based radiopharmaceuticals.

    PubMed

    Wynendaele, Evelien; Bracke, Nathalie; Stalmans, Sofie; De Spiegeleer, Bart

    2014-01-01

    Radiolabelled peptides and proteins have recently gained great interest as theranostics, due to their numerous and considerable advantages over small (organic) molecules. Developmental procedures of these radiolabelled biomolecules start with the radiolabelling process, greatly defined by the amino acid composition of the molecule and the radionuclide used. Depending on the radionuclide selection, radiolabelling starting materials are whether or not essential for efficient radiolabelling, resulting in direct or indirect radioiodination, radiometal-chelate coupling, indirect radiofluorination or (3)H/(14)C-labelling. Before preclinical investigations are performed, quality control analyses of the synthesized radiopharmaceutical are recommended to eliminate false positive or negative functionality results, e.g. changed receptor binding properties due to (radiolabelled) impurities. Therefore, radionuclidic, radiochemical and chemical purity are investigated, next to the general peptide attributes as described in the European and the United States Pharmacopeia. Moreover, in vitro and in vivo stability characteristics of the peptides and proteins also need to be explored, seen their strong sensitivity to proteinases and peptidases, together with radiolysis and trans-chelation phenomena of the radiopharmaceuticals. In vitro biomedical characterization of the radiolabelled peptides and proteins is performed by saturation, kinetic and competition binding assays, analyzing KD, Bmax, kon, koff and internalization properties, taking into account the chemical and metabolic stability and adsorption events inherent to peptides and proteins. In vivo biodistribution can be adapted by linker, chelate or radionuclide modifications, minimizing normal tissue (e.g. kidney and liver) radiation, and resulting in favorable dosimetry analyses. Finally, clinical trials are initiated, eventually leading to the marketing of radiolabelled peptides and proteins for PET/SPECT-imaging and therapy

  14. CO Binding and Ligand Discrimination in Human Myeloperoxidase†

    PubMed Central

    Murphy, Emma J.; Maréchal, Amandine; Segal, Anthony W.; Rich, Peter R.

    2015-01-01

    Despite the fact that ferrous myeloperoxidase (MPO) can bind both O2 and NO, its ability to bind CO has been questioned. UV/visible spectroscopy was used to confirm that CO induces small spectral shifts in ferrous MPO, and Fourier transform infrared difference spectroscopy showed definitively that these arose from formation of a heme ferrous–CO compound. Recombination rates after CO photolysis were monitored at 618 and 645 nm as a function of CO concentration and pH. At pH 6.3, kon and koff were 0.14 mM−1·s−1 and 0.23 s−1, respectively, yielding an unusually high KD of 1.6 mM. This affinity of MPO for CO is 10 times weaker than its affinity for O2. The observed rate constant for CO binding increased with increasing pH and was governed by a single protonatable group with a pKa of 7.8. Fourier transform infrared spectroscopy revealed two different conformations of bound CO with frequencies at 1927 and 1942 cm−1. Their recombination rate constants were identical, indicative of two forms of bound CO that are in rapid thermal equilibrium rather than two distinct protein populations with different binding sites. The ratio of bound states was pH-dependent (pKa ≈ 7.4) with the 1927 cm−1 form favored at high pH. Structural factors that account for the ligand-binding properties of MPO are identified by comparisons with published data on a range of other ligand-binding heme proteins, and support is given to the recent suggestion that the proximal His336 in MPO is in a true imidazolate state. PMID:20146436

  15. Molecular analysis of the Na+ channel blocking actions of the novel class I anti-arrhythmic agent RSD 921.

    PubMed

    Pugsley, M K; Goldin, A L

    1999-05-01

    RSD 921 is a novel, structurally unique, class I Na+ channel blocking drug under development as a local anaesthetic agent and possibly for the treatment of cardiac arrhythmias. The effects of RSD 921 on wild-type heart, skeletal muscle, neuronal and non-inactivating IFMQ3 mutant neuronal Na+ channels expressed in Xenopus laevis oocytes were examined using a two-electrode voltage clamp. RSD 921 produced similarly potent tonic block of all three wild-type channel isoforms, with EC50 values between 35 and 47 microM, whereas the EC50 for block of the IFMQ3 mutant channel was 110+5.5 microM. Block of Na+ channels by RSD 921 was concentration and use-dependent, with marked frequency-dependent block of heart channels and mild frequency-dependent block of skeletal muscle, wild-type neuronal and IFMQ3 mutant channels. RSD 921 produced a minimal hyperpolarizing shift in the steady-state voltage-dependence of inactivation of all three wild-type channel isoforms. Open channel block of the IFMQ3 mutant channel was best fit with a first order blocking scheme with k(on) equal to 0.11+/-0.012x10(6) M(-1) s(-1) and k(off) equal to 12.5+/-2.5 s(-1), resulting in KD of 117+/-31 microM. Recovery from open channel block occurred with a time constant of 14+/-2.7 s(-1). These results suggest that RSD 921 preferentially interacts with the open state of the Na+ channel, and that the drug may produce potent local anaesthetic or anti-arrhythmic action under conditions of shortened action potentials, such as during anoxia or ischaemia.

  16. Prediction of Rate Constant for Supramolecular Systems with Multiconfigurations.

    PubMed

    Guo, Tao; Li, Haiyan; Wu, Li; Guo, Zhen; Yin, Xianzhen; Wang, Caifen; Sun, Lixin; Shao, Qun; Gu, Jingkai; York, Peter; Zhang, Jiwen

    2016-02-25

    The control of supramolecular systems requires a thorough understanding of their dynamics, especially on a molecular level. It is extremely difficult to determine the thermokinetic parameters of supramolecular systems, such as drug-cyclodextrin complexes with fast association/dissociation processes by experimental techniques. In this paper, molecular modeling combined with novel mathematical relationships integrating the thermodynamic/thermokinetic parameters of a series of isomeric multiconfigurations to predict the overall parameters in a range of pH values have been employed to study supramolecular dynamics at the molecular level. A suitable form of Eyring's equation was derived and a two-stage model was introduced. The new approach enabled accurate prediction of the apparent dissociation/association (k(off)/k(on)) and unbinding/binding (k-r/kr) rate constants of the ubiquitous multiconfiguration complexes of the supramolecular system. The pyronine Y (PY) was used as a model system for the validation of the presented method. Interestingly, the predicted k(off) value ((40 ± 1) × 10(5) s(-1), 298 K) of PY is largely in agreement with that previously determined by fluorescence correlation spectroscopy ((5 ± 3) × 10(5) s(-1), 298 K). Moreover, the k(off)/k(on) and k-r/kr for flurbiprofen-β-cylcodextrin and ibuprofen-β-cyclodextrin systems were also predicted and suggested that the association processes are diffusion-controlled. The methodology is considered to be especially useful in the design and selection of excipients for a supramolecular system with preferred association and dissociation rate constants and understanding their mechanisms. It is believed that this new approach could be applicable to a wide range of ligand-receptor supramolecular systems and will surely help in understanding their complex mechanism.

  17. Investigation on the Characteristics of Pellet Ablation in a Toroidal Plasma

    NASA Astrophysics Data System (ADS)

    Sato, K. N.; Sakakita, H.; Fujita, H.

    2003-06-01

    Characteristics of a cloud ablated from an ice pellet has been investigated in detail in the JIPP T-IIU tokamak plasma by utilizing a new scheme of pellet injection system, "the injection-angle controllable system". A long "helical tail" of ablation light has been observed using CCD cameras and a high speed framing photograph in the case of on-axis and off-axis injection with the injection angle smaller than a certain value. The direction of the helical tail is found to be independent to that of the total magnetic field lines of the torus. From the experiments with the combination of two toroildal filed directions and two plasma current directions, it is considered that the tail seems to rotate, in most cases, to the electron diamagnetic direction poloidally, and to the opposite to the plasma current direction toroidally. Consideration on various cross sections including charge exchange, ionization and elastic collisions leads us to the conclusion that the tail-shaped phenomena may come from the situation of charge exchange equilibrium of hydrogen ions and neutrals at extremely high density regime in the cloud. The relation of ablation behavior with plasma potential and rotation has also been studied. Potential measurements of pellet-injected plasmas using heavy ion beam probe (HIBP) method were carried out for the first time. In the case of an injection angle to be anti-parallel to the electron diamagnetic direction in the poloidal plane, the result shows that the direction of potential change is negative, and consequently the potential after the injection should be negative because it has been measured to be negative in usual ohmic plasmas without pellet injection. Thus, the direction of the "tail" structure seems to be consistent to that of the plasma potential measured, if it is considered that tail structure may be caused by the effect of the plasma potential and the rotation.

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

    Sawada, Y.; Kawai, R.; McManaway, M.

    (3H)Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model and a classical compartmental pharmacokinetic model were compared. The models included different assumptions for transport across the blood-brain barrier (BBB);more » estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis of the data is inappropriate due to the high tissue-loss rate constant for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using (18F)CF and positron emission tomography.« less

  19. 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

  20. Probing of miniPEGγ-PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy.

    PubMed

    Dutta, Samrat; Armitage, Bruce A; Lyubchenko, Yuri L

    2016-03-15

    Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplexes. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. J. Org. Chem. 2011, 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γ(MP)PNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that the γ(MP)PNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ≈ 0.030 ± 0.01 s⁻¹ for γ(MP)PNA-DNA hybrid duplex vs 0.375 ± 0.18 s⁻¹ for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γ(MP)PNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γ(MP)PNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes.

  1. Electrostatic redesign of the [myoglobin, cytochrome b5] interface to create a well-defined docked complex with rapid interprotein electron transfer.

    PubMed

    Xiong, Peng; Nocek, Judith M; Griffin, Amanda K K; Wang, Jingyun; Hoffman, Brian M

    2009-05-27

    Cyt b(5) is the electron-carrier "repair" protein that reduces met-Mb and met-Hb to their O(2)-carrying ferroheme forms. Studies of electron transfer (ET) between Mb and cyt b(5) revealed that they react on a "Dynamic Docking" (DD) energy landscape on which binding and reactivity are uncoupled: binding is weak and involves an ensemble of nearly isoenergetic configurations, only a few of which are reactive; those few contribute negligibly to binding. We set the task of redesigning the surface of Mb so that its reaction with cyt b(5) instead would occur on a conventional "simple docking" (SD) energy landscape, on which a complex exhibits a well-defined (set of) reactive binding configuration(s), with binding and reactivity thus no longer being decoupled. We prepared a myoglobin (Mb) triple mutant (D44K/D60K/E85K; Mb(+6)) substituted with Zn-deuteroporphyrin and monitored cytochrome b(5) (cyt b(5)) binding and electron transfer (ET) quenching of the (3)ZnMb(+6) triplet state. In contrast, to Mb(WT), the three charge reversals around the "front-face" heme edge of Mb(+6) have directed cyt b(5) to a surface area of Mb adjacent to its heme, created a well-defined, most-stable structure that supports good ET pathways, and apparently coupled binding and ET: both K(a) and k(et) are increased by the same factor of approximately 2 x 10(2), creating a complex that exhibits a large ET rate constant, k(et) = 10(6 1) s(-1), and is in slow exchange (k(off) < k(et)). In short, these mutations indeed appear to have created the sought-for conversion from DD to simple docking (SD) energy landscapes.

  2. (2S,4S)-4-Fluoro-1-{[(2-hydroxy-1,1-dimethylethyl)amino]acetyl}-pyrrolidine-2-carbonitrile monobenzenesulfonate (TS-021) is a selective and reversible dipeptidyl peptidase IV inhibitor.

    PubMed

    Tajima, Atsushi; Yamamoto, Koji; Kozakai, Akinori; Okumura-Kitajima, Lisa; Mita, Yasuo; Kitano, Kiyokazu; Jingu, Shigeji; Nakaike, Shiro

    2011-03-25

    The incretin hormone glucagon-like peptide-1 (GLP-1) has significant roles in the regulation of postprandial glucose metabolism, and the active form of GLP-1 is rapidly degraded by dipeptidyl peptidase (DPP)-IV. Therefore, DPP-IV inhibition is a promising approach for the treatment of type 2 diabetes. In the present study, we investigated the character of a DPP-IV inhibitor, TS-021, (2S, 4S)-4-fluoro-1-{[(2-hydroxy-1,1-dimethylethyl)amino]acetyl}-pyrrolidine-2-carbonitrile monobenzenesulfonate both in vitro and in vivo. TS-021 inhibits DPP-IV activity in human plasma with an IC(50) value of 5.34nM. In kinetics experiments, TS-021 had a relatively higher dissociation rate constant, with a k(off) value of 1.09×10(-3)s, despite exhibiting a potent human plasma DPP-IV inhibition activity with a K(i) value of 4.96nM. TS-021 exhibited significant inhibition selectivity against DPP-8 (>600 fold), DPP-9 (>1200 fold) and other peptidases examined (>15,000 fold). In normal rats, dogs and monkeys, a single oral dose of TS-021 exhibited favorable pharmacokinetic profiles. In Zucker fatty (fa/fa) rats, a rat model of obesity and impaired glucose tolerance, the oral administration of TS-021 resulted in the suppression of plasma DPP-IV activity and an increase in the active form of GLP-1. Furthermore, TS-021 exhibited a significant improvement in glucose tolerance by increasing the plasma insulin level during oral glucose tolerance tests at doses of 0.02-0.5mg/kg. These results suggest that TS-021 is a selective and reversible dipeptidyl peptidase IV inhibitor and has excellent characteristics as an oral anti-diabetic agent for postprandial hyperglycemia in patients with impaired glucose tolerance or type 2 diabetes. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Visualization of the activation of the histamine H3 receptor (H3R) using novel fluorescence resonance energy transfer biosensors and their potential application to the study of H3R pharmacology.

    PubMed

    Liu, Ying; Zeng, Hong; Pediani, John D; Ward, Richard J; Chen, Lu-Yao; Wu, Nan; Ma, Li; Tang, Mei; Yang, Yang; An, Su; Guo, Xiao-Xi; Hao, Qian; Xu, Tian-Rui

    2018-06-01

    Activation of the histamine-3 receptor (H3R) is involved in memory processes and cognitive action, while blocking H3R activation can slow the progression of neurological disorders, such as Alzheimer's disease, schizophrenia and narcolepsy. To date, however, no direct way to examine the activation of H3R has been utilized. Here, we describe a novel biosensor that can visualize the activation of H3R through an intramolecular fluorescence resonance energy transfer (FRET) signal. To achieve this, we constructed an intramolecular H3R FRET sensor with cyan fluorescent protein (CFP) attached at the C terminus and yellow fluorescent protein (YFP) inserted into the third intracellular loop. The sensor was found to internalize normally on agonist treatment. We measured FRET signals between the donor CFP and the acceptor YFP in living cells in real time, the results of which indicated that H3R agonist treatment (imetit or histamine) increases the FRET signal in a time- and concentration-dependent manner with Kon and Koff values consistent with published data and which maybe correlated with decreasing cAMP levels and the promotion of ERK1/2 phosphorylation. The FRET signal was inhibited by H3R antagonists, and the introduction of mutations at F419A, F423A, L426A and L427A, once again, the promotion of ERK1/2 phosphorylation, was diminished. Thus, we have built a H3R biosensor which can visualize the activation of receptor through real-time structure changes and which can obtain pharmacological kinetic data at the same time. The FRET signals may allow the sensor to become a useful tool for screening compounds and optimizing useful ligands. © 2018 Federation of European Biochemical Societies.

  4. Viral RNAi suppressor reversibly binds siRNA to outcompete Dicer and RISC via multiple-turnover

    PubMed Central

    Rawlings, Renata A.; Krishnan, Vishalakshi; Walter, Nils G.

    2011-01-01

    RNA interference (RNAi) is a conserved gene regulatory mechanism employed by most eukaryotes as a key component of their innate immune response against viruses and retrotransposons. During viral infection, the RNase III-type endonuclease Dicer cleaves viral double-stranded RNA into small interfering RNAs (siRNAs), 21–24 nucleotides in length, and helps load them into the RNA-induced silencing complex (RISC) to guide cleavage of complementary viral RNA. As a countermeasure, many viruses have evolved viral RNA silencing suppressor (RSS) proteins that tightly, and presumably quantitatively, bind siRNAs to thwart RNAi-mediated degradation. Viral RSS proteins also act across kingdoms as potential immunosuppressors in gene therapeutic applications. Here we report fluorescence quenching and electrophoretic mobility shift assays that probe siRNA binding by the dimeric RSS p19 from Carnation Italian Ringspot Virus (CIRV), as well as by human Dicer and RISC assembly complexes. We find that the siRNA:p19 interaction is readily reversible, characterized by rapid binding ((1.69 ± 0.07)×108 M−1s−1) and marked dissociation (koff = 0.062 ± 0.002 s−1). We also observe that p19 efficiently competes with recombinant Dicer and inhibits formation of RISC-related assembly complexes found in human cell extract. Computational modeling based on these results provides evidence for the transient formation of a ternary complex between siRNA, human Dicer, and p19. An expanded model of RNA silencing indicates that multiple-turnover by reversible binding of siRNAs potentiates the efficiency of the suppressor protein. Our predictive model is expected to be applicable to the dosing of p19 as a silencing suppressor in viral gene therapy. PMID:21354178

  5. AZD8797 is an allosteric non-competitive modulator of the human CX3CR1 receptor.

    PubMed

    Cederblad, Linda; Rosengren, Birgitta; Ryberg, Erik; Hermansson, Nils-Olov

    2016-03-01

    The chemokine receptor CX3CR1 has been implicated as an attractive therapeutic target in several diseases, including atherosclerosis and diabetes. However, there has been a lack of non-peptide CX3CR1 inhibitors to substantiate these findings. A selective small-molecule inhibitor of CX3CR1, AZD8797, was recently reported and we present here an in-depth in vitro characterization of that molecule. In a flow adhesion assay, AZD8797 antagonized the natural ligand, fractalkine (CX3CL1), in both human whole blood (hWB) and in a B-lymphocyte cell line with IC50 values of 300 and 6 nM respectively. AZD8797 also prevented G-protein activation in a [(35)S]GTPγS (guanosine 5'-[γ-thio]triphosphate) accumulation assay. In contrast, dynamic mass redistribution (DMR) experiments revealed a weak Gαi-dependent AZD8797 agonism. Additionally, AZD8797 positively modulated the CX3CL1 response at sub-micromolar concentrations in a β-arrestin recruitment assay. In equilibrium saturation binding experiments, AZD8797 reduced the maximal binding of (125)I-CX3CL1 without affecting Kd. Kinetic experiments, determining the kon and koff of AZD8797, demonstrated that this was not an artefact of irreversible or insurmountable binding, thus a true non-competitive mechanism. Finally we show that both AZD8797 and GTPγS increase the rate with which CX3CL1 dissociates from CX3CR1 in a similar manner, indicating a connection between AZD8797 and the CX3CR1-bound G-protein. Collectively, these data show that AZD8797 is a non-competitive allosteric modulator of CX3CL1, binding CX3CR1 and effecting G-protein signalling and β-arrestin recruitment in a biased way. © 2016 Authors.

  6. A chimeric anti-CEA antibody with heavy interchain disulfide bonds deleted: molecular characterization and biodistributions in normal and tumor bearing mice.

    PubMed

    Neumaier, M; Gaida, F J; Lewis, M R; Hefta, L J; Shively, L E; Raubitschek, A; Shively, J E

    1999-01-01

    We have deleted the interchain disulfide bonds in a chimeric anti-CEA antibody (chT84.66) by mutating two cysteines in the heavy chain to glycine residues. The resulting antibody delta SSchT84.66 was expressed in high yield in a bioreactor and purified to homogeneity in a single step on an anti-idiotypic antibody affinity column. The molecular size of the antibody was 150 kDa as judged by gel filtration, SDS gel electrophoresis under non-reducing conditions, and MALDI-TOF/MS. The 150 kDa antibody had nearly identical kinetic (Kon = 1.53 x 10(6) M-1 s-1, .koff = 1.14 x 10(-5) s-1) and affinity constants (Kaff = 1.34 x 10(11) M-1) compared to the parent murine (Kaff = 1.25 x 10(11) M-1) and chimeric (Kaff = 1.16 x 10(11) M-1) antibodies when tested on biosensor chips. When delta SSchT84.66 was conjugated to the isothiocynato derivative of DTPA, radiolabeled with 111In, and injected into either normal or nude mice bearing tumor xenografts, it gave nearly identical biodistributions to chT84.66. delta SSchT84.66 and chT84.66 antibodies gave a maximum tumor uptake of 48 and 74% ID/g, and tumor to blood ratios of 5.3 and 6.2 at 48 h, respectively. We conclude that delta SSchT84.66 irreversibly associates into H2L2 dimers after concentration, that the dimers are stable under both the in vitro and in vivo conditions used in this study, and the properties of the antibody are virtually indistinguishable from the parent chT84.66 antibody.

  7. Improved antibody-based ricin neutralization by affinity maturation is correlated with slower off-rate values.

    PubMed

    Rosenfeld, Ronit; Alcalay, Ron; Mechaly, Adva; Lapidoth, Gideon; Epstein, Eyal; Kronman, Chanoch; J Fleishman, Sarel; Mazor, Ohad

    2017-09-01

    While potent monoclonal antibodies against ricin were introduced over the years, the question whether increasing antibody affinity enables better toxin neutralization was not fully addressed yet. The aim of this study was to characterize the contribution of antibody affinity to the ricin neutralization potential of the antibody. cHD23 monoclonal antibody that targets the toxin B-subunit and interferes with its binding to membranal receptors, was isolated. In order to create antibody clones with improved affinity toward ricin, a scFv-phage display library containing mutated versions of the variable regions of cHD23 was constructed and clones with improved binding of ricin were isolated. Structural modeling of these mutants suggests that the inserted mutations may increase the antibody conformational flexibility thus improving its ability to bind ricin. While it was found that the selected clones exhibited improved neutralization of ricin, the correlation between the KD values and potency was only minor (r = 0.55). However, a positive correlation (r = 0.84) exist between the off-rate values (koff) of the affinity matured clones and their ability to neutralize ricin. As cell membranes display inordinately large amounts of potential surface binding sites for ricin, it is suggested that antibodies with improved off-rate values block the ability of the toxin to bind to target receptors, in a highly efficient manner. Currently, antibody-based therapy is the most effective treatment for ricin intoxication and it is anticipated that the findings of this study will provide useful information and a possible strategy to design an improved antibody-based therapy for the toxin. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Viral RNAi suppressor reversibly binds siRNA to outcompete Dicer and RISC via multiple turnover.

    PubMed

    Rawlings, Renata A; Krishnan, Vishalakshi; Walter, Nils G

    2011-04-29

    RNA interference is a conserved gene regulatory mechanism employed by most eukaryotes as a key component of their innate immune response to viruses and retrotransposons. During viral infection, the RNase-III-type endonuclease Dicer cleaves viral double-stranded RNA into small interfering RNAs (siRNAs) 21-24 nucleotides in length and helps load them into the RNA-induced silencing complex (RISC) to guide the cleavage of complementary viral RNA. As a countermeasure, many viruses have evolved viral RNA silencing suppressors (RSS) that tightly, and presumably quantitatively, bind siRNAs to thwart RNA-interference-mediated degradation. Viral RSS proteins also act across kingdoms as potential immunosuppressors in gene therapeutic applications. Here we report fluorescence quenching and electrophoretic mobility shift assays that probe siRNA binding by the dimeric RSS p19 from Carnation Italian Ringspot Virus, as well as by human Dicer and RISC assembly complexes. We find that the siRNA:p19 interaction is readily reversible, characterized by rapid binding [(1.69 ± 0.07) × 10(8) M(-)(1) s(-1)] and marked dissociation (k(off)=0.062 ± 0.002 s(-1)). We also observe that p19 efficiently competes with recombinant Dicer and inhibits the formation of RISC-related assembly complexes found in human cell extract. Computational modeling based on these results provides evidence for the transient formation of a ternary complex between siRNA, human Dicer, and p19. An expanded model of RNA silencing indicates that multiple turnover by reversible binding of siRNAs potentiates the efficiency of the suppressor protein. Our predictive model is expected to be applicable to the dosing of p19 as a silencing suppressor in viral gene therapy. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. The Chemical Basis of Thiol Addition to Nitro-conjugated Linoleic Acid, a Protective Cell-signaling Lipid*♦

    PubMed Central

    Turell, Lucía; Vitturi, Darío A.; Coitiño, E. Laura; Lebrato, Lourdes; Möller, Matías N.; Sagasti, Camila; Salvatore, Sonia R.; Woodcock, Steven R.; Alvarez, Beatriz; Schopfer, Francisco J.

    2017-01-01

    Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and β-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the β- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to β-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA

  10. Molecular analysis of the Na+ channel blocking actions of the novel class I anti-arrhythmic agent RSD 921

    PubMed Central

    Pugsley, Michael K; Goldin, Alan L

    1999-01-01

    RSD 921 is a novel, structurally unique, class I Na+ channel blocking drug under development as a local anaesthetic agent and possibly for the treatment of cardiac arrhythmias. The effects of RSD 921 on wild-type heart, skeletal muscle, neuronal and non-inactivating IFMQ3 mutant neuronal Na+ channels expressed in Xenopus laevis oocytes were examined using a two-electrode voltage clamp.RSD 921 produced similarly potent tonic block of all three wild-type channel isoforms, with EC50 values between 35 and 47 μM, whereas the EC50 for block of the IFMQ3 mutant channel was 110±5.5 μM.Block of Na+ channels by RSD 921 was concentration and use-dependent, with marked frequency-dependent block of heart channels and mild frequency-dependent block of skeletal muscle, wild-type neuronal and IFMQ3 mutant channels.RSD 921 produced a minimal hyperpolarizing shift in the steady-state voltage-dependence of inactivation of all three wild-type channel isoforms.Open channel block of the IFMQ3 mutant channel was best fit with a first order blocking scheme with kon equal to 0.11±0.012×106 M−1 s−1 and koff equal to 12.5±2.5 s−1, resulting in KD of 117±31 μM. Recovery from open channel block occurred with a time constant of 14±2.7 s−1.These results suggest that RSD 921 preferentially interacts with the open state of the Na+ channel, and that the drug may produce potent local anaesthetic or anti-arrhythmic action under conditions of shortened action potentials, such as during anoxia or ischaemia. PMID:10369450

  11. Revealing kinetics and state-dependent binding properties of IKur-targeting drugs that maximize atrial fibrillation selectivity

    NASA Astrophysics Data System (ADS)

    Ellinwood, Nicholas; Dobrev, Dobromir; Morotti, Stefano; Grandi, Eleonora

    2017-09-01

    The KV1.5 potassium channel, which underlies the ultra-rapid delayed-rectifier current (IKur) and is predominantly expressed in atria vs. ventricles, has emerged as a promising target to treat atrial fibrillation (AF). However, while numerous KV1.5-selective compounds have been screened, characterized, and tested in various animal models of AF, evidence of antiarrhythmic efficacy in humans is still lacking. Moreover, current guidelines for pre-clinical assessment of candidate drugs heavily rely on steady-state concentration-response curves or IC50 values, which can overlook adverse cardiotoxic effects. We sought to investigate the effects of kinetics and state-dependent binding of IKur-targeting drugs on atrial electrophysiology in silico and reveal the ideal properties of IKur blockers that maximize anti-AF efficacy and minimize pro-arrhythmic risk. To this aim, we developed a new Markov model of IKur that describes KV1.5 gating based on experimental voltage-clamp data in atrial myocytes from patient right-atrial samples in normal sinus rhythm. We extended the IKur formulation to account for state-specificity and kinetics of KV1.5-drug interactions and incorporated it into our human atrial cell model. We simulated 1- and 3-Hz pacing protocols in drug-free conditions and with a [drug] equal to the IC50 value. The effects of binding and unbinding kinetics were determined by examining permutations of the forward (kon) and reverse (koff) binding rates to the closed, open, and inactivated states of the KV1.5 channel. We identified a subset of ideal drugs exhibiting anti-AF electrophysiological parameter changes at fast pacing rates (effective refractory period prolongation), while having little effect on normal sinus rhythm (limited action potential prolongation). Our results highlight that accurately accounting for channel interactions with drugs, including kinetics and state-dependent binding, is critical for developing safer and more effective pharmacological anti

  12. Mechanism-based pharmacokinetic-pharmacodynamic modeling of the antinociceptive effect of buprenorphine in healthy volunteers.

    PubMed

    Yassen, Ashraf; Olofsen, Erik; Romberg, Raymonda; Sarton, Elise; Danhof, Meindert; Dahan, Albert

    2006-06-01

    The objective of this investigation was to characterize the pharmacokinetic-pharmacodynamic relation of buprenorphine's antinociceptive effect in healthy volunteers. Data on the time course of the antinociceptive effect after intravenous administration of 0.05-0.6 mg/70 kg buprenorphine in healthy volunteers was analyzed in conjunction with plasma concentrations by nonlinear mixed-effects analysis. A three-compartment pharmacokinetic model best described the concentration time course. Four structurally different pharmacokinetic-pharmacodynamic models were evaluated for their appropriateness to describe the time course of buprenorphine's antinociceptive effect: (1) E(max) model with an effect compartment model, (2) "power" model with an effect compartment model, (3) receptor association-dissociation model with a linear transduction function, and (4) combined biophase equilibration/receptor association-dissociation model with a linear transduction function. The latter pharmacokinetic-pharmacodynamic model described the time course of effect best and was used to explain time dependencies in buprenorphine's pharmacodynamics. The model converged, yielding precise estimation of the parameters characterizing hysteresis and the relation between relative receptor occupancy and antinociceptive effect. The rate constant describing biophase equilibration (k(eo)) was 0.00447 min(-1) (95% confidence interval, 0.00299-0.00595 min(-1)). The receptor dissociation rate constant (k(off)) was 0.0785 min(-1) (95% confidence interval, 0.0352-0.122 min(-1)), and k(on) was 0.0631 ml . ng(-1) . min(-1) (95% confidence interval, 0.0390-0.0872 ml . ng(-1) . min(-1)). This is consistent with observations in rats, suggesting that the rate-limiting step in the onset and offset of the antinociceptive effect is biophase distribution rather than slow receptor association-dissociation. In the dose range studied, no saturation of receptor occupancy occurred explaining the lack of a ceiling effect

  13. Mechanism-based PK/PD modeling of the respiratory depressant effect of buprenorphine and fentanyl in healthy volunteers.

    PubMed

    Yassen, A; Olofsen, E; Romberg, R; Sarton, E; Teppema, L; Danhof, M; Dahan, A

    2007-01-01

    The objective of this study was to characterize the pharmacokinetic/pharmacodynamic (PK/PD) relationship of buprenorphine and fentanyl for the respiratory depressant effect in healthy volunteers. Data on the time course of the ventilatory response at a fixed P(ET)CO(2) of 50 mm Hg and P(ET)O(2) of 110 mm Hg following intravenous administration of buprenorphine and fentanyl were obtained from two phase I studies (50 volunteers received buprenorphine: 0.05-0.6 mg/70 kg and 24 volunteers received fentanyl: 0.075-0.5 mg/70 kg). The PK/PD correlations were analyzed using nonlinear mixed effects modeling. A two- and three-compartment pharmacokinetic model characterized the time course of fentanyl and buprenorphine concentration, respectively. Three structurally different PK/PD models were evaluated for their appropriateness to describe the time course of respiratory depression: (1) a biophase distribution model with a fractional sigmoid E(max) pharmacodynamic model, (2) a receptor association/dissociation model with a linear transduction function, and (3) a combined biophase distribution-receptor association/dissociation model with a linear transduction function. The results show that for fentanyl hysteresis is entirely determined by the biophase distribution kinetics, whereas for buprenorphine hysteresis is caused by a combination of biophase distribution kinetics and receptor association/dissociation kinetics. The half-time values of biophase equilibration (t(1/2, k(eo))) were 16.4 and 75.3 min for fentanyl and buprenorphine, respectively. In addition, for buprenorphine, the value of k(on) was 0.246 ml/ng/min and the value of k(off) was 0.0102 min(-1). The concentration-effect relationship of buprenorphine was characterized by a ceiling effect at higher concentrations (intrinsic activity alpha=0.56, 95% confidence interval (CI): 0.50-0.62), whereas fentanyl displayed full respiratory depressant effect (alpha=0.91, 95% CI: 0.19-1.62).

  14. Solution behavior of iron(III) and iron(II) porphyrins in DMSO and reaction with superoxide. Effect of neighboring positive charge on thermodynamics, kinetics and nature of iron-(su)peroxo product.

    PubMed

    Duerr, K; Troeppner, O; Olah, J; Li, J; Zahl, A; Drewello, T; Jux, N; Harvey, J N; Ivanović-Burmazović, I

    2012-01-14

    The solution behavior of iron(III) and iron(II) complexes of 5(4),10(4),15(4),20(4)-tetra-tert-butyl-5,10,15,20-tetraphenylporphyrin (H(2)tBuTPP) and the reaction with superoxide (KO(2)) in DMSO have been studied in detail. Applying temperature and pressure dependent NMR studies, the thermodynamics of the low-spin/high-spin equilibrium between bis- and mono-DMSO Fe(II) forms have been quantified (K(DMSO) = 0.082 ± 0.002 at 298.2 K, ΔH° = +36 ± 1 kJ mol(-1), ΔS° = +101 ± 4 J K(-1) mol(-1), ΔV° = +16 ± 2 cm(3) mol(-1)). This is a key activation step for substitution and inner-sphere electron transfer. The superoxide binding constant to the iron(II) form of the studied porphyrin complex was found to be (9 ± 0.5) × 10(3) M(-1), and does not change significantly in the presence of the externally added crown ether in DMSO (11 ± 4) × 10(3) M(-1). The rate constants for the superoxide binding (k(on) = (1.30 ± 0.01) × 10(5) M(-1) s(-1)) and release (k(off) = 11.6 ± 0.7 s(-1)) are not affected by the presence of the external crown ether in solution. The resulting iron(II)-superoxide adduct has been characterized (mass spectrometry, EPR, high-pressure UV/Vis spectroscopy) and upon controlled addition of a proton source it regenerates the starting iron(II) complex. Based on DFT calculations, the reaction product without neighboring positive charge has iron(II)-superoxo character in both high-spin side-on and low-spin end-on forms. The results are compared to those obtained for the analogous complex with covalently attached crown ether, and more general conclusions regarding the spin-state equilibrium of iron(II) porphyrins, their reaction with superoxide and the electronic structure of the product species are drawn.

  15. LN-1-255, a penicillanic acid sulfone able to inhibit the class D carbapenemase OXA-48.

    PubMed

    Vallejo, Juan A; Martínez-Guitián, Marta; Vázquez-Ucha, Juan C; González-Bello, Concepción; Poza, Margarita; Buynak, John D; Bethel, Christopher R; Bonomo, Robert A; Bou, German; Beceiro, Alejandro

    2016-08-01

    Carbapenemases are the most important mechanism responsible for carbapenem resistance in Enterobacteriaceae. Among carbapenemases, OXA-48 presents unique challenges as it is resistant to β-lactam inhibitors. Here, we test the capacity of the compound LN-1-255, a 6-alkylidene-2'-substituted penicillanic acid sulfone, to inhibit the activity of the carbapenemase OXA-48. The OXA-48 gene was cloned and expressed in Klebsiella pneumoniae and Escherichia coli in order to obtain MICs in the presence of inhibitors (clavulanic acid, tazobactam and sulbactam) and LN-1-255. OXA-48 was purified and steady-state kinetics was performed with LN-1-255 and tazobactam. The covalent binding mode of LN-1-255 with OXA-48 was studied by docking assays. Both OXA-48-producing clinical and transformant strains displayed increased susceptibility to carbapenem antibiotics in the presence of 4 mg/L LN-1-255 (2-32-fold increased susceptibility) and 16 mg/L LN-1-255 (4-64-fold increased susceptibility). Kinetic assays demonstrated that LN-1-255 is able to inhibit OXA-48 with an acylation efficiency (k2/K) of 10 ± 1 × 10(4) M(-1) s(-1) and a slow deacylation rate (koff) of 7 ± 1 × 10(-4) s(-1). IC50 was 3 nM for LN-1-255 and 1.5 μM for tazobactam. Lastly, kcat/kinact was 500-fold lower for LN-1-255 than for tazobactam. In these studies, carbapenem antibiotics used in combination with LN-1-255 are effective against the carbapenemase OXA-48, an important emerging mechanism of antibiotic resistance. This provides an incentive for further investigations to maximize the efficacy of penicillin sulfone inhibition of class D plasmid-carried Enterobacteriaceae carbapenemases. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Reactivity and dynamics of H2S, NO, and O2 interacting with hemoglobins from Lucina pectinata.

    PubMed

    Ramos-Alvarez, Cacimar; Yoo, Byung-Kuk; Pietri, Ruth; Lamarre, Isabelle; Martin, Jean-Louis; Lopez-Garriga, Juan; Negrerie, Michel

    2013-10-08

    Hemoglobin HbI from the clam Lucina pectinata is involved in H2S transport, whereas homologous heme protein HbII/III is involved in O2 metabolism. Despite similar tertiary structures, HbI and HbII/III exhibit very different reactivity toward heme ligands H2S, O2, and NO. To investigate this reactivity at the heme level, we measured the dynamics of ligand interaction by time-resolved absorption spectroscopy in the picosecond to nanosecond time range. We demonstrated that H2S can be photodissociated from both ferric and ferrous HbI. H2S geminately rebinds to ferric and ferrous out-of-plane iron with time constants (τgem) of 12 and 165 ps, respectively, with very different proportions of photodissociated H2S exiting the protein (24% in ferric and 80% in ferrous HbI). The Gln(E7)His mutation considerably changes H2S dynamics in ferric HbI, indicating the role of Gln(E7) in controling H2S reactivity. In ferric HbI, the rate of diffusion of H2S from the solvent into the heme pocket (kentry) is 0.30 μM(-1) s(-1). For the HbII/III-O2 complex, we observed mainly a six-coordinate vibrationally excited heme-O2 complex with O2 still bound to the iron. This explains the low yield of O2 photodissociation and low koff from HbII/III, compared with those of HbI and Mb. Both isoforms behave very differently with regard to NO and O2 dynamics. Whereas the amplitude of geminate rebinding of O2 to HbI (38.5%) is similar to that of myoglobin (34.5%) in spite of different distal heme sites, it appears to be much larger for HbII/III (77%). The distal Tyr(B10) side chain present in HbII/III increases the energy barrier for ligand escape and participates in the stabilization of bound O2 and NO.

  17. Interaction of nitric oxide with human heme oxygenase-1.

    PubMed

    Wang, Jinling; Lu, Shen; Moënne-Loccoz, Pierre; Ortiz de Montellano, Paul R

    2003-01-24

    NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.

  18. Fevipiprant (QAW039), a Slowly Dissociating CRTh2 Antagonist with the Potential for Improved Clinical Efficacy.

    PubMed

    Sykes, David A; Bradley, Michelle E; Riddy, Darren M; Willard, Elizabeth; Reilly, John; Miah, Asadh; Bauer, Carsten; Watson, Simon J; Sandham, David A; Dubois, Gerald; Charlton, Steven J

    2016-05-01

    Here we describe the pharmacologic properties of a series of clinically relevant chemoattractant receptor-homologous molecules expressed on T-helper type 2 (CRTh2) receptor antagonists, including fevipiprant (NVP-QAW039 or QAW039), which is currently in development for the treatment of allergic diseases. [(3)H]-QAW039 displayed high affinity for the human CRTh2 receptor (1.14 ± 0.44 nM) expressed in Chinese hamster ovary cells, the binding being reversible and competitive with the native agonist prostaglandin D2(PGD2). The binding kinetics of QAW039 determined directly using [(3)H]-QAW039 revealed mean kinetic on (kon) and off (koff) values for QAW039 of 4.5 × 10(7)M(-1)min(-1)and 0.048 minute(-1), respectively. Importantly, thekoffof QAW039 (half-life = 14.4 minutes) was >7-fold slower than the slowest reference compound tested, AZD-1981. In functional studies, QAW039 behaved as an insurmountable antagonist of PGD2-stimulated [(35)S]-GTPγS activation, and its effects were not fully reversed by increasing concentrations of PGD2after an initial 15-minute incubation period. This behavior is consistent with its relatively slow dissociation from the human CRTh2 receptor. In contrast for the other ligands tested this time-dependent effect on maximal stimulation was fully reversed by the 15-minute time point, whereas QAW039's effects persisted for >180 minutes. All CRTh2 antagonists tested inhibited PGD2-stimulated human eosinophil shape change, but importantly QAW039 retained its potency in the whole-blood shape-change assay relative to the isolated shape change assay, potentially reflective of its relatively slower off rate from the CRTh2 receptor. QAW039 was also a potent inhibitor of PGD2-induced cytokine release in human Th2 cells. Slow CRTh2 antagonist dissociation could provide increased receptor coverage in the face of pathologic PGD2concentrations, which may be clinically relevant. Copyright © 2016 by The American Society for Pharmacology and Experimental

  19. A fluorescent approach for identifying P2X1 ligands

    PubMed Central

    Ruepp, Marc-David; Brozik, James A.; de Esch, Iwan J.P.; Farndale, Richard W.; Murrell-Lagnado, Ruth D.; Thompson, Andrew J.

    2015-01-01

    There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 106 M−1 s−1) and koff (0.136 s−1) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology

  20. A fluorescent approach for identifying P2X1 ligands.

    PubMed

    Ruepp, Marc-David; Brozik, James A; de Esch, Iwan J P; Farndale, Richard W; Murrell-Lagnado, Ruth D; Thompson, Andrew J

    2015-11-01

    There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 10(6) M(-1) s(-1)) and koff (0.136 s(-1)) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'. Copyright

  1. Dissecting EB1-microtubule interactions from every direction: using single-molecule visualization and static and dynamic binding measurements

    NASA Astrophysics Data System (ADS)

    Lopez, Benjamin

    2015-03-01

    EB1 is an important microtubule associating protein (MAP) that acts as a master coordinator of protein activity at the growing plus-end of the microtubule. We can recapitulate the plus-end binding behavior of EB1 along the entire length of a static microtubule using microtubules polymerized in the presence of the nonhydrolyzable GTP analogs GMPCPP and GTP γS instead of GTP. Through the use of single-molecule TIRF imaging we find that EB1 is highly dynamic (with a sub-second characteristic binding lifetime) and continuously diffusive while bound to the microtubule. We measure the diffusion coefficient, D, through linear fitting to mean-squared displacement of individually labeled proteins, and the binding lifetime, τ, by fitting a single exponential decay to the probability distribution of trajectory lifetimes. In agreement with measurements of other diffusive MAPs, we find that D increases and τ decreases with increasing ionic strength. We also find that D is sensitive to the choice of GTP analog: EB1 proteins bound to GTP γS polymerized microtubules have a D half of that found with GMPCPP polymerized microtubules. To compare these single-molecule measurements to the bulk binding behavior of EB1, we use TIRF imaging to measure the intensity of microtubules coated with EB1-GFP as a function of EB1 concentration. We find that EB1 binding is cooperative and both the quantity of EB1 bound and the dissociation constant are sensitive to GTP analog and ionic concentration. The correlation between binding affinity and D and the cooperative nature of EB1-microtubule binding leads to a decrease in D with increasing EB1 concentration. Interestingly, we also find an increase in τ at high EB1 concentrations, consistent with attractive EB1-microtubule interactions driving the cooperativity. To further understand the nature of the cooperativity we estimate the interaction energy by measuring the association and dissociation rates (kon and koff respectively) at different

  2. p-( sup 125 I)iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor

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

    Gerhardt, M.A.; Wade, S.M.; Neubig, R.R.

    1990-08-01

    The binding properties of p-(125I)iodoclonidine (( 125I)PIC) to human platelet membranes and the functional characteristics of PIC are reported. (125I)PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific (125I)PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. (125I)PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist (3H) bromoxidine (UK14,304), which representedmore » approximately 40% of the sites bound by the antagonist (3H)yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of (125I)PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for (125I)PIC binding was stereoselective. Competition for (3H)bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for (3H)yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. (125I)PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM.« less

  3. Eccentric circummeatal based flap with limited urethral mobilization: An easy technique for distal hypospadias repair.

    PubMed

    Ekinci, Saniye; Çiftçi, Arbay Özden; Karnak, İbrahim; Şenocak, Mehmet Emin

    2016-04-01

    patients were treated using dilatation, fistula repair, meatoplasty, and secondary repair with the same technique, respectively. Eventually all patients had a vertical slit-like meatus on the tip of a natural looking glans. The most commonly used distal hypospadias repair techniques are glanular approximation, meatal advancement and glanuloplasty, Koff, Mathieu, Thiersch-Duplay procedure, tubularized incised plate repairs, and modifications of these techniques. Cosmetic and functional results and complication rates of ECMB-LUM technique are comparable with those of the commonly used techniques. Copyright © 2015 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

  4. Tactics for preclinical validation of receptor-binding radiotracers

    PubMed Central

    Lever, Susan Z.; Fan, Kuo-Hsien; Lever, John R.

    2016-01-01

    Introduction Aspects of radiopharmaceutical development are illustrated through preclinical studies of [125I]-(E)-1-(2-(2,3-dihydrobenzofuran-5-yl)ethyl)-4-(iodoallyl)piperazine ([125I]-E-IA- BF-PE-PIPZE), a radioligand for sigma-1 (σ1) receptors, coupled with examples from the recent literature. Findings are compared to those previously observed for [125I]-(E)-1-(2-(2,3-dimethoxy-5-yl)ethyl)-4-(iodoallyl)piperazine ([125I]-E-IA-DM-PE-PIPZE). Methods Syntheses of E-IA-BF-PE-PIPZE and [125I]-E-IA-BF-PE-PIPZE were accomplished by standard methods. In vitro receptor binding studies and autoradiography were performed, and binding potential was predicted. Measurements of lipophilicity and protein binding were obtained. In vivo studies were conducted in mice to evaluate radioligand stability, as well as specific binding to σ1 sites in brain, brain regions and peripheral organs in the presence and absence of potential blockers. Results E-IA-BF-PE-PIPZE exhibited high affinity and selectivity for σ1 receptors (Ki = 0.43 ± 0.03 nM, σ2 / σ1 = 173). [125I]-E-IA-BF-PE-PIPZE was prepared in good yield and purity, with high specific activity. Radioligand binding provided dissociation (koff) and association (kon) rate constants, along with a measured Kd of 0.24 ± 0.01 nM and Bmax of 472 ± 13 fmol / mg protein. The radioligand proved suitable for quantitative autoradiography in vitro using brain sections. Moderate lipophilicity, Log D7.4 2.69 ± 0.28, was determined, and protein binding was 71 ± 0.3%. In vivo, high initial whole brain uptake, > 6% injected dose / g, cleared slowly over 24 h. Specific binding represented 75% to 93% of total binding from 15 min to 24 h. Findings were confirmed and extended by regional brain biodistribution. Radiometabolites were not observed in brain (1%). Conclusions Substitution of dihydrobenzofuranylethyl for dimethoxyphenethyl increased radioligand affinity for σ1 receptors by 16-fold. While high specific binding to σ1 receptors was

  5. Iodination of (Tyr11)somatostatin yields a super high affinity ligand for somatostatin receptors in GH4C1 pituitary cells

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

    Presky, D.H.; Schonbrunn, A.

    1988-11-01

    GH4C1 cells are a clonal strain of rat pituitary tumor cells which contain high affinity receptors for the inhibitory neuropeptide somatostatin (SRIF). In contrast to other peptides that bind to specific receptors on these cells, receptor-bound (125I-Tyr1)SRIF does not undergo rapid endocytosis. Rather, partial degradation to 125I-tyrosine occurs concomitantly with the dissociation of (125I-Tyr1)SRIF from cell surface receptors. In this study we characterize the binding, biological activity and receptor-mediated degradation of (125I-Tyr11)SRIF, a SRIF analog that is radiolabeled in the center of the molecule. The binding of trace concentrations of (125I-Tyr11)SRIF (less than 50 pM) required 6 hr to reachmore » equilibrium at 37 degrees compared with the 60 min required for (125I-Tyr1)SRIF. Analysis of the kinetics of (125I- Tyr11)SRIF binding showed that the rate constant for association (kon = 1.7 x 10(8) M-8min-1) was similar to that for (125I-Tyr1)SRIF (0.8 x 10(8) M-1min-1). However, the two radioligands exhibited markedly different dissociation kinetics; the koff for (125I-Tyr11)SRIF was 0.002 min-1 compared with the value of 0.02 min-1 for (125I-Tyr1) SRIF. In agreement with its much slower rate of dissociation, (125I-Tyr11)SRIF bound to the SRIF receptor with higher affinity (Kd = 70 pM) than did (125I-Tyr1)SRIF (Kd = 350 pM). However, the apparent ED50 for (I-Tyr11)SRIF to inhibit cAMP accumulation (1.9 +/- 0.4 nM) was greater than the ED50 for SRIF (0.19 +/- 0.04 nM). The low potency of (I-Tyr11)SRIF probably resulted from the fact that subsaturating concentrations of this peptide did not achieve equilibrium binding during the 30-min incubation used to assay biological activity. As previously reported for (125I-Tyr1)SRIF, receptor-bound (125I-Tyr11)SRIF was not internalized and was released from the cells as a mixture of intact (125I-Tyr11)SRIF (30%) and the degradation product 125I-tyrosine (65%).« less

  6. Quantitative analysis of protein-ligand interactions by NMR.

    PubMed

    Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

    2016-08-01

    Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

  7. A Human Anti-Insulin IgG Autoantibody Apparently Arises Through Clonal Selection from an Insulin-Specific “Germ-Line” Natural Antibody Template

    PubMed Central

    Ichiyoshi, Yuji; Zhou, Min; Casali, Paolo

    2015-01-01

    We analyzed the structural correlates underlying the insulin-dependent selection of the specific anti-insulin IgG1 κ mAb13-producing cell clone, derived from a patient with insulin-dependent diabetes mellitus treated with recombinant human insulin. First, we cloned the germ-line genes that putatively gave rise to the expressed VH and Vκ segments and used them to generate the full (unmutated) “germ-line revertant” of the “wild-type” (somatically mutated) mAb13, using recombinant PCR methods and an in vitro human Cγ1 and Cκ expression system. The full “germ-line revertant” bound insulin specifically and in a dose-saturable fashion, but with a relative avidity (Avrel) more than three-fold lower than that of its wild-type counterpart (Avrel, 1.69 × 10−8 vs 4.91 × 10−9 g/μl). Second, we established, by reassorting wild-type and germ-line revertant forms of the mAb13 VH and Vκ segments, that the increased Avrel for insulin of mAb13 when compared with its full “germ-line revertant” counterpart was entirely dependent on the mutations in the VH not those in the Vκ chain. Third, we determined, by site-directed mutagenesis experiments, that of the three mutations in the mAb13 VH segment (Ser→Gly, Ser→Thr, and Ser→Arg at positions 31, 56, and 58, respectively), only Arg58 was crucial in increasing the mAb13 Avrel (from 1.44 × 10−8 to 5.14 × 10−9 g/μl) and affinity (Kd, from 189 to 59 nM) for insulin. The affinity enhancement mediated by the VH segment Arg58 residue reflected about a threefold decrease in dissociation rate constant (Koff, from 4.92 × 10−3 to 1.54 × 10−3 s−1)but not an increase in association rate constant (Kon, from 2.60 × 104 to 2.61 × 104 M−1 s−1), and it contrasted with the complete loss of insulin binding resulting from the substitution of the VH segment Asn52 by Lys. The present findings suggest that human insulin, a self Ag, has the potential to recruit a natural autoantibody-producing cell precursor

  8. Nanointaglio fabrication of optical lipid multilayer diffraction gratings with applications in biosensing

    NASA Astrophysics Data System (ADS)

    Lowry, Troy Warren

    supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Presented next is a nanointaglio based method for quantitative measurements of lipid-protein interactions and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1. Optical diffraction gratings composed of lipids are printed on surfaces using nanointaglio, resulting in lipid multilayer gratings. Exposure of lipid multilayer gratings to Sar1 results in the inflation of lipid multilayers into unilamellar structures, the kinetics of which can be detected in a label-free manner by monitoring the diffraction of white light through an optical microscope. Local variations in lipid multilayer volume on the surface can be used to vary substrate availability in a microarray format, allowing kinetic and thermodynamic data to be obtained from a single experiment without the need for varying enzyme concentration. A quantitative model is developed and fits to the data allow measurements of both binding affinity (KD) and kinetics (kon and koff). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1 induced inflation of single bilayers from surface supported multilayers, the semi-cylindrical grating lines are observed to remodel into semi-spherical buds when a critical radius of curvature equal to 300 nm is reached, which is explained in terms of a Rayleigh type instability.