The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

PubMed

Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

2016-04-01

Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

PubMed Central

Bai, Erdeni; Rosell, Federico I.; Lige, Bao; Mauk, Marcia R.; Lelj-Garolla, Barbara; Moore, Geoffrey R.; Mauk, A. Grant

2006-01-01

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (KA) of 10(±7)×106, 5.7(±3)×106, 2.0(±2)×106 and 2.0(±3)×104 M−1 for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×106, 3.2(±2)×104, 1.76(±1)×105 and 1.5(±2)×103 M−1 respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents. PMID:16928194

Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1

USDA-ARS?s Scientific Manuscript database

Aldehyde dehydrogenase 1 (ALDH1) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH1 activity in part by increasing NADH binding affinity to the enzyme thus reducing activity. By using time-resolved fluorescence spectroscopy, we have resolved t...

Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels

PubMed Central

Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L.

2014-01-01

SUMMARY AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine and phenylalanine which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate and serine. AvGluR1 LBD crystal structures reveal a novel scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, while in the alanine, methionine and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl− lowers affinity for these ligands, but not for glutamate, aspartate or for phenylalanine which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure based studies on iGluR-ligand interactions. PMID:23434404

Revised Model of Calcium and Magnesium Binding to the Bacterial Cell Wall

PubMed Central

Thomas, Kieth J.; Rice, Charles V.

2014-01-01

Metals bind to the bacterial cell wall yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane (lipoteichoic acid) or covalently bound to the cell wall (wall teichoic acid). The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, contradicting previous reports that calcium binding is 100% dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger and previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, meaning that the binding affinity decreases as more ions become bound to the sample. For Ca2+, Region I has a KA = (1.0 ± 0.2) × 106 M−1 and Region II has a KA = (0.075 ± 0.058) × 106 M−1. For Mg2+, KA1 = (1.5 ± 0.1) × 106 and KA2 = (0.17 ± 0.10) × 106. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 µmol/mg and 0.67 ± 0.03 µmol/mg for Ca2+ and Mg2+ respectively. These data contradict the current paradigm of there being a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent, suggesting a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations. PMID:25315444

Characterization of a conformationally sensitive murine monoclonal antibody directed to the metal ion-dependent adhesion site face of integrin CD11b.

PubMed

Li, Rui; Haruta, Ikuko; Rieu, Philippe; Sugimori, Takashi; Xiong, Jian-Ping; Arnaout, M Amin

2002-02-01

Integrin binding to physiologic ligands requires divalent cations and an inside-out-driven switch of the integrin to a high-affinity state. Divalent cations at the metal ion-dependent adhesion site (MIDAS) face of the alpha subunit-derived A domain provide a direct bridge between ligands and the integrin, and it has been proposed that activation dependency is caused by reorientation of the surrounding residues relative to the metal ion, forming an optimal binding interface. To gain more insight into the functional significance of the protein movements on the MIDAS face, we raised and characterized a murine mAb 107 directed against the MIDAS face of the A domain from integrin CD11b. We find that mAb 107 behaves as a ligand mimic. It binds in a divalent-cation-dependent manner to solvent-exposed residues on the MIDAS face of CD11b, blocks interaction of 11bA or the holoreceptor with ligands, and inhibits spreading and phagocytosis by human neutrophils. However, in contrast to physiologic ligands, mAb 107 preferentially binds to the inactive low-affinity form of the integrin, suggesting that its antagonistic effects are exerted in part by stabilizing the receptor in the low-affinity state. These data support a functional relevance of the protein movements on the MIDAS face and suggest that stabilizing the A domain in the low-affinity state may have therapeutic benefit.

Immobilized metal ion affinity electrophoresis. A study with several model proteins containing histidine.

PubMed

Goubran-Botros, H; Nanak, E; Abdul Nour, J; Birkenmeir, G; Vijayalakshmi, M A

1992-04-24

Immobilized metal ion affinity electrophoresis (IMA-Elec) is one among the many methods derived from the immobilized metal ion affinity chromatography. Two approaches for incorporating the metal ligand, were studied. One was in the form of insoluble particulate material based on Sepharose 6B and the other in the form of soluble polymer based on polyethylene glycol (PEG) 5000. Both the polymers coupled with iminodiacetate and metallized with copper or zinc were used as ligands, incorporated into soluble agarose as the electrophoretic gel. Several histidine-containing model proteins were studied with both the systems and their metal binding strengths were determined as the dissociation constants, Kd. The results clearly demonstrated that the mechanism of protein recognition by immobilized copper or zinc via the accessible histidyl residues was maintained in the IMA-Elec system. Proteins with increasing numbers of histidine residues showed increasing binding strength (lower Kd values). While this basic mechanism was conserved, the supporting polymers (Sepharose 6B and the PEG 5000) showed significant differences in the metal binding to the protein. The polysaccharide Sepharose 6B enhanced the binding strength compared with PEG 5000. The optimum electrophoretic parameters were determined to be current intensities up to 20 mA and pH ca. 7.0. At pH greater than 8.0, a significant decrease in the affinity was observed, this decrease being greater with PEG 5000 than Sepharose 6B as supporting material.

Cation specific binding with protein surface charges

PubMed Central

Hess, Berk; van der Vegt, Nico F. A.

2009-01-01

Biological organization depends on a sensitive balance of noncovalent interactions, in particular also those involving interactions between ions. Ion-pairing is qualitatively described by the law of “matching water affinities.” This law predicts that cations and anions (with equal valence) form stable contact ion pairs if their sizes match. We show that this simple physical model fails to describe the interaction of cations with (molecular) anions of weak carboxylic acids, which are present on the surfaces of many intra- and extracellular proteins. We performed molecular simulations with quantitatively accurate models and observed that the order K+ < Na+ < Li+ of increasing binding affinity with carboxylate ions is caused by a stronger preference for forming weak solvent-shared ion pairs. The relative insignificance of contact pair interactions with protein surfaces indicates that thermodynamic stability and interactions between proteins in alkali salt solutions is governed by interactions mediated through hydration water molecules. PMID:19666545

Comparative serum albumin interactions and antitumor effects of Au(III) and Ga(III) ions.

PubMed

Sarioglu, Omer Faruk; Ozdemir, Ayse; Karaboduk, Kuddusi; Tekinay, Turgay

2015-01-01

In the present study, interactions of Au(III) and Ga(III) ions on human serum albumin (HSA) were studied comparatively via spectroscopic and thermal analysis methods: UV-vis absorbance spectroscopy, fluorescence spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and isothermal titration calorimetry (ITC). The potential antitumor effects of these ions were studied on MCF-7 cells via Alamar blue assay. It was found that both Au(III) and Ga(III) ions can interact with HSA, however; Au(III) ions interact with HSA more favorably and with a higher affinity. FT-IR second derivative analysis results demonstrated that, high concentrations of both metal ions led to a considerable decrease in the α-helix content of HSA; while Au(III) led to around 5% of decrease in the α-helix content at 200μM, it was around 1% for Ga(III) at the same concentration. Calorimetric analysis gave the binding kinetics of metal-HSA interactions; while the binding affinity (Ka) of Au(III)-HSA binding was around 3.87×10(5)M(-1), it was around 9.68×10(3)M(-1) for Ga(III)-HSA binding. Spectroscopy studies overall suggest that both metal ions have significant effects on the chemical structure of HSA, including the secondary structure alterations. Antitumor activity studies on MCF7 tumor cell line with both metal ions revealed that, Au(III) ions have a higher antiproliferative activity compared to Ga(III) ions. Copyright © 2014 Elsevier GmbH. All rights reserved.

Protein scaffolds for selective enrichment of metal ions

DOEpatents

He, Chuan; Zhou, Lu; Bosscher, Michael

2016-02-09

Polypeptides comprising high affinity for the uranyl ion are provided. Methods for binding uranyl using such proteins are likewise provided and can be used, for example, in methods for uranium purification or removal.

Reversible cobalt ion binding to imidazole-modified nanopipettes

PubMed Central

Sa, Niya; Fu, Yaqin; Baker, Lane A.

2010-01-01

In this report, we demonstrate that quartz nanopipettes modified with an imidazole-terminated silane respond to metal ions (Co2+) in solution. The response of nanopipettes is evaluated through examination of the ion current rectification response. By cycling nanopipettes between solutions of different pH, adsorbed Co2+ can be released from the nanopipette surface, to regenerate binding sites of the nanopipette. These results demonstrate that rectification-based sensing strategies for nanopore sensors can benefit from selection of recognition elements with intermediate binding affinities, such that reversible responses to be attained. PMID:21090777

Reversible cobalt ion binding to imidazole-modified nanopipettes.

PubMed

Sa, Niya; Fu, Yaqin; Baker, Lane A

2010-12-15

In this report, we demonstrate that quartz nanopipettes modified with an imidazole-terminated silane respond to metal ions (Co(2+)) in solution. The response of nanopipettes is evaluated through examination of the ion current rectification ratio. When nanopipettes are cycled between solutions of different pH, adsorbed Co(2+) can be released from the nanopipette surface, to regenerate binding sites of the nanopipette. These results demonstrate that rectification-based sensing strategies for nanopore sensors can benefit from selection of recognition elements with intermediate binding affinities, such that reversible responses can be attained.

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

Beaumont, K.; Vaughn, D.A.; Fanestil, D.D.

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. The authors report here that ({sup 3}H)metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. ({sup 3}H)Metolazone bound with high affinity to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or not binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated withmore » their clinical potency. Halide anions specifically inhibited high-affinity binding of ({sup 3}H)metolazone to this site. ({sup 3})Metolazone also bound with lower affinity to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had K{sub i} values of 0.6-10 {mu}M for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity ({sup 3}H)metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.« less

Sodium ion modulates D2 receptor characteristics of dopamine agonist and antagonist binding sites in striatum and retina

PubMed Central

Makman, Maynard H.; Dvorkin, B.; Klein, Patrice N.

1982-01-01

Sodium ion (Na+) influences binding of both dopamine agonists and antagonists to D2 receptors in striatum and retina. Also, Na+ markedly potentiates the loss of high-affinity agonist binding due to the GTP analogue p[NH]ppG. 2-Amino-6, 7-dihydroxy-1,2,3,4-tetrahydro[5,8-3H]naphthalene ([3H]ADTN) binds exclusively to an agonist conformation of D2 receptor in both striatum and retina, distinct from the antagonist conformation labeled by [3H]spiroperidol or [3H]domperidone in striatum or by [3H]spiroperidol in retina. Na+ is not required for interaction of [3H]ADTN or antagonist radioligand sites with the selective D2 agonist LY-141865, the D2 antagonist domperidone, or nonselective dopamine agonists or antagonists; however, Na+ is necessary for high affinity interaction of those radioligand sites with the D2 antagonists molindone and metoclopramide. With Na+ present, striatal sites for [3H]ADTN, [3H]spiroperidol, and [3H]domperidone have similar affinities for antagonists but only [3H]ADTN sites have high affinity for agonists. Na+ further decreases the low affinity of dopamine agonists for [3H]spiroperidol binding sites. Also, Na+ enhances [3H]spiroperidol and decreases [3H]ADTN binding. Na+ alone causes bound [3H]ADTN to dissociate from at least 30% of striatal and 50% of retinal sites, and with Na+ present [3H]ADTN rapidly dissociates from the remaining sites upon addition of p[NH]ppG. It is proposed that D2 receptors in striatum and retina exist in distinct but interconvertible conformational states, with different properties depending on the presence or absence of Na+ and of guanine nucleotide. PMID:6213964

The Role and Specificity of the Catalytic and Regulatory Cation-binding Sites of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae*

PubMed Central

Juárez, Oscar; Shea, Michael E.; Makhatadze, George I.; Barquera, Blanca

2011-01-01

The Na+-translocating NADH:quinone oxidoreductase is the entry site for electrons into the respiratory chain and the main sodium pump in Vibrio cholerae and many other pathogenic bacteria. In this work, we have employed steady-state and transient kinetics, together with equilibrium binding measurements to define the number of cation-binding sites and characterize their roles in the enzyme. Our results show that sodium and lithium ions stimulate enzyme activity, and that Na+-NQR enables pumping of Li+, as well as Na+ across the membrane. We also confirm that the enzyme is not able to translocate other monovalent cations, such as potassium or rubidium. Although potassium is not used as a substrate, Na+-NQR contains a regulatory site for this ion, which acts as a nonessential activator, increasing the activity and affinity for sodium. Rubidium can bind to the same site as potassium, but instead of being activated, enzyme turnover is inhibited. Activity measurements in the presence of both sodium and lithium indicate that the enzyme contains at least two functional sodium-binding sites. We also show that the binding sites are not exclusively responsible for ion selectivity, and other steps downstream in the mechanism also play a role. Finally, equilibrium-binding measurements with 22Na+ show that, in both its oxidized and reduced states, Na+-NQR binds three sodium ions, and that the affinity for sodium is the same for both of these states. PMID:21652714

Metal Binding Studies and EPR Spectroscopy of the Manganese Transport Regulator MntR†

PubMed Central

Golynskiy, Misha V.; Gunderson, William A.; Hendrich, Michael P.; Cohen, Seth M.

2007-01-01

Manganese transport regulator (MntR) is a member of the diphtheria toxin repressor (DtxR) family of transcription factors that is responsible for manganese homeostasis in Bacillus subtilis. Prior biophysical studies have focused on the metal-mediated DNA binding of MntR [Lieser, S. A., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2003) Biochemistry 42, 12634-12642], as well as metal stabilization of the MntR structure [Golynskiy, M. V., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2005) Biochemistry 44, 3380-3389], but only limited data on the metal-binding affinities for MntR are available. Herein, the metal-binding affinities of MntR were determined by using electron paramagnetic resonance (EPR) spectroscopy, as well as competition experiments with the fluorimetric dyes Fura-2 and Mag-fura-2. MntR was not capable of competing with Fura-2 for the binding of transition metal ions. Therefore, the metal-binding affinities and stoichiometries of Mag-fura-2 for Mn2+, Co2+, Ni2+, Zn2+, and Cd2+ were determined and utilized in MntR/Mag-fura-2 competition experiments. The measured Kd values for MntR metal binding are comparable to those reported for DtxR metal binding [Kd from 10-7 to 10-4 M; D’Aquino, J. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 18408-18413], AntR [a homologue from Bacillus anthracis; Sen, K. I. et al. (2006) Biochemistry 45, 4295-4303], and generally follow the Irving-Williams series. Direct detection of the dinuclear Mn2+ site in MntR with EPR spectroscopy is presented, and the exchange interaction was determined, J = -0.2 cm-1. This value is lower in magnitude than most known dinuclear Mn2+ sites in proteins and synthetic complexes and is consistent with a dinuclear Mn2+ site with a longer Mn···Mn distance (4.4 Å) observed in some of the available crystal structures. MntR is found to have a surprisingly low binding affinity (∼160 μM) for its cognate metal ion Mn2+. Moreover, the results of DNA binding studies in the presence of limiting metal ion concentrations were found to be consistent with the measured metal-binding constants. The metal-binding affinities of MntR reported here help to elucidate the regulatory mechanism of this metal-dependent transcription factor. PMID:17176058

Chronic Beryllium Disease: Revealing the Role of Beryllium Ion and Small Peptides Binding to HLA-DP2

PubMed Central

Petukh, Marharyta; Wu, Bohua; Stefl, Shannon; Smith, Nick; Hyde-Volpe, David; Wang, Li; Alexov, Emil

2014-01-01

Chronic Beryllium (Be) Disease (CBD) is a granulomatous disorder that predominantly affects the lung. The CBD is caused by Be exposure of individuals carrying the HLA-DP2 protein of the major histocompatibility complex class II (MHCII). While the involvement of Be in the development of CBD is obvious and the binding site and the sequence of Be and peptide binding were recently experimentally revealed [1], the interplay between induced conformational changes and the changes of the peptide binding affinity in presence of Be were not investigated. Here we carry out in silico modeling and predict the Be binding to be within the acidic pocket (Glu26, Glu68 and Glu69) present on the HLA-DP2 protein in accordance with the experimental work [1]. In addition, the modeling indicates that the Be ion binds to the HLA-DP2 before the corresponding peptide is able to bind to it. Further analysis of the MD generated trajectories reveals that in the presence of the Be ion in the binding pocket of HLA-DP2, all the different types of peptides induce very similar conformational changes, but their binding affinities are quite different. Since these conformational changes are distinctly different from the changes caused by peptides normally found in the cell in the absence of Be, it can be speculated that CBD can be caused by any peptide in presence of Be ion. However, the affinities of peptides for Be loaded HLA-DP2 were found to depend of their amino acid composition and the peptides carrying acidic group at positions 4 and 7 are among the strongest binders. Thus, it is proposed that CBD is caused by the exposure of Be of an individual carrying the HLA-DP2*0201 allele and that the binding of Be to HLA-DP2 protein alters the conformational and ionization properties of HLA-DP2 such that the binding of a peptide triggers a wrong signaling cascade. PMID:25369028

Affinity monolith chromatography: A review of principles and recent analytical applications

PubMed Central

Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

2012-01-01

Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

Ion-binding properties of the ClC chloride selectivity filter

PubMed Central

Lobet, Séverine; Dutzler, Raimund

2006-01-01

The ClC channels are members of a large protein family of chloride (Cl−) channels and secondary active Cl− transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl− ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl− channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction. PMID:16341087

Price To Be Paid for Two-Metal Catalysis: Magnesium Ions That Accelerate Chemistry Unavoidably Limit Product Release from a Protein Kinase

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

Jacobsen, Douglas M.; Bao, Zhao-Qin; O'’Brien, Patrick

Incorporation of divalent metal ions into an active site is a fundamental catalytic tool used by diverse enzymes. Divalent cations are used by protein kinases to both stabilize ATP binding and accelerate chemistry. Kinetic analysis establishes that Cyclin-dependent kinase 2 (CDK2) requires simultaneous binding of two Mg 2+ ions for catalysis of phosphoryl transfer. This tool, however, comes with a price: the rate-acceleration effects are opposed by an unavoidable rate-limiting consequence of the use of two Mg 2+ ions by CDK2. The essential metal ions stabilize ADP product binding and limit the overall rate of the reaction. We demonstrate thatmore » product release is rate limiting for activated CDK2 and evaluate the effects of the two catalytically essential Mg 2+ ions on the stability of the ADP product within the active site. We present two new crystal structures of CDK2 bound to ADP showing how the phosphate groups can be coordinated by either one or two Mg 2+ ions, with the occupancy of one site in a weaker equilibrium. Molecular dynamics simulations indicate that ADP phosphate mobility is more restricted when ADP is coordinated by two Mg 2+ ions compared to one. The structural similarity between the rigid ADP·2Mg product and the cooperatively assembled transition state provides a mechanistic rational for the rate-limiting ADP release that is observed. We demonstrate that although the simultaneous binding of two Mg 2+ ions is essential for efficient phosphoryl transfer, the presence of both Mg 2+ ions in the active site also cooperatively increases ADP affinity and opposes its release. Evolution of protein kinases must have involved careful tuning of the affinity for the second Mg 2+ ion in order to balance the needs to stabilize the chemical transition state and allow timely product release. The link between Mg 2+ site affinity and activity presents a chemical handle that may be used by regulatory factors as well as explain some mutational effects.« less

Interactions between Hofmeister anions and the binding pocket of a protein.

PubMed

Fox, Jerome M; Kang, Kyungtae; Sherman, Woody; Héroux, Annie; Sastry, G Madhavi; Baghbanzadeh, Mostafa; Lockett, Matthew R; Whitesides, George M

2015-03-25

This paper uses the binding pocket of human carbonic anhydrase II (HCAII, EC 4.2.1.1) as a tool to examine the properties of Hofmeister anions that determine (i) where, and how strongly, they associate with concavities on the surfaces of proteins and (ii) how, upon binding, they alter the structure of water within those concavities. Results from X-ray crystallography and isothermal titration calorimetry show that most anions associate with the binding pocket of HCAII by forming inner-sphere ion pairs with the Zn(2+) cofactor. In these ion pairs, the free energy of anion-Zn(2+) association is inversely proportional to the free energetic cost of anion dehydration; this relationship is consistent with the mechanism of ion pair formation suggested by the "law of matching water affinities". Iodide and bromide anions also associate with a hydrophobic declivity in the wall of the binding pocket. Molecular dynamics simulations suggest that anions, upon associating with Zn(2+), trigger rearrangements of water that extend up to 8 Å away from their surfaces. These findings expand the range of interactions previously thought to occur between ions and proteins by suggesting that (i) weakly hydrated anions can bind complementarily shaped hydrophobic declivities, and that (ii) ion-induced rearrangements of water within protein concavities can (in contrast with similar rearrangements in bulk water) extend well beyond the first hydration shells of the ions that trigger them. This study paints a picture of Hofmeister anions as a set of structurally varied ligands that differ in size, shape, and affinity for water and, thus, in their ability to bind to—and to alter the charge and hydration structure of—polar, nonpolar, and topographically complex concavities on the surfaces of proteins.

Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels.

PubMed

Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L

2013-03-05

AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl(-) lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Stable coordination of the inhibitory Ca2+ ion at MIDAS in integrin CD11b/CD18 by an antibody-derived ligand aspartate: Implications for integrin regulation and structure-based drug design

PubMed Central

Mahalingam, Bhuvaneshwari; Ajroud, Kaouther; Alonso, Jose Luis; Anand, Saurabh; Adair, Brian; Horenstein, Alberto L; Malavasi, Fabio; Xiong, Jian-Ping; Arnaout, M. Amin

2011-01-01

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal-ion-dependent-adhesion site (MIDAS) in the integrin A-domain. This interaction stabilizes the A-domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A-domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking monoclonal antibody (mAb) 107 binds MIDAS of integrin CD11b/CD18 A-domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over Mg2+ at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of Ca2+ at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+. Binding of Fab 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that denticity of the ligand Asp/Glu can modify divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. PMID:22095715

Bioinorganic Chemistry of Parkinson's Disease: Affinity and Structural Features of Cu(I) Binding to the Full-Length β-Synuclein Protein.

PubMed

Miotto, Marco C; Pavese, Mayra D; Quintanar, Liliana; Zweckstetter, Markus; Griesinger, Christian; Fernández, Claudio O

2017-09-05

Alterations in the levels of copper in brain tissue and formation of α-synuclein (αS)-copper complexes might play a key role in the amyloid aggregation of αS and the onset of Parkinson's disease (PD). Recently, we demonstrated that formation of the high-affinity Cu(I) complex with the N-terminally acetylated form of the protein αS substantially increases and stabilizes local conformations with α-helical secondary structure and restricted motility. In this work, we performed a detailed NMR-based structural characterization of the Cu(I) complexes with the full-length acetylated form of its homologue β-synuclein (βS), which is colocalized with αS in vivo and can bind copper ions. Our results show that, similarly to αS, the N-terminal region of βS constitutes the preferential binding interface for Cu(I) ions, encompassing two independent and noninteractive Cu(I) binding sites. According to these results, βS binds the metal ion with higher affinity than αS, in a coordination environment that involves the participation of Met-1, Met-5, and Met-10 residues (site 1). Compared to αS, the shift of His from position 50 to 65 in the N-terminal region of βS does not change the Cu(I) affinity features at that site (site 2). Interestingly, the formation of the high-affinity βS-Cu(I) complex at site 1 in the N-terminus promotes a short α-helix conformation that is restricted to the 1-5 segment of the AcβS sequence, which differs with the substantial increase in α-helix conformations seen for N-terminally acetylated αS upon Cu(I) complexation. Our NMR data demonstrate conclusively that the differences observed in the conformational transitions triggered by Cu(I) binding to AcαS and AcβS find a correlation at the level of their backbone dynamic properties; added to the potential biological implications of these findings, this fact opens new avenues of investigations into the bioinorganic chemistry of PD.

Hemoglobin Rahere, a human hemoglobin variant with amino acid substitution at the 2,3-diphosphoglycerate binding site. Functional consequences of the alteration and effects of bezafibrate on the oxygen bindings.

PubMed

Sugihara, J; Imamura, T; Nagafuchi, S; Bonaventura, J; Bonaventura, C; Cashon, R

1985-09-01

We encountered an abnormal hemoglobin (Rahere), with a threonine residue replacing the beta 82 (EF6) lysine residue at the binding site of 2,3-diphosphoglycerate, which was responsible for overt erythrocytosis in two individuals of a Japanese family. Hemoglobin Rahere shows a lower oxygen affinity on the binding of 2,3-diphosphoglycerate or chloride ions than hemoglobin A. Although a decrease in the positive charge density at the binding sites of 2,3-diphosphoglycerate in hemoglobin Rahere apparently shifts the allosteric equilibrium toward the low affinity state, it greatly diminishes the cofactor effects by anions. The oxygen affinity of the patient's erythrocytes is substantially lowered by the presence of bezafibrate, which combines with sites different from those of 2,3-diphosphoglycerate in either hemoglobin Rahere or hemoglobin A.

Hemoglobin Rahere, a human hemoglobin variant with amino acid substitution at the 2,3-diphosphoglycerate binding site. Functional consequences of the alteration and effects of bezafibrate on the oxygen bindings.

PubMed Central

Sugihara, J; Imamura, T; Nagafuchi, S; Bonaventura, J; Bonaventura, C; Cashon, R

1985-01-01

We encountered an abnormal hemoglobin (Rahere), with a threonine residue replacing the beta 82 (EF6) lysine residue at the binding site of 2,3-diphosphoglycerate, which was responsible for overt erythrocytosis in two individuals of a Japanese family. Hemoglobin Rahere shows a lower oxygen affinity on the binding of 2,3-diphosphoglycerate or chloride ions than hemoglobin A. Although a decrease in the positive charge density at the binding sites of 2,3-diphosphoglycerate in hemoglobin Rahere apparently shifts the allosteric equilibrium toward the low affinity state, it greatly diminishes the cofactor effects by anions. The oxygen affinity of the patient's erythrocytes is substantially lowered by the presence of bezafibrate, which combines with sites different from those of 2,3-diphosphoglycerate in either hemoglobin Rahere or hemoglobin A. PMID:3930571

Copper(II) ions and the Alzheimer's amyloid-β peptide: Affinity and stoichiometry of binding

NASA Astrophysics Data System (ADS)

Tõugu, Vello; Friedemann, Merlin; Tiiman, Ann; Palumaa, Peep

2014-10-01

Deposition of amyloid beta (Aβ) peptides into amyloid plaques is the hallmark of Alzheimer's disease. According to the amyloid cascade hypothesis this deposition is an early event and primary cause of the disease, however, the mechanisms that cause this deposition remain elusive. An increasing amount of evidence shows that the interactions of biometals can contribute to the fibrillization and amyloid formation by amyloidogenic peptides. From different anions the copper ions deserve the most attention since it can contribute not only toamyloid formation but also to its toxicity due to the generation of ROS. In this thesis we focus on the affinity and stoichiometry of copper(II) binding to the Aβ molecule.

Sequence-specific binding of counterions to B-DNA

PubMed Central

Denisov, Vladimir P.; Halle, Bertil

2000-01-01

Recent studies by x-ray crystallography, NMR, and molecular simulations have suggested that monovalent counterions can penetrate deeply into the minor groove of B form DNA. Such groove-bound ions potentially could play an important role in AT-tract bending and groove narrowing, thereby modulating DNA function in vivo. To address this issue, we report here 23Na magnetic relaxation dispersion measurements on oligonucleotides, including difference experiments with the groove-binding drug netropsin. The exquisite sensitivity of this method to ions in long-lived and intimate association with DNA allows us to detect sequence-specific sodium ion binding in the minor groove AT tract of three B-DNA dodecamers. The sodium ion occupancy is only a few percent, however, and therefore is not likely to contribute importantly to the ensemble of B-DNA structures. We also report results of ion competition experiments, indicating that potassium, rubidium, and cesium ions bind to the minor groove with similarly weak affinity as sodium ions, whereas ammonium ion binding is somewhat stronger. The present findings are discussed in the light of previous NMR and diffraction studies of sequence-specific counterion binding to DNA. PMID:10639130

Uncertainty analysis of the nonideal competitive adsorption-donnan model: effects of dissolved organic matter variability on predicted metal speciation in soil solution.

PubMed

Groenenberg, Jan E; Koopmans, Gerwin F; Comans, Rob N J

2010-02-15

Ion binding models such as the nonideal competitive adsorption-Donnan model (NICA-Donnan) and model VI successfully describe laboratory data of proton and metal binding to purified humic substances (HS). In this study model performance was tested in more complex natural systems. The speciation predicted with the NICA-Donnan model and the associated uncertainty were compared with independent measurements in soil solution extracts, including the free metal ion activity and fulvic (FA) and humic acid (HA) fractions of dissolved organic matter (DOM). Potentially important sources of uncertainty are the DOM composition and the variation in binding properties of HS. HS fractions of DOM in soil solution extracts varied between 14 and 63% and consisted mainly of FA. Moreover, binding parameters optimized for individual FA samples show substantial variation. Monte Carlo simulations show that uncertainties in predicted metal speciation, for metals with a high affinity for FA (Cu, Pb), are largely due to the natural variation in binding properties (i.e., the affinity) of FA. Predictions for metals with a lower affinity (Cd) are more prone to uncertainties in the fraction FA in DOM and the maximum site density (i.e., the capacity) of the FA. Based on these findings, suggestions are provided to reduce uncertainties in model predictions.

Decorin is a Zn(2+) Metalloprotein

NASA Technical Reports Server (NTRS)

Yang, Vivian W.-C.; LaBrenz, Steven R.; Rosenberg, Lawrence C.; McQuillan, David; Hoeoek, Magnus

1998-01-01

Decorin is ubiquitously distributed in the extracellular matrix of mammals and a member of the proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. We here demonstrate that decorin extracted from bovine tissues under denaturing conditions or produced in recombinant "native" form by cultured mammalian cells, has a high affinity for Zn(2+). Binding of Zn(2+) to decorin is demonstrated by Zn(2+) chelating chromatography and equilibrium dialyses. The Zn(2+) binding sites are localized to the N-terminal domain of the core protein that contains 4 Cys residues in the spacing reminiscent of a Zn finger. A recombinant 41 amino acid long peptide representing the N-terminal domain of decorin has full Zn(2+) binding activity and binds two Zn(2+) ions with an average K(D) of 3 x 10(exp -7) M. Biglycan, a proteoglycan that is structurally closely related to decorin contains a similar high affinity Zn(2+) binding segment, whereas the structurally more distantly related proteoglycans, epiphycan and osteoglycin, did not bind Zn(2+) with high affinity.

Role of Conserved Glycine in Zinc-dependent Medium Chain Dehydrogenase/Reductase Superfamily*

PubMed Central

Tiwari, Manish Kumar; Singh, Raushan Kumar; Singh, Ranjitha; Jeya, Marimuthu; Zhao, Huimin; Lee, Jung-Kul

2012-01-01

The medium-chain dehydrogenase/reductase (MDR) superfamily consists of a large group of enzymes with a broad range of activities. Members of this superfamily are currently the subject of intensive investigation, but many aspects, including the zinc dependence of MDR superfamily proteins, have not yet have been adequately investigated. Using a density functional theory-based screening strategy, we have identified a strictly conserved glycine residue (Gly) in the zinc-dependent MDR superfamily. To elucidate the role of this conserved Gly in MDR, we carried out a comprehensive structural, functional, and computational analysis of four MDR enzymes through a series of studies including site-directed mutagenesis, isothermal titration calorimetry, electron paramagnetic resonance (EPR), quantum mechanics, and molecular mechanics analysis. Gly substitution by other amino acids posed a significant threat to the metal binding affinity and activity of MDR superfamily enzymes. Mutagenesis at the conserved Gly resulted in alterations in the coordination of the catalytic zinc ion, with concomitant changes in metal-ligand bond length, bond angle, and the affinity (Kd) toward the zinc ion. The Gly mutants also showed different spectroscopic properties in EPR compared with those of the wild type, indicating that the binding geometries of the zinc to the zinc binding ligands were changed by the mutation. The present results demonstrate that the conserved Gly in the GHE motif plays a role in maintaining the metal binding affinity and the electronic state of the catalytic zinc ion during catalysis of the MDR superfamily enzymes. PMID:22500022

Cooperative binding modes of Cu(II) in prion protein

NASA Astrophysics Data System (ADS)

Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

2007-03-01

The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

Absence of ion-binding affinity in the putatively inactivated low-[K+] structure of the KcsA potassium channel.

PubMed

Boiteux, Céline; Bernèche, Simon

2011-01-12

Potassium channels are membrane proteins that selectively conduct K(+) across cellular membranes. The narrowest part of their pore, the selectivity filter, is responsible for distinguishing K(+) from Na(+), and can also act as a gate through a mechanism known as C-type inactivation. It has been proposed that a conformation of the KcsA channel obtained by crystallization in presence of low concentration of K(+) (PDB 1K4D) could correspond to the C-type inactivated state. Here, we show using molecular mechanics simulations that such conformation has little ion-binding affinity and that ions do not contribute to its stability. The simulations suggest that, in this conformation, the selectivity filter is mostly occupied by water molecules. Whether such ion-free state of the KcsA channel is physiologically accessible and representative of the inactivated state of eukaryotic channels remains unclear. Copyright © 2011 Elsevier Ltd. All rights reserved.

Metal binding proteins, recombinant host cells and methods

DOEpatents

Summers, Anne O.; Caguiat, Jonathan J.

2004-06-15

The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

Na+ Interactions with the Neutral Amino Acid Transporter ASCT1*

PubMed Central

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

2014-01-01

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

The energy and work of a ligand-gated ion channel

PubMed Central

Auerbach, Anthony

2015-01-01

Ligand-gated ion channels are allosteric membrane proteins that isomerize between C(losed) and O(pen) conformations. A difference in affinity for ligands in the two shapes influences the C↔O ‘gating’ equilibrium constant. The energies associated with adult-type mouse neuromuscular nicotinic acetylcholine receptor-channel (AChR) gating have been measured by using single-channel electrophysiology. Without ligands the free energy, enthalpy and entropy of gating are ΔG0=+8.4, ΔH0=+10.9 and ΔS0=+2.4 kcal/mol (−100 mV, 23 °C). Many mutations throughout the protein change ΔG0, including natural ones that cause disease. Agonists and most mutations change approximately independently the ground state energy difference, so it is possible to forecast and engineer AChR responses simply by combining perturbations. The free energy of the low↔high affinity change for the neurotransmitter at each of two functionally-equivalent binding sites is ΔGBACh=−5.1 kcal/mol. ΔGBACh is set mainly by interactions of ACh with just three binding site aromatic groups. For a series of structurally-related agonists there is a correlation between the energies of low- and high-affinity binding, which implies that gating commences with the formation of the low affinity complex. Brief, intermediate states in binding and gating have been detected. Several proposals for the nature of the gating transition state energy landscape and the isomerization mechanism are discussed. PMID:23357172

High-aluminum-affinity silica is a nanoparticle that seeds secondary aluminosilicate formation.

PubMed

Jugdaohsingh, Ravin; Brown, Andy; Dietzel, Martin; Powell, Jonathan J

2013-01-01

Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7) we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP). Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m(2) g(-1) and it competes effectively with transferrin for Al(III) binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP) with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III) ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III) species binding monomeric silica to form early phase, non-toxic aluminosilicates.

High-Aluminum-Affinity Silica Is a Nanoparticle That Seeds Secondary Aluminosilicate Formation

PubMed Central

Jugdaohsingh, Ravin; Brown, Andy; Dietzel, Martin; Powell, Jonathan J.

2013-01-01

Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7) we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP). Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m2 g-1 and it competes effectively with transferrin for Al(III) binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP) with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III) ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III) species binding monomeric silica to form early phase, non-toxic aluminosilicates. PMID:24349573

Role of hydrogen bonding in ligand interaction with the N-methyl-D-aspartate receptor ion channel

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

Leeson, P.D.; Carling, R.W.; James, K.

1990-05-01

Displacement of (3H)MK-801 (dizocilpine, 1) binding to rat brain membranes has been used to evaluate the affinities of novel dibenzocycloalkenimines related to 1 for the ion channel binding site (also known as the phencyclidine or PCP receptor) on the N-methyl-D-aspartate (NMDA) subtype of excitory amino acid receptor. In common with many other agents having actions in the central nervous system, these compounds contain a hydrophobic aromatic moiety and a basic nitrogen atom. The conformational rigidity of these ligands provides a unique opportunity to evaluate the importance of specific geometrical properties that influence active-site recognition, in particular the role of themore » nitrogen atom in hydrogen-bonding interactions. The relative affinities (IC50s) of hydrocarbon-substituted analogues of 1 and ring homologated cyclooctenimines illustrate the importance of size-limited hydrophobic binding of both aryl rings and of the quaternary C-5 methyl group. Analysis of the binding of a series of the 10 available structurally rigid dibenzoazabicyclo(x.y.z)alkanes, by using molecular modeling techniques, uncovered a highly significant correlation between affinity and a proposed ligand-active site hydrogen bonding vector (r = 0.950, p less than 0.001). These results are used to generate a pharmacophore of the MK-801 recognition site/PCP receptor, which accounts for the binding of all of the known ligands.« less

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

USDA-ARS?s Scientific Manuscript database

ALDH2 catalyzes oxidation of toxic aldehydes to their corresponding carboxylic acids. Magnesium ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements have monitored the blue shift of the NADH fluorescence spectrum to elucidate the extent of...

Coordination Environment of a Site-Bound Metal Ion in the Hammerhead Ribozyme Determined by 15N and 2H ESEEM Spectroscopy

PubMed Central

Vogt, Matthew; Lahiri, Simanti; Hoogstraten, Charles G.; Britt, R. David; DeRose, Victoria J.

2010-01-01

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a Kd,app < 10 µM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin–echo envelope modulation) spectroscopy. Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with 15N guanine. The coordinated metal ion retains four water ligands as measured by 2H ESEEM spectroscopy. The results presented here show that a functionally important, specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength. Although it has a relatively high thermodynamic affinity, this ion remains partially hydrated and is chelated to the RNA by just two ligands. PMID:17177426

Equilibrium binding behavior of magnesium to wall teichoic acid.

PubMed

Thomas, Kieth J; Rice, Charles V

2015-10-01

Peptidoglycan and teichoic acids are the major cell wall components of Gram-positive bacteria that obtain and sequester metal ions required for biochemical processes. The delivery of metals to the cytoplasmic membrane is aided by anionic binding sites within the peptidoglycan and along the phosphodiester polymer of teichoic acid. The interaction with metals is a delicate balance between the need for attraction and ion diffusion to the membrane. Likewise, metal chelation from the extracellular fluid must initially have strong binding energetics that weaken within the cell wall to enable ion release. We employed atomic absorption and equilibrium dialysis to measure the metal binding capacity and metal binding affinity of wall teichoic acid and Mg2+. Data show that Mg2+ binds to WTA with a 1:2Mg2+ to phosphate ratio with a binding capacity of 1.27 μmol/mg. The affinity of Mg2+ to WTA was also found to be 41×10(3) M(-1) at low metal concentrations and 1.3×10(3) M(-1) at higher Mg2+ concentrations due to weakening electrostatic effects. These values are lower than the values describing Mg2+ interactions with peptidoglycan. However, the binding capacity of WTA is 4 times larger than peptidoglycan. External WTA initially binds metals with positive cooperativity, but metal binding switches to negative cooperativity, whereas interior WTA binds metals with only negative cooperativity. The relevance of this work is to describe changes in metal binding behavior depending on environment. When metals are sparse, chelation is strong to ensure survival yet the binding weakens when essential minerals are abundant. Copyright © 2015 Elsevier B.V. All rights reserved.

Density functional theory and conductivity studies of boron-based anion receptors

DOE PAGES

Leung, Kevin; Chaudhari, Mangesh I.; Rempe, Susan B.; ...

2015-07-10

Anion receptors that bind strongly to fluoride anions in organic solvents can help dissolve the lithium fluoride discharge products of primary carbon monofluoride (CFx) batteries, thereby preventing the clogging of cathode surfaces and improving ion conductivity. The receptors are also potentially beneficial to rechargeable lithium ion and lithium air batteries. We apply Density Functional Theory (DFT) to show that an oxalate-based pentafluorophenyl-boron anion receptor binds as strongly, or more strongly, to fluoride anions than many phenyl-boron anion receptors proposed in the literature. Experimental data shows marked improvement in electrolyte conductivity when this oxalate anion receptor is present. The receptor ismore » sufficiently electrophilic that organic solvent molecules compete with F – for boron-site binding, and specific solvent effects must be considered when predicting its F – affinity. To further illustrate the last point, we also perform computational studies on a geometrically constrained boron ester that exhibits much stronger gas-phase affinity for both F – and organic solvent molecules. After accounting for specific solvent effects, however, its net F – affinity is about the same as the simple oxalate-based anion receptor. Lastly, we propose that LiF dissolution in cyclic carbonate organic solvents, in the absence of anion receptors, is due mostly to the formation of ionic aggregates, not isolated F – ions.« less

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

USDA-ARS?s Scientific Manuscript database

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ...

[Relation between location of elements in periodic table and affinity for the malignant tumor (author's transl)].

PubMed

Ando, A; Hisada, K; Ando, I

1977-10-01

Affinity of many inorganic compounds for the malignant tumor was examined, using the rats which were subcutaneously transplanted with Yoshida sarcoma. And the relations between the uptake rate into the malignant tumor and in vitro binding power to the protein were investigated in these compounds. In these experiments, the bipositive ions and anions had not affinity for the tumor tissue with a few exceptions. On the other hand, Hg, Au and Bi, which have strong binding power to the protein, showed high uptake rate into the malignant tumor. As Hg++, Au+ and Bi+++ are soft acids according to classification of Lewis acids, it was thought that these elements would bind strongly to soft base (R-SH, R-S-) present in the tumor tissue. In many hard acids (according to classification of Lewis acids), the uptake rate into the tumor was shown as a function of ionic potentials (valency/ionic radii) of the metal ions. It is presumed that the chemical bond of these hard acids in the tumor tissue is ionic bond to hard base (R-COO-, R-PO3(2-), R-SO3-, R-NH2).

Probing the binding affinity of amyloids to reduce toxicity of oligomers in diabetes

PubMed Central

Smaoui, Mohamed Raef; Orland, Henri; Waldispühl, Jérôme

2015-01-01

Motivation: Amyloids play a role in the degradation of β-cells in diabetes patients. In particular, short amyloid oligomers inject themselves into the membranes of these cells and create pores that disrupt the strictly controlled flow of ions through the membranes. This leads to cell death. Getting rid of the short oligomers either by a deconstruction process or by elongating them into longer fibrils will reduce this toxicity and allow the β-cells to live longer. Results: We develop a computational method to probe the binding affinity of amyloid structures and produce an amylin analog that binds to oligomers and extends their length. The binding and extension lower toxicity and β-cell death. The amylin analog is designed through a parsimonious selection of mutations and is to be administered with the pramlintide drug, but not to interact with it. The mutations (T9K L12K S28H T30K) produce a stable native structure, strong binding affinity to oligomers, and long fibrils. We present an extended mathematical model for the insulin–glucose relationship and demonstrate how affecting the concentration of oligomers with such analog is strictly coupled with insulin release and β-cell fitness. Availability and implementation: SEMBA, the tool to probe the binding affinity of amyloid proteins and generate the binding affinity scoring matrices and R-scores is available at: http://amyloid.cs.mcgill.ca Contact: jeromew@cs.mcgill.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25777526

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications.

PubMed

Kimoto, Michiko; Nakamura, Mana; Hirao, Ichiro

2016-09-06

A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G-C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF165 unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF165 RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF165 Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF165 and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF165 DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF165. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular Dynamics Simulations of Protein-Ligand Complexes in Near Physiological Conditions

NASA Astrophysics Data System (ADS)

Wambo, Thierry Oscar

Proteins are important molecules for their key functions. However, under certain circumstances, the function of these proteins needs to be regulated to keep us healthy. Ligands are small molecules often used to modulate the function of proteins. The binding affinity is a quantitative measure of how strong the ligand will modulate the function of the protein: a strong binding affinity will highly impact the performance of the protein. It becomes clear that it is critical to have appropriate techniques to accurately compute the binding affinity. The most difficult task in computer simulations is how to efficiently sample the space spanned by the ligand during the binding process. In this work, we have developed some schemes to compute the binding affinity of a ligand to a protein, and of a metal ion to a protein. Application of these techniques to some complexes yield results in agreement with experimental values. These methods are a brute force approach and make no assumption other than that the complexes are governed by the force field used. Specifically, we computed the free energy of binding between (1) human carbonic anhydrase II and the drug acetazolamide (hcaII-AZM), (2) human carbonic anhydrase II and the zinc ion (hcaII-Zinc), and (3) beta-lactoglobulin and five fatty acids complexes (BLG-FAs). We found the following free energies of binding in unit of kcal/mol: -12.96 +/-2.44 (-15.74) for hcaII-Zinc complex, -5.76+/-0.76 (-5.57) for BLG-OCA , -4.44+/-1.08 (-5.22) for BLG-DKA,-6.89+/-1.25 (-7.24) for BLG-DAO, -8.57+/-0.82 (-8.14) for BLG-MYR, -8.99+/-0.87 (-8.72) for BLG-PLM, and -11.87+/-1.8 (-10.8) for hcaII-AZM. The values inside the parentheses are experimental results. The simulations and quantitative analysis of each system provide interesting insights into the interactions between each entity and helps us to better understand the dynamics of these systems.

Cation binding to a Bacillus (1,3-1,4)-beta-glucanase. Geometry, affinity and effect on protein stability.

PubMed

Keitel, T; Meldgaard, M; Heinemann, U

1994-05-15

The hybrid Bacillus (1,3-1,4)-beta-glucanase H(A16-M), consisting of 16 N-terminal amino acids derived from the mature form of the B. amyloliquefaciens enzyme and of 198 C-proximal amino acids from the B. macerans enzyme, binds a calcium ion at a site at its molecular surface remote from the active center [T. Keitel, O. Simon, R. Borriss & U. Heinemann (1993) Proc. Natl Acad. Sci. USA 90, 5287-5291]. X-ray diffraction analysis at 0.22-nm resolution of crystals grown in the absence of calcium and in the presence of EDTA shows this site to be occupied by a sodium ion. Whereas the calcium ion has six oxygen atoms in its coordination sphere, two of which are from water molecules, sodium is fivefold coordinated with a fifth ligand belonging to a symmetry-related protein molecule in the crystal lattice. The affinity of H(A16-M) for calcium over sodium has been determined calorimetrically. Calcium binding stabilizes the native three-dimensional structure of the protein as shown by guanidinium chloride unfolding and thermal inactivation experiments. The enhanced enzymic activity of Bacillus beta-glucanases at elevated temperatures in the presence of calcium ions is attributed to a general stabilizing effect by the cation.

Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

PubMed

Hu, Meng-Xin; Li, Xiang; Li, Ji-Nian; Huang, Jing-Jing; Ren, Ge-Rui

2018-02-23

Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m 2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

PubMed

Abdullah, Nazish; Padmanarayana, Murugesh; Marty, Naomi J; Johnson, Colin P

2014-01-21

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Ion-binding properties of Calnuc, Ca2+ versus Mg2+--Calnuc adopts additional and unusual Ca2+-binding sites upon interaction with G-protein.

PubMed

Kanuru, Madhavi; Samuel, Jebakumar J; Balivada, Lavanya M; Aradhyam, Gopala K

2009-05-01

Calnuc is a novel, highly modular, EF-hand containing, Ca(2+)-binding, Golgi resident protein whose functions are not clear. Using amino acid sequences, we demonstrate that Calnuc is a highly conserved protein among various organisms, from Ciona intestinalis to humans. Maximum homology among all sequences is found in the region that binds to G-proteins. In humans, it is known to be expressed in a variety of tissues, and it interacts with several important protein partners. Among other proteins, Calnuc is known to interact with heterotrimeric G-proteins, specifically with the alpha-subunit. Herein, we report the structural implications of Ca(2+) and Mg(2+) binding, and illustrate that Calnuc functions as a downstream effector for G-protein alpha-subunit. Our results show that Ca(2+) binds with an affinity of 7 mum and causes structural changes. Although Mg(2+) binds to Calnuc with very weak affinity, the structural changes that it causes are further enhanced by Ca(2+) binding. Furthermore, isothermal titration calorimetry results show that Calnuc and the G-protein bind with an affinity of 13 nm. We also predict a probable function for Calnuc, that of maintaining Ca(2+) homeostasis in the cell. Using Stains-all and terbium as Ca(2+) mimic probes, we demonstrate that the Ca(2+)-binding ability of Calnuc is governed by the activity-based conformational state of the G-protein. We propose that Calnuc adopts structural sites similar to the ones seen in proteins such as annexins, c2 domains or chromogrannin A, and therefore binds more calcium ions upon binding to Gialpha. With the number of organelle-targeted G-protein-coupled receptors increasing, intracellular communication mediated by G-proteins could become a new paradigm. In this regard, we propose that Calnuc could be involved in the downstream signaling of G-proteins.

Melanin-Based Coatings as Lead-Binding Agents

PubMed Central

Sono, Karin; Lye, Diane; Moore, Christine A.; Boyd, W. Christopher; Gorlin, Thomas A.; Belitsky, Jason M.

2012-01-01

Interactions between metal ions and different forms of melanin play significant roles in melanin biochemistry. The binding properties of natural melanin and related synthetic materials can be exploited for nonbiological applications, potentially including water purification. A method for investigating metal ion-melanin interactions on solid support is described, with lead as the initial target. 2.5 cm discs of the hydrophobic polymer PVDF were coated with synthetic eumelanin from the tyrosinase-catalyzed polymerization of L-dopa, and with melanin extracted from human hair. Lead (Pb2+) binding was quantified by atomic absorption spectroscopy (flame mode), and the data was well fit by the Langmuir model. Langmuir affinities ranged from 3.4 · 103 to 2.2 · 104 M−1. At the maximum capacity observed, the synthetic eumelanin coating bound ~9% of its mass in lead. Binding of copper (Cu2+), zinc (Zn2+), and cadmium (Cd2+) to the synthetic-eumelanin-coated discs was also investigated. Under the conditions tested, the Langmuir affinities for Zn2+, Cd2+, and Cu2+ were 35%, 53%, and 77%, respectively, of the Langmuir affinity for Pb2+. The synthetic-eumelanin-coated discs have a slightly higher capacity for Cu2+ on a per mole basis than for Pb2+, and lower capacities for Cd2+ and Zn2+. The system described can be used to address biological questions and potentially be applied toward melanin-based water purification. PMID:22611345

Structural Basis of Low-Affinity Nickel Binding to the Nickel-Responsive Transcription Factor NikR from Escherichia coli†‡

PubMed Central

2010-01-01

Escherichia coli NikR regulates cellular nickel uptake by binding to the nik operon in the presence of nickel and blocking transcription of genes encoding the nickel uptake transporter. NikR has two binding affinities for the nik operon: a nanomolar dissociation constant with stoichiometric nickel and a picomolar dissociation constant with excess nickel [Bloom, S. L., and Zamble, D. B. (2004) Biochemistry 43, 10029−10038; Chivers, P. T., and Sauer, R. T. (2002) Chem. Biol. 9, 1141−1148]. While it is known that the stoichiometric nickel ions bind at the NikR tetrameric interface [Schreiter, E. R., et al. (2003) Nat. Struct. Biol. 10, 794−799; Schreiter, E. R., et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13676−13681], the binding sites for excess nickel ions have not been fully described. Here we have determined the crystal structure of NikR in the presence of excess nickel to 2.6 Å resolution and have obtained nickel anomalous data (1.4845 Å) in the presence of excess nickel for both NikR alone and NikR cocrystallized with a 30-nucleotide piece of double-stranded DNA containing the nik operon. These anomalous data show that excess nickel ions do not bind to a single location on NikR but instead reveal a total of 22 possible low-affinity nickel sites on the NikR tetramer. These sites, for which there are six different types, are all on the surface of NikR, and most are found in both the NikR alone and NikR−DNA structures. Using a combination of crystallographic data and molecular dynamics simulations, the nickel sites can be described as preferring octahedral geometry, utilizing one to three protein ligands (typically histidine) and at least two water molecules. PMID:20704276

The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

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

Humphreys, C.J.

1989-01-01

The plasmalemmal serotonin transporter uses transmembrane gradients of Na{sup +}, Cl{sup {minus}} and K{sup +} to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na{sup +}. Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na{sup +} and Cl{sup {minus}}, the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized,more » purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na{sup +} binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl{sup {minus}}. Cl{sup {minus}} enhances the transporters affinity for imipramine, as well as for Na{sup +}. At concentrations in the range of its K{sub M} for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na{sup +}-independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both ({sup 3}H)imipramine binding and ({sup 3}H)serotonin transport.« less

Dihydrogenphosphate recognition: Assistance from the acidic OH moiety of the anion

NASA Astrophysics Data System (ADS)

Das, Rituraj; Pathak, Nibedan; Choudhury, Samarjit; Borah, Suchibrata; Mahanta, Sanjeev Pran

2017-11-01

The binding affinity of the acidic hydrogen i.e. OH moiety of dihydrogenphosphate was investigated with receptors having competent H-bond donor and H-bond acceptor component. Three derivatives of 2, 3-dipyrrol-2‧-ylquinoxaline substituted with H-bond acceptor moiety at pyrrole α- positions were synthesized and their dihydrogenphosphate affinity was studied. All the three receptors shows general affinity towards fluoride, acetate and cyanide ions in DMSO solution. Interestingly, formyl substitution at both the pyrrole α-positions of 2, 3-dipyrrol-2‧-ylquinoxaline leads to binding of H2PO4-. 1H-NMR study rules out the involvement of the H-bond donor unit of the receptor in the biding event and reveals that the binding occurs predominantly via the Osbnd H⋯O interaction between the acidic OH moiety of the anion and the Cdbnd O of the formyl group of the receptor.

Graphane versus graphene: a computational investigation of the interaction of nucleobases, aminoacids, heterocycles, small molecules (CO2, H2O, NH3, CH4, H2), metal ions and onium ions.

PubMed

Umadevi, Deivasigamani; Narahari Sastry, G

2015-11-11

Graphane has emerged as a two-dimensional hydrocarbon with interesting physical properties and potential applications. Understanding the interaction of graphane with various molecules and ions is crucial to appreciate its potential applications. We investigated the interaction of nucleobases, aminoacids, saturated and unsaturated heterocycles, small molecules, metal ions and onium ions with graphane by using density functional theory calculations. The preferred orientations of these molecules and ions on the graphane surface have been analysed. The binding energies of graphane with these molecules have been compared with the corresponding binding energies of graphene. Our results reveal that graphane forms stable complexes with all the molecules and ions yet showing lesser binding affinity when compared to graphene. As an exemption, the preferential strong binding of H2O with graphane than graphene reveals the fact that graphane is more hydrophilic than graphene. Charge transfer between graphane and the molecules and ions have been found to be an important factor in determining the binding strength of the complexes. The effect of the interaction of these molecules and ions on the HOMO-LUMO energy gap of graphane has also been investigated.

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium.

PubMed

Cheng, Huaixu; Luo, Zhidan; Lu, Mingsheng; Gao, Song; Wang, Shujun

2017-05-01

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium ions for thermostability, and is a promising alternative to commercially available α-amylases to increase the efficiency of industrial processes like the liquefaction of starch. We analyzed the amino acid sequence of this α-amylase by sequence alignments and structural modeling, and found that this α-amylase closely resembles the α-amylase from Pyrococcus woesei. The gene of this α-amylase was cloned in Escherichia coli and the recombinant α-amylase was overexpressed and purified with a combined renaturation-purification procedure. We confirmed thermostability and exogenous calcium ion independency of the recombinant α-amylase and further investigated the mechanism of the independency using biochemical approaches. The results suggested that the α-amylase has a high calcium ion binding affinity that traps a calcium ion that would not dissociate at high temperatures, providing a direct explanation as to why the addition of calcium ions is not required for thermostability. Understanding of the mechanism offers a strong base on which to further engineer properties of this α-amylase for better potential applications in industrial processes.

Interaction of ibogaine with human α3β4-nicotinic acetylcholine receptors in different conformational states

PubMed Central

Arias, Hugo R.; Rosenberg, Avraham; Targowska-Duda, Katarzyna M.; Feuerbach, Dominik; Yuan, Xiao Juan; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W.

2015-01-01

The interaction of ibogaine and phencyclidine (PCP) with human (h) α3β4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca2+ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (±)-epibatidine-induced Ca2+ influx in hα3β4 AChRs with ~9-fold higher potency than that for PCP, (b) [3H]ibogaine binds to a single site in the hα3β4 AChR ion channel with relatively high affinity (Kd = 0.46 ± 0.06 µM), and ibogaine inhibits [3H]ibogaine binding to the desensitized hα3β4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [3H]ibogaine binding to the hα3β4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6′) and valine/phenylalanine (position 13′) rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time. PMID:20684041

Interaction of ibogaine with human alpha3beta4-nicotinic acetylcholine receptors in different conformational states.

PubMed

Arias, Hugo R; Rosenberg, Avraham; Targowska-Duda, Katarzyna M; Feuerbach, Dominik; Yuan, Xiao Juan; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W

2010-09-01

The interaction of ibogaine and phencyclidine (PCP) with human (h) alpha3beta4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca2+ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (+/-)-epibatidine-induced Ca2+ influx in h(alpha)3beta4 AChRs with approximately 9-fold higher potency than that for PCP, (b) [3H]ibogaine binds to a single site in the h(alpha)3beta4 AChR ion channel with relatively high affinity (Kd = 0.46 +/- 0.06 microM), and ibogaine inhibits [3H]ibogaine binding to the desensitized h(alpha)3beta4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [3H]ibogaine binding to the h(alpha)3beta4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6') and valine/phenylalanine (position 13') rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time.

Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

PubMed

Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

2015-01-01

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

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

PubMed Central

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

2013-01-01

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

New tris(dopamine) derivative as an iron chelator. Synthesis, solution thermodynamic stability, and antioxidant research.

PubMed

Zhang, Qingchun; Jin, Bo; Shi, Zhaotao; Wang, Xiaofang; Lei, Shan; Tang, Xingyan; Liang, Hua; Liu, Qiangqiang; Gong, Mei; Peng, Rufang

2017-06-01

A new tris(dopamine) derivative, containing three dopamine chelate moieties which were attached to a trimesic acid molecular scaffold, has been prepared and fully characterized by NMR, FTIR and HRMS. The solution thermodynamic stability of the chelator with Fe(III), Mg(II), Zn(II) and Fe(II) ions was investigated. Results demonstrated that the chelator exhibited effective binding ability and improved selectivity to Fe(III) ion. The chelator possessed affinity similar to that of diethylenetriaminepentaacetic acid chelator for Fe(III) ion. The high affinity could be attributed to the favorable geometric arrangement between the chelator and Fe(III) ion coordination preference. The chelator also exhibited high antioxidant activity and nontoxicity to neuron-like rat pheochromocytoma cells. Hence, the chelator could be used as chelating agent for iron overload situations without depleting essential metal ions, such as Mg(II) and Zn(II) ions. Copyright © 2017. Published by Elsevier Inc.

Hemoglobin

DTIC Science & Technology

1993-03-08

affinity, which is less at low levels of hemoglobin saturation, increases markedly as fractional saturation increases. Thus, high affinity for 02 at... diphosphoglycerate (2,3-DPG), and carbon dioxide (Co 2). Since they are linked to 02 binding, they are called oxygen-linked effectors. The oxygen...hemoglobin molecule because of the negative charge of the ions. 2,3- Diphosphoglycerate is a molecule formed during the breakdown of sugar in normal human

Inhibition of ferric ion to oxalate oxidase shed light on the substrate binding site.

PubMed

Pang, Yu; Lan, Wanjun; Huang, Xuelei; Zuo, Guanke; Liu, Hui; Zhang, Jingyan

2015-10-01

Oxalate oxidase (OxOx), a well known enzyme catalyzes the cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide, however its catalytic process is not well understood. To define the substrate binding site, interaction of Fe(3+) ions with OxOx was systemically investigated using biochemical method, circular dichrosim spectroscopy, microscale thermophoresis, and computer modeling. We demonstrated that Fe(3+) is a non-competitive inhibitor with a milder binding affinity to OxOx, and the secondary structure of the OxOx was slightly altered upon its binding. On the basis of the structural properties of the OxOx and its interaction with Fe(3+) ions, two residue clusters of OxOx were assigned as potential Fe(3+) binding sites, the mechanism of the inhibition of Fe(3+) was delineated. Importantly, the residues that interact with Fe(3+) ions are involved in the substrate orienting based on computer docking. Consequently, the interaction of OxOx with Fe(3+) highlights insight into substrate binding site in OxOx.

Ion imprinted polymeric nanoparticles for selective separation and sensitive determination of zinc ions in different matrices

NASA Astrophysics Data System (ADS)

Shamsipur, Mojtaba; Rajabi, Hamid Reza; Pourmortazavi, Seied Mahdi; Roushani, Mahmoud

2014-01-01

Preparation of Zn2+ ion-imprinted polymer (Zn-IIP) nanoparticles is presented in this report. The Zn-IIP nanoparticles are prepared by dissolving stoichiometric amounts of zinc nitrate and selected chelating ligand, 3,5,7,20,40-pentahydroxyflavone, in 15 mL ethanol-acetonitrile (2:1; v/v) mixture as a porogen solvent in the presence of ethylene glycol-dimethacrylate (EGDMA) as cross-linking, methacrylic acid (MAA) as functional monomer, and 2,2-azobisisobutyronitrile (AIBN) as initiator. After polymerization, Cavities in the polymer particles corresponding to the Zn2+ ions were created by leaching the polymer in HCl aqueous solution. The synthesized IIPs were characterized by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, fluorescence spectroscopy and thermal analysis techniques. Also, the pH range for rebinding of Zn2+ ion on the IIP and equilibrium binding time were optimized, using flame atomic absorption spectrometry. In selectivity study, it was found that imprinting results increased affinity of the material toward Zn2+ ion over other competitor metal ions with the same charge and close ionic radius. The prepared IIPs were repeatedly used and regenerated for six times without any significant decrease in polymer binding affinities. Finally, the prepared sorbent was successfully applied to the selective recognition and determination of zinc ion in different real samples.

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

PubMed Central

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

2017-01-01

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

Binding of perlecan to transthyretin in vitro.

PubMed Central

Smeland, S; Kolset, S O; Lyon, M; Norum, K R; Blomhoff, R

1997-01-01

Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein-glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions. PMID:9307034

UO₂²⁺ uptake by proteins: understanding the binding features of the super uranyl binding protein and design of a protein with higher affinity.

PubMed

Odoh, Samuel O; Bondarevsky, Gary D; Karpus, Jason; Cui, Qiang; He, Chuan; Spezia, Riccardo; Gagliardi, Laura

2014-12-17

The capture of uranyl, UO2(2+), by a recently engineered protein (Zhou et al. Nat. Chem. 2014, 6, 236) with high selectivity and femtomolar sensitivity has been examined by a combination of density functional theory, molecular dynamics, and free-energy simulations. It was found that UO2(2+) is coordinated to five carboxylate oxygen atoms from four amino acid residues of the super uranyl binding protein (SUP). A network of hydrogen bonds between the amino acid residues coordinated to UO2(2+) and residues in its second coordination sphere also affects the protein's uranyl binding affinity. Free-energy simulations show how UO2(2+) capture is governed by the nature of the amino acid residues in the binding site, the integrity and strength of the second-sphere hydrogen bond network, and the number of water molecules in the first coordination sphere. Alteration of any of these three factors through mutations generally results in a reduction of the binding free energy of UO2(2+) to the aqueous protein as well as of the difference between the binding free energies of UO2(2+) and other ions (Ca(2+), Cu(2+), Mg(2+), and Zn(2+)), a proxy for the protein's selectivity over these ions. The results of our free-energy simulations confirmed the previously reported experimental results and allowed us to discover a mutant of SUP, specifically the GLU64ASP mutant, that not only binds UO2(2+) more strongly than SUP but that is also more selective for UO2(2+) over other ions. The predictions from the computations were confirmed experimentally.

Understanding Ion Binding Affinity and Selectivity in β-Parvalbumin Using Molecular Dynamics and Mean Spherical Approximation Theory.

PubMed

Kucharski, Amir N; Scott, Caitlin E; Davis, Jonathan P; Kekenes-Huskey, Peter M

2016-08-25

Parvalbumin (PV) is a globular calcium (Ca(2+))-selective protein expressed in a variety of biological tissues. Our computational studies of the rat β-parvalbumin (β-PV) isoform seek to elucidate the molecular thermodynamics of Ca(2+) versus magnesium (Mg(2+)) binding at the protein's two EF-hand motifs. Specifically, we have utilized molecular dynamics (MD) simulations and a mean-field electrolyte model (mean spherical approximation (MSA) theory) to delineate how the EF-hand scaffold controls the "local" thermodynamics of Ca(2+) binding selectivity over Mg(2+). Our MD simulations provide the probability density of metal-chelating oxygens within the EF-hand scaffolds for both Ca(2+) and Mg(2+), as well the conformational strain induced by Mg(2+) relative to Ca(2+) binding. MSA theory utilizes the binding domain oxygen and charge distributions to predict the chemical potential of ion binding, as well as their corresponding concentrations within the binding domain. We find that the electrostatic and steric contributions toward ion binding were similar for Mg(2+) and Ca(2+), yet the latter was 5.5 kcal/mol lower in enthalpy when internal strain within the EF hand was considered. We therefore speculate that beyond differences in dehydration energies for the Ca(2+) versus Mg(2+), strain induced in the β-PV EF hand by cation binding significantly contributes to the nearly 10,000-fold difference in binding affinity reported in the literature. We further complemented our analyses of local factors governing cation binding selectivity with whole-protein (global) contributions, such as interhelical residue-residue contacts and solvent exposure of hydrophobic surface. These contributions were found to be comparable for both Ca(2+)- and Mg(2+)-bound β-PV, which may implicate local factors, EF-hand strain, and dehydration, in providing the primary means of selectivity. We anticipate these methods could be used to estimate metal binding thermodynamics across a broad range of PV sequence homologues and EF-hand-containing, Ca(2+) binding proteins.

Understanding the Role of Metal Ions in RNA Folding and Function: Lessons from RNase P, a Ribonucleoprotein Enzyme

NASA Astrophysics Data System (ADS)

Harris, Michael E.; Christian, Eric L.

There is a large and rapidly growing literature relating RNA function to metal ion identity and concentration; however, due to the complexity and large number of interactions it remains a significant experimental challenge to tie the interactions of individual ions to specific aspects of RNA function. Investigation of the ribonculeopro-tein enzyme RNase P function has assisted in defining characteristics of RNA—metal ion interactions and provided a useful model system for understanding RNA catalysis and ribonucleoprotein assembly. The goal of this chapter is to review progress in understanding the physical basis of functional metal ion interactions with P RNA and relate this progress to the development of our understanding of RNA metal ion interactions in general. The research results reviewed here encompass: (1) Determination of the contribution of divalent metal ion binding to specific aspects of enzyme function, (2) Identification of individual metal ion binding sites in P RNA and their contribution to function, and (3) The effect of protein binding on RNA—metal ion affinity.

Binding of Alkali Metal Ions with 1,3,5-Tri(phenyl)benzene and 1,3,5-Tri(naphthyl)benzene: The Effect of Phenyl and Naphthyl Ring Substitution on Cation-π Interactions Revealed by DFT Study.

PubMed

Mirchi, Ali; Sizochenko, Natalia; Dinadayalane, Tandabany; Leszczynski, Jerzy

2017-11-22

The effect of substitution of phenyl and naphthyl rings to benzene was examined to elucidate the cation-π interactions involving alkali metal ions with 1,3,5-tri(phenyl)benzene (TPB) and 1,3,5-tri(naphthyl)benzene (TNB). Benzene, TPB, and four TNB isomers (with ααα, ααβ, αββ, and βββ types of fusion) and their complexes with Li + , Na + , K + , Rb + , and Cs + were optimized using DFT approach with B3LYP and M06-2X functionals in conjunction with the def2-QZVP basis set. Higher relative stability of β,β,β-TNB over α,α,α-TNB can be attributed to peri repulsion, which is defined as the nonbonding repulsive interaction between substituents in the 1- and the 8-positions on the naphthalene core. Binding energies, distances between ring centroid and the metal ions, and the distance to metal ions from the center of other six-membered rings were compared for all complexes. Our computational study reveals that the binding affinity of alkali metal cations increases significantly with the 1,3,5-trisubstitution of phenyl and naphthyl rings to benzene. The detailed computational analyses of geometries, partial charges, binding energies, and ligand organization energies reveal the possibility of favorable C-H···M + interactions when a α-naphthyl group exists in complexes of TNB structures. Like benzene-alkali metal ion complexes, the binding affinity of metal ions follows the order: Li + > Na + > K + > Rb + > Cs + for any considered 1,3,5-trisubstituted benzene systems. In case of TNB, we found that the strength of interactions increases as the fusion point changes from α to β position of naphthalene.

Synthesis, characterization and application of ion imprinted polymeric nanobeads for highly selective preconcentration and spectrophotometric determination of Ni2 + ion in water samples

NASA Astrophysics Data System (ADS)

Rajabi, Hamid Reza; Razmpour, Saham

2016-01-01

Here, the researchers report on the synthesis of ion imprinted polymeric (IIP) nanoparticles using a thermal polymerization strategy, and their usage for the separation of Ni2 + ion from water samples. The prepared Ni-IIP was characterized by colorimetry, FT-IR spectroscopy, and scanning electron microscopy. It was found that the particle size of the prepared particle to be 50-70 nm in diameter with the highly selective binding capability for Ni2 + ion, with reasonable adsorption and desorption process. After preconcentration, bound ions can be eluted with an aqueous solution of hydrochloric acid, after their complexation with dimethylglyoxime, these ions can be quantified by UV-Vis absorption spectrophotometry. The effect of various parameters on the extraction efficiency including pH of sample solution, adsorption and leaching times, initial sample volume, concentration and volume of eluent were investigated. In selectivity study, it was found that imprinting causes increased affinity of the prepared IIP toward Ni2 + ion over other ions such as Na+, K+, Ag+, Co2 +, Cu2 +, Cd2 +, Hg2 +, Pb2 +, Zn2 +, Mn2 +, Mg2 +, Cr3 +, and Fe3 +. The prepared IIP can be used and regenerated for at least eight times without any significant decrease in binding affinities. The prepared IIP is considered to be promising and selective sorbent for solid-phase extraction and preconcentration of Ni2 + ion from different water samples.

Modulation of Conformational Equilibria in the S-Adenosylmethionine (SAM) II Riboswitch by SAM, Mg(2+), and Trimethylamine N-Oxide.

PubMed

McPhie, Peter; Brown, Patrick; Chen, Bin; Dayie, Theodore K; Minton, Allen P

2016-09-13

The dependence of the conformation of the S-adenosylmethionine (SAM) II riboswitch on the concentration of added Mg(2+) ions and SAM, individually and in mixtures, was monitored by circular dichroism (CD) spectroscopy and by measurement of the diffusion coefficient. The results are analyzed in the context of two complementary quantitative models, both of which are consistent with a single underlying physical model. Magnesium binding sites in the open state have an affinity on average higher than the affinity of those in the compact state, but formation of the compact state is accompanied by an increase in the number of binding sites. Consequently, at low Mg(2+) concentrations, Mg(2+) binds preferentially to the open state, favoring its formation, but at high concentrations, Mg(2+) binds preferentially to the compact state. The affinity of the riboswitch for SAM increases drastically with an increased level of binding of Mg(2+) to the compact pseudoknot conformation. The effect of increasing concentrations of trimethylamine N-oxide (TMAO), a well-studied molecular crowding agent, on the conformation of the riboswitch and its affinity for SAM were also monitored by CD spectroscopy and measurement of diffusion. In the absence of added Mg(2+), high concentrations of TMAO were found to induce a conformational change compatible with the formation of the pseudoknot form but have only a small effect on the affinity of the RNA for SAM.

DNA sensing by a Eu-binding peptide containing a proflavine unit.

PubMed

Ancel, Laetitia; Gateau, Christelle; Lebrun, Colette; Delangle, Pascale

2013-01-18

Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.

Analysis of the actions of the novel dopamine receptor-directed compounds (S)-OSU6162 and ACR16 at the D2 dopamine receptor

PubMed Central

Kara, Elodie; Lin, Hong; Svensson, Kjell; Johansson, Anette M; Strange, Philip G

2010-01-01

BACKGROUND AND PURPOSE The two phenylpiperidines, OSU6162 and ACR16, have been proposed as novel drugs for the treatment of brain disorders, including schizophrenia and Huntington's disease, because of their putative dopamine stabilizing effects. Here we evaluated the activities of these compounds in a range of assays for the D2 dopamine receptor in vitro. EXPERIMENTAL APPROACH The affinities of these compounds for the D2 dopamine receptor were evaluated in competition with [3H]spiperone and [3H]NPA. Agonist activity of these compounds was evaluated in terms of their ability to stimulate [35S]GTPγS binding. KEY RESULTS Both compounds had low affinities for inhibition of [3H]spiperone binding (pKi vs. [3H]spiperone, ACR16: <5, OSU6162: 5.36). Neither compound was able to stimulate [35S]GTPγS binding when assayed in the presence of Na+ ions, but if the Na+ ions were removed, both compounds were low-affinity, partial agonists (Emax relative to dopamine: ACR16: 10.2%, OSU6162:54.3%). Schild analysis of the effects of OSU6162 to inhibit dopamine-stimulated [35S]GTPγS binding indicated Schild slopes of ∼0.9, suggesting little deviation from competitive inhibition. OSU6162 was, however, able to accelerate [3H]NPA dissociation from D2 dopamine receptors, indicating some allosteric effects of this compound. CONCLUSIONS AND IMPLICATIONS The two phenylpiperidines were low-affinity, low-efficacy partial agonists at the D2 dopamine receptor in vitro, possibly exhibiting some allosteric effects. Comparing their in vitro and in vivo effects, the in vitro affinities were a reasonable guide to potencies in vivo. However, the lack of in vitro–in vivo correlation for agonist efficacy needs to be further addressed. PMID:20804495

High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses.

PubMed

Nürnberger, T; Nennstiel, D; Jabs, T; Sacks, W R; Hahlbrock, K; Scheel, D

1994-08-12

An oligopeptide of 13 amino acids (Pep-13) identified within a 42 kDa glycoprotein elicitor from P. mega-sperma was shown to be necessary and sufficient to stimulate a complex defense response in parsley cells comprising H+/Ca2+ influxes, K+/Cl- effluxes, an oxidative burst, defense-related gene activation, and phytoalexin formation. Binding of radiolabeled Pep-13 to parsley microsomes and protoplasts was specific, reversible, and saturable. Identical structural features of Pep-13 were found to be responsible for specific binding and initiation of all plant responses analyzed. The high affinity binding site recognizing the peptide ligand (KD = 2.4 nM) may therefore represent a novel class of receptors in plants, and the rapidly induced ion fluxes may constitute elements of the signal transduction cascade triggering pathogen defense in plants.

Highly sensitive and selective detection of Al(III) ions in aqueous buffered solution with fluorescent peptide-based sensor.

PubMed

In, Byunggyu; Hwang, Gi Won; Lee, Keun-Hyeung

2016-09-15

A fluorescent sensor based on a tripeptide (SerGluGlu) with a dansyl fluorophore detected selectively Al(III) among 16 metal ions in aqueous buffered solutions without any organic cosolvent. The peptide-based sensor showed a highly sensitive turn on response to aluminium ion with high binding affinity (1.84×10(4)M(-1)) in aqueous buffered solutions. The detection limit (230nM, 5.98ppb) of the peptide-based sensor was much lower than the maximum allowable level (7.41μM) of aluminium ions in drinking water demanded by EPA. The binding mode of the peptide sensor with aluminium ions was characterized using ESI mass spectrometry, NMR titration, and pH titration experiments. Copyright © 2016 Elsevier Ltd. All rights reserved.

Free energy calculations of glycosaminoglycan-protein interactions.

PubMed

Gandhi, Neha S; Mancera, Ricardo L

2009-10-01

Glycosaminoglycans (GAGs) are complex highly charged linear polysaccharides that have a variety of roles in biological processes. We report the first use of molecular dynamics (MD) free energy calculations using the MM/PBSA method to investigate the binding of GAGs to protein molecules, namely the platelet endothelial cell adhesion molecule 1 (PECAM-1) and annexin A2. Calculations of the free energy of the binding of heparin fragments of different sizes reveal the existence of a region of low GAG-binding affinity in domains 5-6 of PECAM-1 and a region of high affinity in domains 2-3, consistent with experimental data and ligand-protein docking studies. A conformational hinge movement between domains 2 and 3 was observed, which allows the binding of heparin fragments of increasing size (pentasaccharides to octasaccharides) with an increasingly higher binding affinity. Similar simulations of the binding of a heparin fragment to annexin A2 reveal the optimization of electrostatic and hydrogen bonding interactions with the protein and protein-bound calcium ions. In general, these free energy calculations reveal that the binding of heparin to protein surfaces is dominated by strong electrostatic interactions for longer fragments, with equally important contributions from van der Waals interactions and vibrational entropy changes, against a large unfavorable desolvation penalty due to the high charge density of these molecules.

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

PubMed

Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

2014-11-01

Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

Physiological sodium concentrations enhance the iodide affinity of the Na+/I- symporter

NASA Astrophysics Data System (ADS)

Nicola, Juan P.; Carrasco, Nancy; Mario Amzel, L.

2014-06-01

The Na+/I- symporter (NIS) mediates active I- transport—the first step in thyroid hormonogenesis—with a 2Na+:1I- stoichiometry. NIS-mediated 131I- treatment of thyroid cancer post-thyroidectomy is the most effective targeted internal radiation cancer treatment available. Here to uncover mechanistic information on NIS, we use statistical thermodynamics to obtain Kds and estimate the relative populations of the different NIS species during Na+/anion binding and transport. We show that, although the affinity of NIS for I- is low (Kd=224 μM), it increases when Na+ is bound (Kd=22.4 μM). However, this Kd is still much higher than the submicromolar physiological I- concentration. To overcome this, NIS takes advantage of the extracellular Na+ concentration and the pronounced increase in its own affinity for I- and for the second Na+ elicited by binding of the first. Thus, at physiological Na+ concentrations, ~79% of NIS molecules are occupied by two Na+ ions and ready to bind and transport I-.

The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region

NASA Astrophysics Data System (ADS)

Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus; Kristensen, Lise Baadsgaard; Gerbola, Alexander; Gajhede, Michael; Jørgensen, Flemming Steen; Olsen, Lars; Kastrup, Jette Sandholm

2017-04-01

Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine.

The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region.

PubMed

Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus; Kristensen, Lise Baadsgaard; Gerbola, Alexander; Gajhede, Michael; Jørgensen, Flemming Steen; Olsen, Lars; Kastrup, Jette Sandholm

2017-04-07

Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine.

The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region

PubMed Central

Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus; Kristensen, Lise Baadsgaard; Gerbola, Alexander; Gajhede, Michael; Jørgensen, Flemming Steen; Olsen, Lars; Kastrup, Jette Sandholm

2017-01-01

Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine. PMID:28387240

A Secondary Structural Transition in the C-helix Promotes Gating of Cyclic Nucleotide-regulated Ion Channels*

PubMed Central

Puljung, Michael C.; Zagotta, William N.

2013-01-01

Cyclic nucleotide-regulated ion channels bind second messengers like cAMP to a C-terminal domain, consisting of a β-roll, followed by two α-helices (B- and C-helices). We monitored the cAMP-dependent changes in the structure of the C-helix of a C-terminal fragment of HCN2 channels using transition metal ion FRET between fluorophores on the C-helix and metal ions bound between histidine pairs on the same helix. cAMP induced a change in the dimensions of the C-helix and an increase in the metal binding affinity of the histidine pair. cAMP also caused an increase in the distance between a fluorophore on the C-helix and metal ions bound to the B-helix. Stabilizing the C-helix of intact CNGA1 channels by metal binding to a pair of histidines promoted channel opening. These data suggest that ordering of the C-helix is part of the gating conformational change in cyclic nucleotide-regulated channels. PMID:23525108

Variation in One Residue Associated with the Metal Ion-Dependent Adhesion Site Regulates αIIbβ3 Integrin Ligand Binding Affinity

PubMed Central

Wu, Xue; Xiu, Zhilong; Li, Guohui; Luo, Bing-Hao

2013-01-01

The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS) divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS) of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion. PMID:24116162

Monitoring binding affinity between drug and α1-acid glycoprotein in real time by Venturi easy ambient sonic-spray ionization mass spectrometry.

PubMed

Liu, Ning; Lu, Xin; Yang, YuHan; Yao, Chen Xi; Ning, BaoMing; He, Dacheng; He, Lan; Ouyang, Jin

2015-10-01

A new approach for monitoring the binding affinity between drugs and alpha 1-acid glycoprotein in real time was developed based on a combination of drug-protein reaction followed by Venturi easy ambient sonic-spray ionization mass spectrometry determination of the free drug concentrations. A known basic drug, propranolol was used to validate the new built method. Binding constant values calculated by venturi easy ambient sonic-spray ionization mass spectrometry was in good accordance with a traditional ultrafiltration combined with high performance liquid chromatography method. Then six types of basic drugs were used as the samples to conduct the real time analysis. Upon injection of alpha 1-acid glycoprotein to the drug mixture, the ion chromatograms were extracted to show the changes in the free drug concentrations in real time. By observing the drop-out of six types of drugs during the whole binding reaction, the binding affinities of different drugs were distinguished. A volume shift validating experiment and an injection delay correcting experiment were also performed to eliminate extraneous factors and verify the reliability of our experiment. Therefore, the features of Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS) and the experimental results indicate that our technique is likely to become a powerful tool for monitoring drug-AGP binding affinity in real time. Copyright © 2015 Elsevier B.V. All rights reserved.

Ca(2+) -complex stability of GAPAGPLIVPY peptide in gas and aqueous phase, investigated by affinity capillary electrophoresis and molecular dynamics simulations and compared to mass spectrometric results.

PubMed

Nachbar, Markus; El Deeb, Sami; Mozafari, Mona; Alhazmi, Hassan A; Preu, Lutz; Redweik, Sabine; Lehmann, Wolf Dieter; Wätzig, Hermann

2016-03-01

Strong, sequence-specific gas-phase bindings between proline-rich peptides and alkaline earth metal ions in nanoESI-MS experiments were reported by Lehmann et al. (Rapid Commun. Mass Spectrom. 2006, 20, 2404-2410), however its relevance for physiological-like aqueous phase is uncertain. Therefore, the complexes should also be studied in aqueous solution and the relevance of the MS method for binding studies be evaluated. A mobility shift ACE method was used for determining the binding between the small peptide GAPAGPLIVPY and various metal ions in aqueous solution. The findings were compared to the MS results and further explained using computational methods. While the MS data showed a strong alkaline earth ion binding, the ACE results showed nonsignificant binding. The proposed vacuum state complex also decomposed during a molecular dynamic simulation in aqueous solution. This study shows that the formed stable peptide-metal ion adducts in the gas phase by ESI-MS does not imply the existence of analogous adducts in the aqueous phase. Comparing peptide-metal ion interaction under the gaseous MS and aqueous ACE conditions showed huge difference in binding behavior. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Further insights into the metal ion binding abilities and the metalation pathway of a plant metallothionein from Musa acuminata

PubMed Central

Cabral, Augusto C. S.; Jakovleska, Jovana; Deb, Aniruddha; Penner-Hahn, James E.; Pecoraro, Vincent L.

2017-01-01

The superfamily of metallothioneins (MTs) combines a diverse group of metalloproteins, sharing the characteristics of rather low molecular weight and high cysteine content. The latter provides MTs with the capability to coordinate thiophilic metal ions, in particular those with a d10 electron configuration. The sub-family of plant MT3 proteins is only poorly characterized and there is a complete lack of three-dimensional structure information. Building upon our previous results on the Musa acuminata MT3 (musMT3) protein, the focus of the present work is to understand the metal cluster formation process, the role of the single histidine residue present in musMT3, and the metal ion binding affinity. We concentrate our efforts on the coordination of ZnII and CdII ions, using CoII as a spectroscopic probe for ZnII binding. The overall protein-fold is analysed with a combination of limited proteolytic digestion, mass spectrometry, and dynamic light scattering. Histidine coordination of metal ions is probed with extended X-ray absorption fine structure spectroscopy and CoII titration experiments. Initial experiments with isothermal titration calorimetry provide insights into the thermodynamics of metal ion binding. PMID:29218632

High Resolution Features from Low Affinity Interactions: Photoactive Analogs of the Haloether Anesthetics

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

Xi,J.; Liu, R.; Rossi, M.

2006-01-01

The difficulty in obtaining binding target and site information for low-affinity drugs, like the inhaled anesthetics, has limited identification of their molecular effectors. Because such information can be provided by photoactive analogues, we designed, synthesized, and characterized a novel diazirnyl haloether that closely mimics isoflurane, the most widely used clinical general anesthetic. This compound, H-diaziflurane, is a nontoxic, potent anesthetic that potentiates GABA-gated ion channels in primary cultures of hippocampal neurons. Calorimetric and structural characterizations show that H-diaziflurane binds a model anesthetic host protein with similar energetics as isoflurane and forms photoadducts with residues lining the isoflurane binding site. H-diazifluranemore » will be immediately useful for identifying targets and sites important for the molecular pharmacology of the inhaled haloether anesthetics.« less

Efficient fabrication of high-capacity immobilized metal ion affinity chromatographic media: The role of the dextran-grafting process and its manipulation.

PubMed

Zhao, Lan; Zhang, Jingfei; Huang, Yongdong; Li, Qiang; Zhang, Rongyue; Zhu, Kai; Suo, Jia; Su, Zhiguo; Zhang, Zhigang; Ma, Guanghui

2016-03-01

Novel high-capacity Ni(2+) immobilized metal ion affinity chromatographic media were prepared through the dextran-grafting process. Dextran was grafted to an allyl-activated agarose-based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran-grafted agarose microspheres and partly dextran-grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran-grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran-grafted microspheres, while just distributed around the outside of the partly dextran-grafted ones. Flow hydrodynamic properties were improved greatly after the dextran-grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran-grafting process, and partly dextran-grafted Ni(2+) chelating medium had a maximum binding capacity for His-tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high-efficiency and large-scale protein purification. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Zinc(II) binds to the neuroprotective peptide humanin.

PubMed

Armas, Ambar; Sonois, Vanessa; Mothes, Emmanuelle; Mazarguil, Honoré; Faller, Peter

2006-10-01

The abnormal accumulation of the peptide amyloid-beta in the form of senile (or amyloid) plaques is one of the hallmarks of Alzheimer's disease (AD). Zinc ions have been implicated in AD and plaques formation. Recently, the peptide humanin has been discovered. Humanin showed neuroprotective activity against amyloid-beta insults. Here the question investigated is if humanin could interact directly with Zn(II). It is shown that Zn(II) and its substitutes Cd(II)/Co(II) bind to humanin via a thiolate bond from the side chain of the single cysteine at position 8. The low intensity of the d-d bands of Co(II)-humanin indicated an octahedral coordination geometry. Titration experiments suggest that Zn(II) binds to humanin with an apparent affinity in the low muM range. This apparent Zn-binding affinity is in the same order as for amyloid-beta and glutathione and could thus be of physiological relevance.

Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

PubMed Central

Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

2012-01-01

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

Ratiometric and turn-on monitoring for heavy and transition metal ions in aqueous solution with a fluorescent peptide sensor.

PubMed

Joshi, Bishnu Prasad; Park, Junwon; Lee, Wan In; Lee, Keun-Hyeung

2009-05-15

A novel fluorescent peptide sensor containing tryptophan (donor) and dansyl fluorophore (acceptor) was synthesized for monitoring heavy and transition metal (HTM) ions on the basis of metal ion binding motif (Cys-X-X-X-Cys). The peptide probe successfully exhibited a turn on and ratiometric response for several heavy metal ions such as Hg(2+), Cd(2+), Pb(2+), Zn(2+), and Ag(+) in aqueous solution. The enhancements of emission intensity were achieved in the presence of the HTM ions by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The detection limits of the sensor for Cd(2+), Pb(2+), Zn(2+), and Ag(+) were lower than the EPA's drinking water maximum contaminant levels (MCL). We described the fluorescent enhancement, binding affinity, and detection limit of the peptide probe for HTM ions.

Structure-guided design of a high-affinity platelet integrin αIIbβ3 receptor antagonist that disrupts Mg²⁺ binding to the MIDAS.

PubMed

Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J; Filizola, Marta; Springer, Timothy A; Coller, Barry S

2012-03-14

An integrin found on platelets, α(IIb)β(3) mediates platelet aggregation, and α(IIb)β(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the β subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the β(3) subunits. They induce conformational changes in the β(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)β(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)β(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 Å revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the β(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)β(3) antagonists and may offer advantages as a therapeutic agent.

The regulation of integrin function by divalent cations

PubMed Central

Zhang, Kun; Chen, JianFeng

2012-01-01

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca2+-binding motifs that have essential roles in integrin biogenesis. The function of another Ca2+-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions. PMID:22647937

Affinity monolith chromatography: A review of general principles and applications.

PubMed

Li, Zhao; Rodriguez, Elliott; Azaria, Shiden; Pekarek, Allegra; Hage, David S

2017-11-01

Affinity monolith chromatography, or AMC, is a liquid chromatographic method in which the support is a monolith and the stationary phase is a biological-binding agent or related mimic. AMC has become popular for the isolation of biochemicals, for the measurement of various analytes, and for studying biological interactions. This review will examine the principles and applications of AMC. The materials that have been used to prepare AMC columns will be discussed, which have included various organic polymers, silica, agarose, and cryogels. Immobilization schemes that have been used in AMC will also be considered. Various binding agents and applications that have been reported for AMC will then be described. These applications will include the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, dye-ligand affinity chromatography, and immobilized metal-ion affinity chromatography. The use of AMC with chiral stationary phases and as a tool to characterize biological interactions will also be examined. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Binding site and affinity prediction of general anesthetics to protein targets using docking.

PubMed

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G

2012-05-01

The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explored whether a computational method, AutoDock, could serve as such a tool. High-resolution crystal data of water-soluble proteins (cytochrome C, apoferritin, and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus [GLIC]) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (http://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants were compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent cocrystallization data. Docking calculations for 6 general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known 50% effective concentration (EC(50)) values were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC(50) values and octanol/water partition coefficients for the 6 general anesthetics. All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (P = 0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the extracellular domain of GLIC. The predicted affinities correlated significantly with the known EC(50) values for the 6 frequently used anesthetics in GLIC for the site identified in the experimental crystal data (P = 0.006). However, predicted affinities in apoferritin, human serum albumin, and cytochrome C did not correlate with these 6 anesthetics' known experimental EC(50) values. A weak correlation between the predicted affinities and the octanol/water partition coefficients was observed for the sites in GLIC. We demonstrated that anesthetic binding sites and relative affinities can be predicted using docking calculations in an automatic docking server (AutoDock) for both water-soluble and membrane proteins. Correlation of predicted affinity and EC(50) for 6 frequently used general anesthetics was only observed in GLIC, a member of a protein family relevant to anesthetic mechanism.

Binding Site and Affinity Prediction of General Anesthetics to Protein Targets Using Docking

PubMed Central

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G.

2012-01-01

Background The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explore whether a computational method, AutoDock, could serve as such a tool. Methods High-resolution crystal data of water soluble proteins (cytochrome C, apoferritin and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus, GLIC) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (https://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants are compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent co-crystallization data. Docking calculations for six general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known EC50 were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC50s and octanol/water partition coefficients for the six general anesthetics. Results All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (p=0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the extracellular domain of GLIC. The predicted affinities correlated significantly with the known EC50s for the six commonly used anesthetics in GLIC for the site identified in the experimental crystal data (p=0.006). However, predicted affinities in apoferritin, human serum albumin, and cytochrome C did not correlate with these six anesthetics’ known experimental EC50s. A weak correlation between the predicted affinities and the octanol/water partition coefficients was observed for the sites in GLIC. Conclusion We demonstrated that anesthetic binding sites and relative affinities can be predicted using docking calculations in an automatic docking server (Autodock) for both water soluble and membrane proteins. Correlation of predicted affinity and EC50 for six commonly used general anesthetics was only observed in GLIC, a member of a protein family relevant to anesthetic mechanism. PMID:22392968

Cyclic peptide unguisin A is an anion receptor with high affinity for phosphate and pyrophosphate.

PubMed

Daryl Ariawan, A; Webb, James E A; Howe, Ethan N W; Gale, Philip A; Thordarson, Pall; Hunter, Luke

2017-04-05

Unguisin A (1) is a marine-derived, GABA-containing cyclic heptapeptide. The biological function of this flexible macrocycle is obscure. Here we show that compound 1 lacks any detectable activity in antimicrobial growth inhibition assays, a result that runs contrary to a previous report. However, we find that 1 functions as a promiscuous host molecule in a variety of anion-binding interactions, with high affinity particularly for phosphate and pyrophosphate. We also show that a series of rigidified, backbone-fluorinated analogues of 1 displays altered affinity for chloride ions.

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

PubMed

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

2017-06-16

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

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin's oxygen affinity.

PubMed

Cheng, Y; Lin, H; Xue, D; Li, R; Wang, K

2001-02-14

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln(3+) binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln(3+) binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln(3+) binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N(I)), non-cooperative strong sites (N(S)) and non-cooperative weak sites (N(W)) with binding constants in decreasing order: K(I)>K(S)>K(W). The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln(3+) concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln(3+) binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln(3+) concentration. At the range of [Ln(3+)]/[Hb]<2, the marked increase of oxygen affinity (P(50) decrease) with the Ln(3+) concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln(3+) concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln(3+) binding. This was indicated by the changes in secondary structure characterized by the decrease of alpha-helix content.

Spectroscopic characterization of effective components anthraquinones in Chinese medicinal herbs binding with serum albumins

NASA Astrophysics Data System (ADS)

Bi, Shuyun; Song, Daqian; Kan, Yuhe; Xu, Dong; Tian, Yuan; Zhou, Xin; Zhang, Hanqi

2005-11-01

The interactions of serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) with emodin, rhein, aloe-emodin and aloin were assessed employing fluorescence quenching and absorption spectroscopic techniques. The results obtained revealed that there are relatively strong binding affinity for the four anthraquinones with HSA and BSA and the binding constants for the interactions of anthraquinones with HSA or BSA at 20 °C were obtained. Anthraquinone-albumin interactions were studied at different temperatures and in the presence of some metal ions. And the competition binding of anthraquinones with serum albumins was also discussed. The Stern-Volmer curves suggested that the quenching occurring in the reactions was the static quenching process. The binding distances and transfer efficiencies for each binding reactions were calculated according to the Föster theory of non-radiation energy transfer. Using thermodynamic equations, the main action forces of these reactions were also obtained. The reasons of the different binding affinities for different anthraquinone-albumin reactions were probed from the point of view of molecular structures.

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

PubMed

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

2017-08-15

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

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

PubMed Central

2017-01-01

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

Functional Analysis of the Citrate Activator CitO from Enterococcus faecalis Implicates a Divalent Metal in Ligand Binding

PubMed Central

Blancato, Víctor S.; Pagliai, Fernando A.; Magni, Christian; Gonzalez, Claudio F.; Lorca, Graciela L.

2016-01-01

The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC), indicated that CitO has a high affinity for citrate (KD = 1.2 ± 0.2 μM), while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni2+, and Zn2+ to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation. PMID:26903980

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

NASA Technical Reports Server (NTRS)

Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

2003-01-01

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

Reactivity of food phenols with iron and copper ions: binding, dioxygen activation and oxidation mechanisms.

PubMed

Nkhili, Ezzohra; Loonis, Michèle; Mihai, Simona; El Hajji, Hakima; Dangles, Olivier

2014-06-01

In this work, the affinity of common dietary phenols (gallic acid, caffeic acid, catechin, and rutin) for iron and copper ions was quantitatively investigated in neutral phosphate buffer as well as the reactivity of the complexes toward dioxygen. Contrasting behaviors were observed: because of the competing phosphate ions, Fe(III) binding is much slower than Fe(II) binding, which is rapidly followed by autoxidation of Fe(II) into Fe(III). With both ions, O2 consumption and H2O2 production are modest and the phenolic ligands are only slowly oxidized. By contrast, metal-phenol binding is fast with both Cu(I) and Cu(II). With Cu(I), O2 consumption and H2O2 production are very significant and the phenolic ligands are rapidly oxidized into a complex mixture of oligomers. The corresponding mechanism with Cu(II) is hampered by the preliminary rate-determining step of Cu(II) reduction by the phenols. The consequences of these findings for the stability and antioxidant activity of plant phenols are discussed.

Probing the ATP site of GRP78 with nucleotide triphosphate analogs

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

Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun

GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78 ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligandsmore » (ATP analogs) to a receptor (GRP78 ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78 ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg ++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg ++-dependent, as the removal of Mg ++ nearly abolished binding to GRP78 ATPase. The AMPPCP-Mg ++ structure showed evidence for the critical role of Mg ++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg ++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg ++. The 2'-deoxyATP structure showed the conformation of the bound nucleotide flipped out of the active site, explaining the low affinity binding to GRP78 and suggesting that the 2'-OH group is essential for the high affinity binding to GRP78. Altogether, our results demonstrate that GRP78 ATPase possesses nucleotide specificity more relaxed than previously anticipated and can tolerate certain modifications to the nucleobase 7-position and, to a lesser extent, the beta-gamma bridging atom, thereby providing a possible atomic mechanism underlying the transmembrane transport of the ATP analogs.« less

Probing the ATP site of GRP78 with nucleotide triphosphate analogs

DOE PAGES

Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun; ...

2016-05-04

GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78 ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligandsmore » (ATP analogs) to a receptor (GRP78 ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78 ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg ++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg ++-dependent, as the removal of Mg ++ nearly abolished binding to GRP78 ATPase. The AMPPCP-Mg ++ structure showed evidence for the critical role of Mg ++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg ++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg ++. The 2'-deoxyATP structure showed the conformation of the bound nucleotide flipped out of the active site, explaining the low affinity binding to GRP78 and suggesting that the 2'-OH group is essential for the high affinity binding to GRP78. Altogether, our results demonstrate that GRP78 ATPase possesses nucleotide specificity more relaxed than previously anticipated and can tolerate certain modifications to the nucleobase 7-position and, to a lesser extent, the beta-gamma bridging atom, thereby providing a possible atomic mechanism underlying the transmembrane transport of the ATP analogs.« less

Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

PubMed

Wojtas, Magdalena; Hołubowicz, Rafał; Poznar, Monika; Maciejewska, Marta; Ożyhar, Andrzej; Dobryszycki, Piotr

2015-10-27

Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 μM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 μM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

Synthesis of a ratiometric fluorescent peptide sensor for the highly selective detection of Cd2+.

PubMed

Li, Yan; Li, Lianzhi; Pu, Xuewei; Ma, Guolin; Wang, Erqiong; Kong, Jinming; Liu, Zhipeng; Liu, Yangzhong

2012-06-15

A novel ratiometric fluorescent peptidyl chemosensor (Dansyl-Cys-Pro-Gly-Cys-Trp-NH(2), D-P5) for metal ions detection has been synthesized via Fmoc solid-phase peptide synthesis. The chemosensor exhibited a high selectivity for Cd(2+) over other metal ions including competitive transition and Group I and II metal ions in neutral pH. The fluorescence emission intensity of D-P5 was significantly enhanced in the presence of Cd(2+) by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The binding stoichiometry, detection limit, binding affinity, reversibility and pH sensitivity of the sensor for Cd(2+) were investigated. Copyright © 2012 Elsevier Ltd. All rights reserved.

Combined copper/zinc attachment to prion protein

NASA Astrophysics Data System (ADS)

Hodak, Miroslav; Bernholc, Jerry

2013-03-01

Misfolding of prion protein (PrP) is responsible for diseases such as ``mad-cow disease'' in cattle and Creutzfeldt-Jacob in humans. Extensive experimental investigation has established that this protein strongly interacts with copper ions, and this ability has been linked to its still unknown function. Attachment of other metal ions (zinc, iron, manganese) have been demonstrated as well, but none of them could outcompete copper. Recent finding, however, indicates that at intermediate concentrations both copper and zinc ions can attach to the PrP at the octarepeat region, which contains high affinity metal binding sites. Based on this evidence, we have performed density functional theory simulations to investigate the combined Cu/Zn attachment. We consider all previously reported binding modes of copper at the octarepeat region and examine a possibility simultaneous Cu/Zn attachment. We find that this can indeed occur for only one of the known binding sites, when copper changes its coordination mode to allow for attachment of zinc ion. The implications of the simultaneous attachment on neural function remain to be explored.

Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

PubMed Central

Chung, Wai Keen; Freed, Alexander S.; Holstein, Melissa A.; McCallum, Scott A.; Cramer, Steven M.

2010-01-01

NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarse-grained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems. PMID:20837551

Essential slow degrees of freedom in protein-surface simulations: A metadynamics investigation.

PubMed

Prakash, Arushi; Sprenger, K G; Pfaendtner, Jim

2018-03-29

Many proteins exhibit strong binding affinities to surfaces, with binding energies much greater than thermal fluctuations. When modelling these protein-surface systems with classical molecular dynamics (MD) simulations, the large forces that exist at the protein/surface interface generally confine the system to a single free energy minimum. Exploring the full conformational space of the protein, especially finding other stable structures, becomes prohibitively expensive. Coupling MD simulations with metadynamics (enhanced sampling) has fast become a common method for sampling the adsorption of such proteins. In this paper, we compare three different flavors of metadynamics, specifically well-tempered, parallel-bias, and parallel-tempering in the well-tempered ensemble, to exhaustively sample the conformational surface-binding landscape of model peptide GGKGG. We investigate the effect of mobile ions and ion charge, as well as the choice of collective variable (CV), on the binding free energy of the peptide. We make the case for explicitly biasing ions to sample the true binding free energy of biomolecules when the ion concentration is high and the binding free energies of the solute and ions are similar. We also make the case for choosing CVs that apply bias to all atoms of the solute to speed up calculations and obtain the maximum possible amount of information about the system. Copyright © 2017 Elsevier Inc. All rights reserved.

Dynamic ion-ion and water-ion interactions in ion channels.

PubMed Central

Wu, J V

1992-01-01

The dynamic interactions among ions and water molecules in ion channels are treated based on an assumption that ions at binding sites can be knocked off by both transient entering ions and local water molecules. The theory, when applied to a single-site model K+ channel, provides solutions for super- and subsaturations, flux-ratio exponent (n') greater than 1, osmotic streaming current, activity-dependent reversal potentials, and anomalous mole-fraction behavior. The analysis predicts that: (a) the saturation may but, in general, does not follow the Michaelis-Menten relation; (b) streaming current results from imbalanced water-ion knock-off interactions; (c) n' greater than 1 even for single-site channels, but it is unlikely to exceed 1.4 unless the pore is occupied by one or more ion(s); (d) in the calculation involving two permeant ion species with similar radii, the heavier ions show higher affinity; the ion-ion knock-off dissociation from the site is more effective when two interacting ions are identical. Therefore, the "multi-ion behaviors" found in most ion channels are the consequences of dynamic ion-ion and water-ion interactions. The presence of these interactions does not require two or more binding sites in channels. PMID:1376158

In silico design of novel hERG-neutral sildenafil-like PDE5 inhibitors.

PubMed

Kayık, Gülru; Tüzün, Nurcan Ş; Durdagi, Serdar

2017-10-01

Cyclic nucleotide phosphodiesterase enzymes (PDEs) have functions in regulating the levels of intracellular second messengers, 3', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP), via hydrolysis and decomposing mechanisms in cells. They take essential roles in modulating various cellular activities such as memory and smooth muscle functions. PDE type 5 (PDE5) inhibitors enhance the vasodilatory effects of cGMP in the corpus cavernosum and they are used to treat erectile dysfunction. Patch clamp experiments showed that the IC 50 values of the human ether-à-go-go-related gene (hERG1) potassium (K) ion channel blocking affinity of PDE5 inhibitors sildenafil, vardenafil, and tadalafil as 33, 12, and 100 μM, respectively. hERG1 channel is responsible for the regulation of the action potential of human ventricular myocyte by contributing the rapid component of delayed rectifier K + current (I Kr ) component of the cardiac action potential. In this work, interaction patterns and binding affinity predictions of selected PDE5 inhibitors against the hERG1 channel are studied. It is attempted to develop PDE5 inhibitor analogs with lower binding affinity to hERG1 ion channel while keeping their pharmacological activity against their principal target PDE5 using in silico methods. Based on detailed analyses of docking poses and predicted interaction energies, novel analogs of PDE5 inhibitors with lower predicted binding affinity to hERG1 channels without loosing their principal target activity were proposed. Moreover, molecular dynamics (MD) simulations and post-processing MD analyses (i.e. Molecular Mechanics/Generalized Born Surface Area calculations) were performed. Detailed analysis of molecular simulations helped us to better understand the PDE5 inhibitor-target binding interactions in the atomic level. Results of this study can be useful for designing of novel and safe PDE5 inhibitors with enhanced activity and other tailored properties.

Mg2+ ions: do they bind to nucleobase nitrogens?

PubMed Central

Leonarski, Filip; D'Ascenzo, Luigi; Auffinger, Pascal

2017-01-01

Given the many roles proposed for Mg2+ in nucleic acids, it is essential to accurately determine their binding modes. Here, we surveyed the PDB to classify Mg2+ inner-sphere binding patterns to nucleobase imine N1/N3/N7 atoms. Among those, purine N7 atoms are considered to be the best nucleobase binding sites for divalent metals. Further, Mg2+ coordination to N7 has been implied in several ribozyme catalytic mechanisms. We report that Mg2+ assigned near imine nitrogens derive mostly from poor interpretations of electron density patterns and are most often misidentified Na+, K+, NH4+ ions, water molecules or spurious density peaks. Consequently, apart from few documented exceptions, Mg2+ ions do not bind to N7 atoms. Without much of a surprise, Mn2+, Zn2+ and Cd2+, which have a higher affinity for nitrogens, may contact N7 atoms when present in crystallization buffers. In this respect, we describe for the first time a potential Zn2+ ribosomal binding site involving two purine N7 atoms. Further, we provide a set of guidelines to help in the assignment of Mg2+ in crystallographic, cryo-EM, NMR and model building practices and discuss implications of our findings related to ion substitution experiments. PMID:27923930

Ni2+-binding RNA motifs with an asymmetric purine-rich internal loop and a G-A base pair.

PubMed Central

Hofmann, H P; Limmer, S; Hornung, V; Sprinzl, M

1997-01-01

RNA molecules with high affinity for immobilized Ni2+ were isolated from an RNA pool with 50 randomized positions by in vitro selection-amplification. The selected RNAs preferentially bind Ni2+ and Co2+ over other cations from first series transition metals. Conserved structure motifs, comprising about 15 nt, were identified that are likely to represent the Ni2+ binding sites. Two conserved motifs contain an asymmetric purine-rich internal loop and probably a mismatch G-A base pair. The structure of one of these motifs was studied with proton NMR spectroscopy and formation of the G-A pair at the junction of helix and internal loop was demonstrated. Using Ni2+ as a paramagnetic probe, a divalent metal ion binding site near this G-A base pair was identified. Ni2+ ions bound to this motif exert a specific stabilization effect. We propose that small asymmetric purine-rich loops that contain a G-A interaction may represent a divalent metal ion binding site in RNA. PMID:9409620

Manganese Binding Properties of Human Calprotectin Under Conditions of High and Low Calcium: X-ray Crystallographic and Advanced EPR Spectroscopic Analysis

PubMed Central

Gagnon, Derek M.; Brophy, Megan Brunjes; Bowman, Sarah E. J.; Stich, Troy A.; Drennan, Catherine L.; Britt, R. David; Nolan, Elizabeth M.

2015-01-01

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Herein, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimer is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin-echo envelope modulation and electron nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed 15N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by ca. two orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His6 site of human calprotectin. PMID:25597447

Manganese binding properties of human calprotectin under conditions of high and low calcium: X-ray crystallographic and advanced electron paramagnetic resonance spectroscopic analysis.

PubMed

Gagnon, Derek M; Brophy, Megan Brunjes; Bowman, Sarah E J; Stich, Troy A; Drennan, Catherine L; Britt, R David; Nolan, Elizabeth M

2015-03-04

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Herein, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimer is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin-echo envelope modulation and electron-nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed (15)N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by two to three orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His6 site of human calprotectin.

Manganese Binding Properties of Human Calprotectin under Conditions of High and Low Calcium: X-ray Crystallographic and Advanced Electron Paramagnetic Resonance Spectroscopic Analysis

DOE PAGES

Gagnon, Derek M.; Brophy, Megan Brunjes; Bowman, Sarah E. J.; ...

2015-01-18

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Here in this paper, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimermore » is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin–echo envelope modulation and electron–nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed 15N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by two to three orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His 6 site of human calprotectin.« less

Manganese Binding Properties of Human Calprotectin under Conditions of High and Low Calcium: X-ray Crystallographic and Advanced Electron Paramagnetic Resonance Spectroscopic Analysis

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

Gagnon, Derek M.; Brophy, Megan Brunjes; Bowman, Sarah E. J.

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Here in this paper, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimermore » is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin–echo envelope modulation and electron–nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed 15N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by two to three orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His 6 site of human calprotectin.« less

Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants.

PubMed

Kristensen, Anders S; Larsen, Mads B; Johnsen, Laust B; Wiborg, Ove

2004-03-01

The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.

Study of the surface-enhanced Raman spectroscopy of residual impurities in hydroxylamine-reduced silver colloid and the effects of anions on the colloid activity.

PubMed

Dong, Xiao; Gu, Huaimin; Liu, Fangfang

2012-03-01

The paper investigated the residual ions in hydroxylamine-reduced silver colloid (HRSC) and the relationship between the condition of HRSC and the enhanced mechanisms of this colloid. We also detected the SERS of MB and studied the effects of anions on the Raman signal. In the case of HRSC, the bands of residual ions diminish while the bands of Ag-anions increase gradually with increasing the concentrations of Cl(-) and NO(3)(-). It means the affinity of residual ions on the silver surface is weaker than that of Cl(-) and NO(3)(-) and the residual ions are replaced gradually by the added Cl(-) or NO(3)(-). The Raman signal of residual ions can be detected by treatment with anions that do not bind strongly to the silver surface, such as SO(4)(2-). The most intense band of Ag-anions bonds can be also observed when adding weakly binding anions to the colloid. However, the anions which make up the Ag-anions bonds are residual Cl(-) and the effect of weakly binding anions is only to aggregate the silver particles. Residual Cl(-) can be replaced by I(-) which has the highest affinity. From the detection of methylene blue (MB), the effects of anions on the enhancement of Raman signal are discussed in detail, and these findings could make the conditions suitable for detecting analytes in high efficiency. This study will have a profound implication to SERS users about their interpretation of SERS spectra when obtaining these anomalous bands. Copyright © 2011 Elsevier B.V. All rights reserved.

Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P2 affinity, phosphoinositide selectivity, and PI(4,5)P2 pool accessibility

PubMed Central

Rjasanow, Alexandra; Leitner, Michael G.; Thallmair, Veronika; Halaszovich, Christian R.; Oliver, Dominik

2015-01-01

The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K+ channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs. PMID:26150791

Specific ion effects on membrane potential and the permselectivity of ion exchange membranes.

PubMed

Geise, Geoffrey M; Cassady, Harrison J; Paul, Donald R; Logan, Bruce E; Hickner, Michael A

2014-10-21

Membrane potential and permselectivity are critical parameters for a variety of electrochemically-driven separation and energy technologies. An electric potential is developed when a membrane separates electrolyte solutions of different concentrations, and a permselective membrane allows specific species to be transported while restricting the passage of other species. Ion exchange membranes are commonly used in applications that require advanced ionic electrolytes and span technologies such as alkaline batteries to ammonium bicarbonate reverse electrodialysis, but membranes are often only characterized in sodium chloride solutions. Our goal in this work was to better understand membrane behaviour in aqueous ammonium bicarbonate, which is of interest for closed-loop energy generation processes. Here we characterized the permselectivity of four commercial ion exchange membranes in aqueous solutions of sodium chloride, ammonium chloride, sodium bicarbonate, and ammonium bicarbonate. This stepwise approach, using four different ions in aqueous solution, was used to better understand how these specific ions affect ion transport in ion exchange membranes. Characterization of cation and anion exchange membrane permselectivity, using these ions, is discussed from the perspective of the difference in the physical chemistry of the hydrated ions, along with an accompanying re-derivation and examination of the basic equations that describe membrane potential. In general, permselectivity was highest in sodium chloride and lowest in ammonium bicarbonate solutions, and the nature of both the counter- and co-ions appeared to influence measured permselectivity. The counter-ion type influences the binding affinity between counter-ions and polymer fixed charge groups, and higher binding affinity between fixed charge sites and counter-ions within the membrane decreases the effective membrane charge density. As a result permselectivity decreases. The charge density and polarizability of the co-ions also appeared to influence permselectivity leading to ion-specific effects; co-ions that are charge dense and have low polarizability tended to result in high membrane permselectivity.

Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

PubMed Central

Hamouda, Ayman K.; Wang, Ze-Jun; Stewart, Deirdre S.; Jain, Atul D.; Glennon, Richard A.

2015-01-01

Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4β2 and α2β2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αβεδ) and Torpedo (αβγδ) nAChR expressed in Xenopus oocytes with IC50 values of ∼1 μM. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([3H]phencyclidine; IC50 = 4 μM) than in the resting state ([3H]tetracaine; IC50 = 60 μM), whereas it bound with only very low affinity to the ACh binding sites ([3H]ACh, IC50 = 1 mM). Upon irradiation at 312 nm, [3H]dFBr photoincorporated into amino acids within the Torpedo nAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [3H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical α-γ interface containing the transmitter binding sites and at the noncanonical δ-β subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [3H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity. PMID:25870334

Hierarchy and Assortativity as New Tools for Binding-Affinity Investigation: The Case of the TBA Aptamer-Ligand Complex.

PubMed

Cataldo, Rosella; Alfinito, Eleonora; Reggiani, Lino

2017-12-01

Aptamers are single stranded DNA, RNA, or peptide sequences having the ability to bind several specific targets (proteins, molecules as well as ions). Therefore, aptamer production and selection for therapeutic and diagnostic applications is very challenging. Usually, they are generated in vitro, although computational approaches have been recently developed for the in silico production. Despite these efforts, the mechanism of aptamer-ligand formation is not completely clear, and producing high-affinity aptamers is still quite difficult. This paper aims to develop a computational model able to describe aptamer-ligand affinity. Topological tools, such as the conventional degree distribution, the rank-degree distribution (hierarchy), and the node assortativity are employed. In doing so, the macromolecules tertiary-structures are mapped into appropriate graphs. These graphs reproduce the main topological features of the macromolecules, by preserving the distances between amino acids (nucleotides). Calculations are applied to the thrombin binding aptamer (TBA), and the TBA-thrombin complex produced in the presence of Na + or K + . The topological analysis is able to detect several differences between complexes obtained in the presence of the two cations, as expected by previous investigations. These results support graph analysis as a novel computational tool for testing affinity. Otherwise, starting from the graphs, an electrical network can be obtained by using the specific electrical properties of amino acids and nucleobases. Therefore, a further analysis concerns with the electrical response, revealing that the resistance is sensitively affected by the presence of sodium or potassium, thus suggesting resistance as a useful physical parameter for testing binding affinity.

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell.

PubMed

Sahu, Santosh Kumar; Aradhyam, Gopala Krishna; Gummadi, Sathyanarayana N

2009-10-01

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca2+ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca2+/Mg2+ binding to human scramblases and conformational changes taking place in them remains unknown. In the present study, we analyzed the Ca2+ and Mg2+ binding to the calcium binding motifs of hPLSCR1-4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry. The results in this study show that (i) affinities of the peptides are in the order hPLSCR1>hPLSCR3>hPLSCR2>hPLSCR4 for Ca2+ and in the order hPLSCR1>hPLSCR2>hPLSCR3>hPLSCR4 for Mg2+, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca2+ and Mg2+ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families. Based on the above results, we hypothesize that the Ca2+ binding motif of hPLSCR1 is a novel type of Ca2+ binding motif. Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.

Binding patterns of vanadium ions with different valence states to human serum transferrin studied by HPLC/high-resolution ICP-MS.

PubMed

Nagaoka, Megumi Hamano; Yamazaki, Takeshi; Maitani, Tamio

2002-09-06

Vanadium (V) is an essential metal for mammals and has different valence states. In blood, V is bound to serum transferrin (Tf), a glycoprotein which has two metal-binding sites, and carbonate is generally required for the binding. In this study, the binding patterns of V(III), V(IV), and V(V) to human serum Tf (hTf) were analyzed using an HPLC system equipped with an anion-exchange column and directly connected to a high-resolution inductively coupled plasma-mass spectrometer for metal detection (51V). In affinity to hTf, the three ions were ranked V(III)>V(IV)>V(V) in the presence of bicarbonate and V(III) reverse congruent V(IV)>V(V) in the absence. Intermediates in the "open forms" binding to the respective sites were detected at the initial stage. V(IV) and V(V) were bound to the N-lobe site in the "closed form" and "open form," respectively. In the absence of bicarbonate, V ions with respective valence states were bound to hTf in the "open form." In terms of binding to hTf, tri-valent V was most favorable in the presence of bicarbonate.

Binding of calcium and target peptide to calmodulin-like protein CML19, the centrin 2 of Arabidopsis thaliana.

PubMed

La Verde, Valentina; Trande, Matteo; D'Onofrio, Mariapina; Dominici, Paola; Astegno, Alessandra

2018-03-01

Calmodulin-like protein 19 (CML19) is an Arabidopsis centrin that modulates nucleotide excision repair (NER) by binding to RAD4 protein, the Arabidopsis homolog of human Xeroderma pigmentosum complementation group C protein. Although the necessity of CML19 as a part of the RAD4 plant recognition complex for functional NER is known at a cellular level, little is known at a molecular level. Herein, we used a combination of biophysical and biochemical approaches to investigate the structural and ion and target-peptide binding properties of CML19. We found that CML19 possesses four Ca 2+ -specific binding sites, two of high affinity in the N-terminal domain and two of low affinity in the C-terminal domain. Binding of Ca 2+ to CML19 increases its alpha-helix content, stabilizes the tertiary structure, and triggers a conformational change, resulting in the exposure of a hydrophobic patch instrumental for target protein recognition. Using bioinformatics tools we identified a CML19-binding site at the C-terminus of RAD4, and through in vitro binding experiments we analyzed the interaction between a 17-mer peptide representing this site and CML19. We found that the peptide shows a high affinity for CML19 in the presence of Ca 2+ (stoichiometry 1:1) and the interaction primarily involves the C-terminal half of CML19. Copyright © 2017 Elsevier B.V. All rights reserved.

AF64A depletes hippocampal high-affinity choline uptake but does not alter the density of alpha-bungarotoxin binding sites or modify the effect of exogenous choline.

PubMed

Morley, B J; Garner, L L

1990-06-11

Sodium-dependent, high-affinity choline uptake (HACU) and the density of alpha-bungarotoxin (BuTX) receptor-binding sites were measured in the hippocampus following the intraventricular infusion of ethylcholine aziridinium ion (AF64A), a neurotoxin that competes with choline at high-affinity choline transport sites and may result in the degeneration of cholinergic axons. Eight days after the infusion of AF64A into the lateral ventricles (2.5 nmol/side), HACU was depleted by 60% in the hippocampus of experimental animals in comparison with controls, but the density of BuTX-binding sites was not altered. The administration of 15 mg/ml of choline chloride in the drinking water increased the density of BuTX-binding sites, as previously reported by this laboratory. The administration of AF64A did not prevent the effect of exogenous choline on the density of binding sites, nor did choline treatment alter the effect of AF64A on HACU. These data indicate that the density of BuTX-binding sites in the hippocampus is not altered following a substantial decrease in HACU and presumed degeneration of cholinergic axons. Since the effect of exogenous choline was not prevented by AF64A treatment, the data are interpreted to support the hypothesis that the increase in the density of BuTX-binding sites following dietary choline supplementation is attributable to a direct effect of choline on receptor sites.

Negative ions of polyatomic molecules.

PubMed Central

Christophorou, L G

1980-01-01

In this paper general concepts relating to, and recent advances in, the study of negative ions of polyatomic molecules area discussed with emphasis on halocarbons. The topics dealt with in the paper are as follows: basic electron attachment processes, modes of electron capture by molecules, short-lived transient negative ions, dissociative electron attachment to ground-state molecules and to "hot" molecules (effects of temperature on electron attachment), parent negative ions, effect of density, nature, and state of the medium on electron attachment, electron attachment to electronically excited molecules, the binding of attached electrons to molecules ("electron affinity"), and the basic and the applied significance of negative-ion studies. PMID:7428744

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

PubMed

Katti, Sachin; Nyenhuis, Sarah B; Her, Bin; Srivastava, Atul K; Taylor, Alexander B; Hart, P John; Cafiso, David S; Igumenova, Tatyana I

2017-06-27

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca 2+ -dependent manner. In these cases, membrane association is triggered by Ca 2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd 2+ , in lieu of Ca 2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd 2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd 2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd 2+ -complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca 2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca 2+ ion binding to the C2 domain loop regions.

State-dependent block of CNG channels by dequalinium.

PubMed

Rosenbaum, Tamara; Gordon-Shaag, Ariela; Islas, León D; Cooper, Jeremy; Munari, Mika; Gordon, Sharona E

2004-03-01

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.

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

PubMed Central

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

2015-01-01

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

Optimization of Time-Resolved Fluorescence Assay for Detection of Eu-DOTA-labeled Ligand-Receptor Interactions

PubMed Central

De Silva, Channa R.; Vagner, Josef; Lynch, Ronald; Gillies, Robert J.; Hruby, Victor J.

2010-01-01

Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improved sensitivity and affordability in high throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as DTPA derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIA) have not yet been successfully used with more stable chelators, e.g. DOTA derivatives, due to the incomplete release of lanthanide(III) ions from the complex. Here, a modified and an optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA labeled peptides. Complete release of Eu(III) ions from DOTA labeled ligands was observed using hydrochloric acid (2.0 M) prior to the luminescent enhancement step. NDP-α-MSH labeled with Eu(III)-DOTA was synthesized and the binding affinity to cells overexpressing the human melanocortin-4 receptors (hMC4R) was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA labeled heterobivalent peptide to the cells expressing both hMC4R and CCK-2 (Cholecystokinin) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries. PMID:19852924

Energetics of phosphate binding to ammonium and guanidinium containing metallo-receptors in water.

PubMed

Tobey, Suzanne L; Anslyn, Eric V

2003-12-03

The design and synthesis of receptors containing a Cu(II) binding site with appended ammonium groups (1) and guanidinium groups (2), along with thermodynamics analyses of anion binding, are reported. Both receptors 1 and 2 show high affinities (10(4) M(-1)) and selectivities for phosphate over other anions in 98:2 water:methanol at biological pH. The binding of the host-guest pairs is proposed to proceed through ion-pairing interactions between the charged functional groups on both the host and the guest. The affinities and selectivities for oxyanions were determined using UV/vis titration techniques. Additionally, thermodynamic investigations indicate that the 1:phosphate complex is primarily entropy driven, while the 2:phosphate complex displays both favorable enthalpy and entropy changes. The thermodynamic data for binding provide a picture of the roles of the host, guest, counterions, and solvent. The difference in the entropy and enthalpy driving forces for the ammonium and guanidinium containing hosts are postulated to derive primarily from differences in the solvation shell of these two groups.

Application of the Nernst-Planck approach to lead ion exchange in Ca-loaded Pelvetia canaliculata.

PubMed

Costa, Joana F de Sá S; Vilar, Vítor J P; Botelho, Cidália M S; da Silva, Eduardo A B; Boaventura, Rui A R

2010-07-01

Ca-loaded Pelvetia canaliculata biomass was used to remove Pb(2+) in aqueous solution from batch and continuous systems. The physicochemical characterization of algae Pelvetia particles by potentiometric titration and FTIR analysis has shown a gel structure with two major binding groups - carboxylic (2.8 mmol g(-1)) and hydroxyl (0.8 mmol g(-1)), with an affinity constant distribution for hydrogen ions well described by a Quasi-Gaussian distribution. Equilibrium adsorption (pH 3 and 5) and desorption (eluents: HNO(3) and CaCl(2)) experiments were performed, showing that the biosorption mechanism was attributed to ion exchange among calcium, lead and hydrogen ions with stoichiometry 1:1 (Ca:Pb) and 1:2 (Ca:H and Pb:H). The uptake capacity of lead ions decreased with pH, suggesting that there is a competition between H(+) and Pb(2+) for the same binding sites. A mass action law for the ternary mixture was able to predict the equilibrium data, with the selectivity constants alpha(Ca)(H)=9+/-1 and alpha(Ca)(Pb)=44+/-5, revealing a higher affinity of the biomass towards lead ions. Adsorption (initial solution pH 4.5 and 2.5) and desorption (0.3M HNO(3)) kinetics were performed in batch and continuous systems. A mass transfer model using the Nernst-Planck approximation for the ionic flux of each counter-ion was used for the prediction of the ions profiles in batch systems and packed bed columns. The intraparticle effective diffusion constants were determined as 3.73x10(-7)cm(2)s(-1) for H(+), 7.56x10(-8)cm(2)s(-1) for Pb(2+) and 6.37x10(-8)cm(2)s(-1) for Ca(2+). Copyright 2010 Elsevier Ltd. All rights reserved.

Impact of pH on the structure and function of neural cadherin.

PubMed

Jungles, Jared M; Dukes, Matthew P; Vunnam, Nagamani; Pedigo, Susan

2014-12-02

Neural (N-) cadherin is a transmembrane protein within adherens junctions that mediates cell-cell adhesion. It has 5 modular extracellular domains (EC1-EC5) that bind 3 calcium ions between each of the modules. Calcium binding is required for dimerization. N-Cadherin is involved in diverse processes including tissue morphogenesis, excitatory synapse formation and dynamics, and metastasis of cancer. During neurotransmission and tumorigenesis, fluctuations in extracellular pH occur, causing tissue acidosis with associated physiological consequences. Studies reported here aim to determine the effect of pH on the dimerization properties of a truncated construct of N-cadherin containing EC1-EC2. Since N-cadherin is an anionic protein, we hypothesized that acidification of solution would cause an increase in stability of the apo protein, a decrease in the calcium-binding affinity, and a concomitant decrease in the formation of adhesive dimer. The stability of the apo monomer was increased and the calcium-binding affinity was decreased at reduced pH, consistent with our hypothesis. Surprisingly, analytical SEC studies showed an increase in calcium-induced dimerization as solution pH decreased from 7.4 to 5.0. Salt-dependent dimerization studies indicated that electrostatic repulsion attenuates dimerization affinity. These results point to a possible electrostatic mechanism for moderating dimerization affinity of the Type I cadherin family. Extrapolating these results to cell adhesion in vivo leads to the assertion that decreased pH promotes adhesion by N-cadherin, thereby stabilizing synaptic junctions.

Antenna-predominant and male-biased CSP19 of Sesamia inferens is able to bind the female sex pheromones and host plant volatiles.

PubMed

Zhang, Ya-Nan; Ye, Zhan-Feng; Yang, Ke; Dong, Shuang-Lin

2014-02-25

Insect chemosensory proteins (CSPs) are proposed to capture and transport hydrophobic chemicals across the sensillum lymph to olfactory receptors (ORs), but this has not been clarified in moths. In this study, we built on our previously reported segment sequence work and cloned the full length CSP19 gene (SinfCSP19) from the antennae of Sesamia inferens by using rapid amplification of cDNA ends. Quantitative real time-PCR (qPCR) assays indicated that the gene was expressed in a unique profile, i.e. predominant in antennae and significantly higher in male than in female. To explore the function, recombinant SinfCSP19 was expressed in Escherichia coli cells and purified by Ni-ion affinity chromatography. Binding affinities of the recombinant SinfCSP19 with 39 plant volatiles, 3 sex pheromone components and 10 pheromone analogs were measured using fluorescent competitive binding assays. The results showed that 6 plant volatiles displayed high binding affinities to SinfCSP19 (Ki = 2.12-8.75 μM), and more interesting, the 3 sex pheromone components and analogs showed even higher binding to SinfCSP19 (Ki = 0.49-1.78 μM). Those results suggest that SinfCSP19 plays a role in reception of female sex pheromones of S. inferens and host plant volatiles. Copyright © 2013 Elsevier B.V. All rights reserved.

Mechanistic characterization of the HDV genomic ribozyme: a mutant of the C41 motif provides insight into the positioning and thermodynamic linkage of metal ions and protons.

PubMed

Nakano, Shu-ichi; Bevilacqua, Philip C

2007-03-20

Binding of two Mg2+ and two H+ ions influences the self-cleavage activity of the genomic HDV ribozyme. The positioning of these four ligands and their thermodynamic linkage are not fully resolved. Protonated C41 engages in a base triple, whereas protonated C75 has been implicated as an acid-base catalyst in bond cleavage. Prior studies led to the identification of one structural inner-sphere ion and one catalytic outer-sphere ion. In the present study, the contributions of the C41 base triple to the metal ion- and pH-dependence of the reaction are examined. Experiments were conducted on a CG to UA double mutant (DM), which changes the base triple to one involving an unprotonated C41. Below pH 6, the DM has a steeper dependence on pH than the wild-type (WT), consistent with a single protonation misfolding the core; this conclusion is also supported by thermal denaturation studies. Between pH 6 and 8, the WT and DM display nearly identical catalytic metal ion and H+ binding profiles. In contrast, over the same pH range, the WT and DM have distinct structural ion binding profiles; for the WT, binding is favored at lower pH, whereas the DM shows no pH dependence. These data localize the structural ion to the vicinity of the C41 motif. An overall model is presented that accommodates binding affinity, coupling, and positioning of the two metal ions and the two protons within the ribozyme. The data suggest that a protonated base triple allows the WT ribozyme to maintain appreciable activity at acidic pH, which could play an important role in the life cycle of the virus.

Selectivity of externally facing ion-binding sites in the Na/K pump to alkali metals and organic cations

PubMed Central

Ratheal, Ian M.; Virgin, Gail K.; Yu, Haibo; Roux, Benoît; Gatto, Craig; Artigas, Pablo

2010-01-01

The Na/K pump is a P-type ATPase that exchanges three intracellular Na+ ions for two extracellular K+ ions through the plasmalemma of nearly all animal cells. The mechanisms involved in cation selection by the pump's ion-binding sites (site I and site II bind either Na+ or K+; site III binds only Na+) are poorly understood. We studied cation selectivity by outward-facing sites (high K+ affinity) of Na/K pumps expressed in Xenopus oocytes, under voltage clamp. Guanidinium+, methylguanidinium+, and aminoguanidinium+ produced two phenomena possibly reflecting actions at site III: (i) voltage-dependent inhibition (VDI) of outwardly directed pump current at saturating K+, and (ii) induction of pump-mediated, guanidinium-derivative–carried inward current at negative potentials without Na+ and K+. In contrast, formamidinium+ and acetamidinium+ induced K+-like outward currents. Measurement of ouabain-sensitive ATPase activity and radiolabeled cation uptake confirmed that these cations are external K+ congeners. Molecular dynamics simulations indicate that bound organic cations induce minor distortion of the binding sites. Among tested metals, only Li+ induced Na+-like VDI, whereas all metals tested except Na+ induced K+-like outward currents. Pump-mediated K+-like organic cation transport challenges the concept of rigid structural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Furthermore, actions by guanidinium+ derivatives suggest that Na+ binds to site III in a hydrated form and that the inward current observed without external Na+ and K+ represents cation transport when normal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites. PMID:20937860

The Structure of the Metal Transporter Tp34 and its Affinity for Divalent Metal Ions

NASA Astrophysics Data System (ADS)

Knutsen, Gregory; Deka, Ranjit; Brautigam, Chad; Tomchick, Diana; Machius, Mischa; Norgard, Michael

2007-10-01

Tp34 is periplasmic membrane protein of the nonculitvatable spirochete Treponema pallidum, the pathogen of syphillis. It was proposed that Tp34 is a divalent metal transporter, but the identity of the preferred metal ion(s) was unclear. In this study we investigated the ability of divalent metal ions to induce rTp34 dimerization using hydrodynamic techniques and determine the crystal structure of metal bound forms. Using analytical ultracentrifugation sedimentation velocity experiments, we determined that cobalt is superior to nickel at inducing the dimerization of rTp34. rTp34 was crystallized and selected crystals were incubated at a pH 7.5 with CuSO4 and NiSO4. Diffraction experiments were conducted and the processed electron density maps showed that copper was bound to the major metal binding site as well as to three additional minor binding sites. By contrast nickel was only bound to the major metal binding site in one monomer and to three additional minor sites. These results along with previous findings support evidence of Tp34 being involved with metal transport and/or iron utilization.

Covalent modifications of the amyloid beta peptide by hydroxynonenal: Effects on metal ion binding by monomers and insights into the fibril topology.

PubMed

Grasso, G; Komatsu, H; Axelsen, P H

2017-09-01

Amyloid β peptides (Aβ) and metal ions are associated with oxidative stress in Alzheimer's disease (AD). Oxidative stress, acting on ω-6 polyunsaturated fatty acyl chains, produces diverse products, including 4-hydroxy-2-nonenal (HNE), which can covalently modify the Aβ that helped to produce it. To examine possible feedback mechanisms involving Aβ, metal ions and HNE production, the effects of HNE modification and fibril formation on metal ion binding was investigated. Results indicate that copper(II) generally inhibits the modification of His side chains in Aβ by HNE, but that once modified, copper(II) still binds to Aβ with high affinity. Fibril formation protects only one of the three His residues in Aβ from HNE modification, and this protection is consistent with proposed models of fibril structure. These results provide insight into a network of biochemical reactions that may be operating as a consequence of oxidative stress in AD, or as part of the pathogenic process. Copyright © 2016. Published by Elsevier Inc.

Ion adsorption at the rutile-water interface: linking molecular and macroscopic properties.

PubMed

Zhang, Z; Fenter, P; Cheng, L; Sturchio, N C; Bedzyk, M J; Predota, M; Bandura, A; Kubicki, J D; Lvov, S N; Cummings, P T; Chialvo, A A; Ridley, M K; Bénézeth, P; Anovitz, L; Palmer, D A; Machesky, M L; Wesolowski, D J

2004-06-08

A comprehensive picture of the interface between aqueous solutions and the (110) surface of rutile (alpha-TiO2) is being developed by combining molecular-scale and macroscopic approaches, including experimental measurements, quantum calculations, molecular simulations, and Gouy-Chapman-Stern models. In situ X-ray reflectivity and X-ray standing-wave measurements are used to define the atomic arrangement of adsorbed ions, the coordination of interfacial water molecules, and substrate surface termination and structure. Ab initio calculations and molecular dynamics simulations, validated through direct comparison with the X-ray results, are used to predict ion distributions not measured experimentally. Potentiometric titration and ion adsorption results for rutile powders having predominant (110) surface expression provide macroscopic constraints of electrical double layer (EDL) properties (e.g., proton release) which are evaluated by comparison with a three-layer EDL model including surface oxygen proton affinities calculated using ab initio bond lengths and partial charges. These results allow a direct correlation of the three-dimensional, crystallographically controlled arrangements of various species (H2O, Na+, Rb+, Ca2+, Sr2+, Zn2+, Y3+, Nd3+) with macroscopic observables (H+ release, metal uptake, zeta potential) and thermodynamic/electrostatic constraints. All cations are found to be adsorbed as "inner sphere" species bonded directly to surface oxygen atoms, while the specific binding geometries and reaction stoichiometries are dependent on ionic radius. Ternary surface complexes of sorbed cations with electrolyte anions are not observed. Finally, surface oxygen proton affinities computed using the MUSIC model are improved by incorporation of ab initio bond lengths and hydrogen bonding information derived from MD simulations. This multitechnique and multiscale approach demonstrates the compatibility of bond-valence models of surface oxygen proton affinities and Stern-based models of the EDL structure, with the actual molecular interfacial distributions observed experimentally, revealing new insight into EDL properties including specific binding sites and hydration states of sorbed ions, interfacial solvent properties (structure, diffusivity, dielectric constant), surface protonation and hydrolysis, and the effect of solution ionic strength.

Simple method for determining binding energies of fullerene and complex atomic negative ions

NASA Astrophysics Data System (ADS)

Felfli, Zineb; Msezane, Alfred

2017-04-01

A robust potential which embeds fully the vital core polarization interaction has been used in the Regge pole method to explore low-energy electron scattering from C60, Eu and Nb through the total cross sections (TCSs) calculations. From the characteristic dramatically sharp resonances in the TCSs manifesting negative ion formation in these systems, we extracted the binding energies for the C60, Euand Nbanions they are found to be in outstanding agreement with the measured electron affinities of C60, Eu and Nb. Common among these considered systems, including the standard atomic Au is the formation of their ground state negative ions at the second Ramsauer-Townsend (R-T) minima of their TCSs. Indeed, this is a signature of all the fullerenes and complex atoms considered thus far. Shape resonances, R-T minima and binding energies of the resultant anions are presented. This work was supported by U.S. DOE, Basic Energy Sciences, Office of Energy Research.

Synthesis of poly(N-isopropylacrylamide) particles for metal affinity binding of peptides

PubMed Central

Tsai, Hsin-Yi; Lee, Alexander; Peng, Wei; Yates, Matthew Z.

2013-01-01

Temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgel particles with metal affinity ligands were prepared for selective binding of peptides containing the His6-tag (six consecutive histidine residues). The PNIPAM particles were copolymerized with the functional ligand vinylbenzyl iminodiacetic acid (VBIDA) through a two-stage dispersion polymerization using poly(N-vinyl pyrrolidone) (PVP) as a steric stabilizer. The resulting particles were monodisperse in size and colloidally stable over a wide range of temperature and ionic strength due to chemically grafted PVP chains. The particle size was also found to be sensitive to ionic strength and pH of the aqueous environment, likely due to the electrostatic repulsion between ionized VBIDA groups. Divalent nickel ions were chelated to the VBIDA groups, allowing selective metal affinity attachment of a His6-Cys peptide. The peptide was released upon the addition of the competitive ligand imidazole, demonstrating that the peptide attachment to the particles is reversible and selective. PMID:24176889

Nickel binding and [NiFe]-hydrogenase maturation by the metallochaperone SlyD with a single metal-binding site in Escherichia coli.

PubMed

Kaluarachchi, Harini; Altenstein, Matthias; Sugumar, Sonia R; Balbach, Jochen; Zamble, Deborah B; Haupt, Caroline

2012-03-16

SlyD (sensitive to lysis D) is a nickel metallochaperone involved in the maturation of [NiFe]-hydrogenases in Escherichia coli (E. coli) and specifically contributes to the nickel delivery step during enzyme biosynthesis. This protein contains a C-terminal metal-binding domain that is rich in potential metal-binding residues that enable SlyD to bind multiple nickel ions with high affinity. The SlyD homolog from Thermus thermophilus does not contain the extended cysteine- and histidine-rich C-terminal tail of the E. coli protein, yet it binds a single Ni(II) ion tightly. To investigate whether a single metal-binding motif can functionally replace the full-length domain, we generated a truncation of E. coli SlyD, SlyD155. Ni(II) binding to SlyD155 was investigated by using isothermal titration calorimetry, NMR and electrospray ionization mass spectrometry measurements. This in vitro characterization revealed that SlyD155 contains a single metal-binding motif with high affinity for nickel. Structural characterization by X-ray absorption spectroscopy and NMR indicated that nickel was coordinated in an octahedral geometry with at least two histidines as ligands. Heterodimerization between SlyD and another hydrogenase accessory protein, HypB, is essential for optimal hydrogenase maturation and was confirmed for SlyD155 via cross-linking experiments and NMR titrations, as were conserved chaperone and peptidyl-prolyl isomerase activities. Although these properties of SlyD are preserved in the truncated version, it does not modulate nickel binding to HypB in vitro or contribute to the maturation of [NiFe]-hydrogenases in vivo, unlike the full-length protein. This study highlights the importance of the unusual metal-binding domain of E. coli SlyD in hydrogenase biogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.

Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein.

PubMed

Galvão, Carolina W; Pedrosa, Fábio O; Souza, Emanuel M; Yates, M Geoffrey; Chubatsu, Leda S; Steffens, Maria Berenice R

2004-06-01

The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role. The RecX protein of the H. seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA. The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions. The ability of RecX to bind DNA may be relevant to its function in the SOS response.

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

Streltsov, Victor A.; Titmuss, Stephen J.; Epa, V. Chandana

Neurodegeneration observed in Alzheimer disease (AD) is believed to be related to the toxicity from reactive oxygen species (ROS) produced in the brain by the amyloid-{beta} (A{beta}) protein bound primarily to copper ions. The evidence for an oxidative stress role of A{beta}-Cu redox chemistry is still incomplete. Details of the copper binding site in A{beta} may be critical to the etiology of AD. Here we present the structure determined by combining x-ray absorption spectroscopy (XAS) and density functional theory analysis of A{beta} peptides complexed with Cu{sup 2+} in solution under a range of buffer conditions. Phosphate-buffered saline buffer salt (NaCl)more » concentration does not affect the high-affinity copper binding mode but alters the second coordination sphere. The XAS spectra for truncated and full-length A{beta}-Cu{sup 2+} peptides are similar. The novel distorted six-coordinated (3N3O) geometry around copper in the A{beta}-Cu{sup 2+} complexes include three histidines: glutamic, or/and aspartic acid, and axial water. The structure of the high-affinity Cu{sup 2+} binding site is consistent with the hypothesis that the redox activity of the metal ion bound to A{beta} can lead to the formation of dityrosine-linked dimers found in AD.« less

Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy

PubMed Central

KISSELEVA, NATALIA; KHVOROVA, ANASTASIA; WESTHOF, ERIC; SCHIEMANN, OLAV

2005-01-01

Electron paramagnetic resonance (EPR) spectroscopy is used to study the binding of MnII ions to a tertiary stabilized hammer-head ribozyme (tsHHRz) and to compare it with the binding to the minimal hammerhead ribozyme (mHHRz). Continuous wave EPR measurements show that the tsHHRz possesses a single high-affinity MnII binding site with a KD of ≤10 nM at an NaCl concentration of 0.1 M. This dissociation constant is at least two orders of magnitude smaller than the KD determined previously for the single high-affinity MnII site in the mHHRz. In addition, whereas the high-affinity MnII is displaced from the mHHRz upon binding of the aminoglycoside antibiotic neomycin B, it is not from the tsHHRz. Despite these pronounced differences in binding, a comparison between the electron spin echo envelope modulation and hyperfine sublevel correlation spectra of the minimal and tertiary stabilized HHRz demonstrates that the structure of both binding sites is very similar. This suggests that the MnII is located in both ribozymes between the bases A9 and G10.1 of the sheared G · A tandem base pair, as shown previously and in detail for the mHHRz. Thus, the much stronger MnII binding in the tsHHRz is attributed to the interaction between the two external loops, which locks in the RNA fold, trapping the MnII in the tightly bound conformation, whereas the absence of long-range loop–loop interactions in the mHHRz leads to more dynamical and open conformations, decreasing MnII binding. PMID:15611296

Calcium Sensing by Recoverin: Effect of Protein Conformation on Ion Affinity.

PubMed

Timr, Štěpán; Kadlec, Jan; Srb, Pavel; Ollila, O H Samuli; Jungwirth, Pavel

2018-04-05

The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.

Copper binding to soil fulvic and humic acids: NICA-Donnan modeling and conditional affinity spectra.

PubMed

Xu, Jinling; Tan, Wenfeng; Xiong, Juan; Wang, Mingxia; Fang, Linchuan; Koopal, Luuk K

2016-07-01

Binding of Cu(II) to soil fulvic acid (JGFA), soil humic acids (JGHA, JLHA), and lignite-based humic acid (PAHA) was investigated through NICA-Donnan modeling and conditional affinity spectrum (CAS). It is to extend the knowledge of copper binding by soil humic substances (HS) both in respect of enlarging the database of metal ion binding to HS and obtaining a good insight into Cu binding to the functional groups of FA and HA by using the NICA-Donnan model to unravel the intrinsic and conditional affinity spectra. Results showed that Cu binding to HS increased with increasing pH and decreasing ionic strength. The amount of Cu bound to the HAs was larger than the amount bound to JGFA. Milne's generic parameters did not provide satisfactory predictions for the present soil HS samples, while material-specific NICA-Donnan model parameters described and predicted Cu binding to the HS well. Both the 'low' and 'high' concentration fitting procedures indicated a substantial bidentate structure of the Cu complexes with HS. By means of CAS underlying NICA isotherm, which was scarcely used, the nature of the binding at different solution conditions for a given sample and the differences in binding mode were illustrated. It was indicated that carboxylic group played an indispensable role in Cu binding to HS in that the carboxylic CAS had stronger conditional affinity than the phenolic distribution due to its large degree of proton dissociation. The fact was especially true for JGFA and JLHA which contain much larger amount of carboxylic groups, and the occupation of phenolic sites by Cu was negligible. Comparable amounts of carboxylic and phenolic groups on PAHA and JGHA, increased the occupation of phenolic type sites by Cu. The binding strength of PAHA-Cu and JGHA-Cu was stronger than that of JGFA-Cu and JLHA-Cu. The presence of phenolic groups increased the chance of forming more stable complexes, such as the salicylate-Cu or catechol-Cu type structures. Copyright © 2016. Published by Elsevier Inc.

Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy.

PubMed

Anselmetti, Dario; Bartels, Frank Wilco; Becker, Anke; Decker, Björn; Eckel, Rainer; McIntosh, Matthew; Mattay, Jochen; Plattner, Patrik; Ros, Robert; Schäfer, Christian; Sewald, Norbert

2008-02-19

Tunable and switchable interaction between molecules is a key for regulation and control of cellular processes. The translation of the underlying physicochemical principles to synthetic and switchable functional entities and molecules that can mimic the corresponding molecular functions is called reverse molecular engineering. We quantitatively investigated autoinducer-regulated DNA-protein interaction in bacterial gene regulation processes with single atomic force microscopy (AFM) molecule force spectroscopy in vitro, and developed an artificial bistable molecular host-guest system that can be controlled and regulated by external signals (UV light exposure and thermal energy). The intermolecular binding functionality (affinity) and its reproducible and reversible switching has been proven by AFM force spectroscopy at the single-molecule level. This affinity-tunable optomechanical switch will allow novel applications with respect to molecular manipulation, nanoscale rewritable molecular memories, and/or artificial ion channels, which will serve for the controlled transport and release of ions and neutral compounds in the future.

Gas-Phase Analysis of the Complex of Fibroblast GrowthFactor 1 with Heparan Sulfate: A Traveling Wave Ion Mobility Spectrometry (TWIMS) and Molecular Modeling Study

NASA Astrophysics Data System (ADS)

Zhao, Yuejie; Singh, Arunima; Xu, Yongmei; Zong, Chengli; Zhang, Fuming; Boons, Geert-Jan; Liu, Jian; Linhardt, Robert J.; Woods, Robert J.; Amster, I. Jonathan

2017-01-01

Fibroblast growth factors (FGFs) regulate several cellular developmental processes by interacting with cell surface heparan proteoglycans and transmembrane cell surface receptors (FGFR). The interaction of FGF with heparan sulfate (HS) is known to induce protein oligomerization, increase the affinity of FGF towards its receptor FGFR, promoting the formation of the HS-FGF-FGFR signaling complex. Although the role of HS in the signaling pathways is well recognized, the details of FGF oligomerization and formation of the ternary signaling complex are still not clear, with several conflicting models proposed in literature. Here, we examine the effect of size and sulfation pattern of HS upon FGF1 oligomerization, binding stoichiometry and conformational stability, through a combination of ion mobility (IM) and theoretical modeling approaches. Ion mobility-mass spectrometry (IMMS) of FGF1 in the presence of several HS fragments ranging from tetrasaccharide (dp4) to dodecasaccharide (dp12) in length was performed. A comparison of the binding stoichiometry of variably sulfated dp4 HS to FGF1 confirmed the significance of the previously known high-affinity binding motif in FGF1 dimerization, and demonstrated that certain tetrasaccharide-length fragments are also capable of inducing dimerization of FGF1. The degree of oligomerization was found to increase in the presence of dp12 HS, and a general lack of specificity for longer HS was observed. Additionally, collision cross-sections (CCSs) of several FGF1-HS complexes were calculated, and were found to be in close agreement with experimental results. Based on the (CCSs) a number of plausible binding modes of 2:1 and 3:1 FGF1-HS are proposed.

Affinity cleavage at the putative metal-binding site of pigeon liver malic enzyme by the Fe(2+)-ascorbate system.

PubMed

Wei, C H; Chou, W Y; Huang, S M; Lin, C C; Chang, G G

1994-06-28

Pigeon liver malic enzyme was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at neutral pH and 0 or 25 degrees C. Omitting the ascorbate or replacing the ferrous ion with manganese ion did not lead to any inactivation. Manganese, magnesium, zinc, cobalt, or calcium ion at 200 molar excess over ferrous ion offered complete protection of the enzyme from Fe(2+)-induced inactivation. Ni2+ provided partial protection, while Ba2+ or imidazole was ineffective in protection. Addition of 4 mM Mn2+ or 5 mM EDTA into a partially modified enzyme stopped further inactivation of the enzyme. Inclusion of substrates (L-malate or NADP+, singly or in combination) in the incubation mixture did not affect the inactivation rate. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native pigeon liver malic enzyme had a subunit M(r) of 65,000. The inactivated enzyme with residual activity of only 0.3% was cleaved into two fragments with M(r) of 31,000 and 34,000, respectively. The cleavage site was identified as the peptide bond between Asp258 and Ile259. Native pigeon liver malic enzyme was blocked at the N-terminus. Cleavage at the putative metal-binding site exposed a new N-terminus, which was identified to be at the 34-kDa fragment containing the C-terminal half of original sequence 259-557. Our results indicated that Fe2+ catalyzed a specific oxidation of pigeon liver malic enzyme at Asp258 and/or some other essential amino acid residues that caused enzyme inactivation. The modified enzyme was then affinity cleaved at the Mn(2+)-binding site.

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

PubMed Central

Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

1999-01-01

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

Urban Biomining: Biological Extraction of Metals and Materials from Electronics Waste Using a Synthetic Biology Approach

NASA Astrophysics Data System (ADS)

Urbina-Navarrete, J.; Rothschild, L.

2016-12-01

End-of-life electronics waste (e-waste) containing toxic and valuable materials is a rapidly progressing human health and environmental issue. Using synthetic biology tools, we have developed a recycling method for e-waste. Our innovation is to use a recombinant version of a naturally-occurring silica-degrading enzyme to depolymerize the silica in metal- and glass- containing e-waste components, and subsequently, to use engineered bacterial surfaces to bind and separate metals from a solution. The bacteria with bound metals can then be used as "bio-ink" to print new circuits using a novel plasma jet electronics printing technology. Here, we present the results from our initial studies that focus on the specificity of metal-binding motifs for a cognate metal. The candidate motifs that show high affinity and specificity will be engineered into bacterial surfaces for downstream applications in biologically-mediated metal recycling. Since the chemistry and role of Cu in metalloproteins is relatively well-characterized, we are using Cu as a proxy to elucidate metal and biological ligand interactions with various metals in e-waste. We assess the binding parameters of 3 representative classes of Cu-binding motifs using isothermal titration calorimetry; 1) natural motifs found in metalloproteins, 2) consensus motifs, and 3) rationally designed peptides that are predicted, in silico, to bind Cu. Our results indicate that naturally-occurring motifs have relative high affinity and specificity for Cu (association constant for Cu Ka 104 M-1, Zn Ka 103 M-1) when competing ions are present in the aqueous milieu. However, motifs developed through rational design by applying quantum mechanical methods that take into account complexation energies of the elemental binding partners and molecular geometry of the cognate metal, not only show high affinity for the cognate metal (Cu Ka 106 M-1), but they show specificity and discrimination against other metal ions that would be competitors for the same binding sites. This is an initial proof-of-concept study that focuses on Cu-binding; however the overall objective of this research is to have peptides that selectively bind many metals from e-waste and this would allow for the separation of the metals from a solution, at ambient temperatures and under non-toxic conditions.

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence.

PubMed Central

VanScyoc, Wendy S; Sorensen, Brenda R; Rusinova, Elena; Laws, William R; Ross, J B Alexander; Shea, Madeline A

2002-01-01

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition. PMID:12414709

Occupancy of a C2-C2 type 'zinc-finger' protein domain by copper. Direct observation by electrospray ionization mass spectrometry.

PubMed

Hutchens, T W; Allen, M H; Li, C M; Yip, T T

1992-09-07

The metal ion specificity of most 'zinc-finger' metal binding domains is unknown. The human estrogen receptor protein contains two different C2-C2 type 'zinc-finger' sequences within its DNA-binding domain (ERDBD). Copper inhibits the function of this protein by mechanisms which remain unclear. We have used electrospray ionization mass spectrometry to evaluate directly the 71-residue ERDBD (K180-M250) in the absence and presence of Cu(II) ions. The ERDBD showed a high affinity for Cu and was completely occupied with 4 Cu bound; each Cu ion was evidently bound to only two ligand residues (net loss of only 2 Da per bound Cu). The Cu binding stoichiometry was confirmed by atomic absorption. These results (i) provide the first direct physical evidence for the ability of the estrogen receptor DNA-binding domain to bind Cu and (ii) document a twofold difference in the Zn- and Cu-binding capacity. Differences in the ERDBD domain structure with bound Zn and Cu are predicted. Given the relative intracellular contents of Zn and Cu, our findings demonstrate the need to investigate further the Cu occupancy of this and other zinc-finger domains both in vitro and in vivo.

Mitochondrial benzodiazepine receptor linked to inner membrane ion channels by nanomolar actions of ligands.

PubMed Central

Kinnally, K W; Zorov, D B; Antonenko, Y N; Snyder, S H; McEnery, M W; Tedeschi, H

1993-01-01

The mitochrondrial benzodiazepine receptor (mBzR) binds a subset of benzodiazepines and isoquinoline carboxamides with nanomolar affinity and consists of the voltage-dependent anion channel, the adenine nucleotide translocator, and an 18-kDa protein. The effect of ligands of the mBzR on two inner mitochondrial membrane channel activities was determined with patch-clamp techniques. The relative inhibitory potencies of the drugs resemble their binding affinities for the mBzR. Ro5-4864 and protoporphyrin IX inhibit activity of the multiple conductance channel (MCC) and the mitochondrial centum-picosiemen (mCtS) channel activities at nanomolar concentrations. PK11195 inhibits mCtS activity at similar levels. Higher concentrations of protoporphyrin IX induce MCC but possibly not mCtS activity. Clonazepam, which has low affinity for mBzR, is at least 500 times less potent at both channel activities. Ro15-1788, which also has a low mBzR affinity, inhibits MCC at very high concentrations (16 microM). The findings indicate an association of these two channel activities with the proteins forming the mBzR complex and are consistent with an interaction of inner and outer membrane channels. PMID:7679505

Biophysical costs associated with tetrodotoxin resistance in the sodium channel pore of the garter snake, Thamnophis sirtalis.

PubMed

Lee, Chong Hyun; Jones, David K; Ahern, Christopher; Sarhan, Maen F; Ruben, Peter C

2011-01-01

Tetrodotoxin (TTX) is a potent toxin that specifically binds to voltage-gated sodium channels (NaV). TTX binding physically blocks the flow of sodium ions through NaV, thereby preventing action potential generation and propagation. TTX has different binding affinities for different NaV isoforms. These differences are imparted by amino acid substitutions in positions within, or proximal to, the TTX-binding site in the channel pore. These substitutions confer TTX-resistance to a variety of species. The garter snake Thamnophis sirtalis has evolved TTX-resistance over the course of an arms race, allowing some populations of snakes to feed on tetrodotoxic newts, including Taricha granulosa. Different populations of the garter snake have different degrees of TTX-resistance, which is closely related to the number of amino acid substitutions. We tested the biophysical properties and ion selectivity of NaV of three garter snake populations from Bear Lake, Idaho; Warrenton, Oregon; and Willow Creek, California. We observed changes in gating properties of TTX-resistant (TTXr) NaV. In addition, ion selectivity of TTXr NaV was significantly different from that of TTX-sensitive NaV. These results suggest TTX-resistance comes at a cost to performance caused by changes in the biophysical properties and ion selectivity of TTXr NaV.

Binding of ReO[subscript 4];#8722; with an engineered MoO[subscript 4 superscript 2];#8722;-binding protein: towards a new approach in radiopharmaceutical applications

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

Aryal, Baikuntha P.; Brugarolas, Pedro; He, Chuan

2012-05-25

Radiolabeled biomolecules are routinely used for clinical diagnostics. {sup 99m}Tc is the most commonly used radioactive tracer in radiopharmaceuticals. {sup 188}Re and {sup 186}Re are also commonly used as radioactive tracers in medicine. However, currently available methods for radiolabeling are lengthy and involve several steps in bioconjugation processes. In this work we present a strategy to engineer proteins that may selectively recognize the perrhenate (ReO{sub 4}{sup -}) ion as a new way to label proteins. We found that a molybdate (MoO{sub 4}{sup 2-})-binding protein (ModA) from Escherichia coli can bind perrhenate with high affinity. Using fluorescence and isothermal titration calorimetrymore » measurements, we determined the dissociation constant of ModA for ReO{sub 4}{sup -} to be 541 nM and we solved a crystal structure of ModA with a bound ReO{sub 4}{sup -}. On the basis of the structure we created a mutant protein containing a disulfide linkage, which exhibited increased affinity for perrhenate (K{sub d} = 104 nM). High-resolution crystal structures of ModA (1.7 {angstrom}) and A11C/R153C mutant (2.0 {angstrom}) were solved with bound perrhenate. Both structures show that a perrhenate ion occupies the molybdate binding site using the same amino acid residues that are involved in molybdate binding. The overall structure of the perrhenate-bound ModA is unchanged compared with that of the molybdate-bound form. In the mutant protein, the bound perrhenate is further stabilized by the engineered disulfide bond.« less

NEUTRALIZATION OF THE ASPARTIC ACID RESIDUE D367, BUT NOT D454, INHIBITS BINDING OF NA+ TO THE GLUTAMATE-FREE FORM AND CYCLING OF THE GLUTAMATE TRANSPORTER EAAC1

PubMed Central

Tao, Zhen; Zhang, Zhou; Grewer, Christof

2008-01-01

Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na+ ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, D367 and D454, in Na+ cotransport. In order to test the effect of charge neutralization mutations in these positions on Na+ binding to the glutamate-free transporter, we recorded the Na+-induced anion leak current to determine the Km of EAAC1 for Na+. For EAAC1WT, this Km was determined as 120 mM. When the negative charge of D367 was neutralized by mutagenesis to asparagine, Na+ activated the anion leak current with a Km of about 2 M, indicating dramatically impaired Na+ binding to the mutant transporter. In contrast, the Na+ affinity of EAAC1D454N was virtually unchanged compared to the wild type transporter (Km = 90 mM). The reduced occupancy of the Na+ binding site of EAAC1D367N resulted in a dramatic reduction in glutamate affinity (Km = 3.6 mM, 140 mM [Na+]), which could be partially overcome by increasing extracellular [Na+]. In addition to impairing Na+ binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which D367, but not D454 is involved in coordinating the bound Na+ in the glutamate-free transporter form. PMID:16478724

Metal selectivity of the E. coli nickel metallochaperone, SlyD

PubMed Central

Kaluarachchi, Harini; Siebel, Judith F.; Kaluarachchi-Duffy, Supipi; Krecisz, Sandra; Sutherland, Duncan E. K.; Stillman, Martin J.; Zamble, Deborah B.

2012-01-01

SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal-binding capabilities, and previous work demonstrated that the protein can coordinate several types of first row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To further our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals Mn(II), Fe(II), Co(II), Cu(I) and Zn(II) were examined by using a combination of optical spectroscopy and mass spectrometry. SlyD binding to Mn(II) or to Fe(II) ions was not detected but the protein coordinates multiple ions of Co(II), Zn(II) and Cu(I) with appreciable affinities (KD ≤ nM), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is Mn(II), Fe(II) < Co(II) < Ni(II) ~ Zn(II) ≪ Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed. PMID:22047179

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Raman crystallography of RNA.

PubMed

Gong, Bo; Chen, Jui-Hui; Yajima, Rieko; Chen, Yuanyuan; Chase, Elaine; Chadalavada, Durga M; Golden, Barbara L; Carey, Paul R; Bevilacqua, Philip C

2009-10-01

Raman crystallography is the application of Raman spectroscopy to single crystals. This technique has been applied to a variety of protein molecules where it has provided unique information about biopolymer folding, substrate binding, and catalysis. Here, we describe the application of Raman crystallography to functional RNA molecules. RNA represents unique opportunities and challenges for Raman crystallography. One issue that confounds studies of RNA is its tendency to adopt multiple non-functional folds. Raman crystallography has the advantage that it isolates a single state of the RNA within the crystal and can evaluate its fold, metal ion binding properties (ligand identity, stoichiometry, and affinity), proton binding properties (identity, stoichiometry, and affinity), and catalytic potential. In particular, base-specific stretches can be identified and then associated with the binding of metal ions and protons. Because measurements are carried out in the hanging drop at ambient, rather than cryo, conditions and because RNA crystals tend to be approximately 70% solvent, RNA dynamics and conformational changes become experimentally accessible. This review focuses on experimental setup and procedures, acquisition and interpretation of Raman data, and determination of physicochemical properties of the RNA. Raman crystallographic and solution biochemical experiments on the HDV RNA enzyme are summarized and found to be in excellent agreement. Remarkably, characterization of the crystalline state has proven to help rather than hinder functional characterization of functional RNA, most likely because the tendency of RNA to fold heterogeneously is limited in a crystalline environment. Future applications of Raman crystallography to RNA are briefly discussed.

Assessment of the Density Functional Tight Binding Method for Protic Ionic Liquids

PubMed Central

2015-01-01

Density functional tight binding (DFTB), which is ∼100–1000 times faster than full density functional theory (DFT), has been used to simulate the structure and properties of protic ionic liquid (IL) ions, clusters of ions and the bulk liquid. Proton affinities for a wide range of IL cations and anions determined using DFTB generally reproduce G3B3 values to within 5–10 kcal/mol. The structures and thermodynamic stabilities of n-alkyl ammonium nitrate clusters (up to 450 quantum chemical atoms) predicted with DFTB are in excellent agreement with those determined using DFT. The IL bulk structure simulated using DFTB with periodic boundary conditions is in excellent agreement with published neutron diffraction data. PMID:25328497

Structural, energetic, spectroscopic and QTAIM analyses of cation-π interactions involving mono- and bi-cyclic ring fused benzene systems.

PubMed

Hassan, Ayorinde; Dinadayalane, Tandabany C; Grabowski, Sławomir J; Leszczynski, Jerzy

2013-12-28

The effect of increasing the number of monocyclic six-membered rings or bicyclic rings of bicyclo[2.1.1]hexenyl fused to benzene on cation-π interactions involving alkali metal ions (Li(+), Na(+), and K(+)) has been investigated. The binding energy data at the B3LYP/6-311+G(2d,2p) level clearly indicate that the binding affinity of the metal ion with benzene is enhanced by increasing the number of rings fused irrespective of a monocyclic or a bicyclic ring. Calculated binding energies are in good agreement with the available experimental results. The binding strength of cations with ligands decreases in the order Li(+) > Na(+) > K(+). Our study establishes that trisannelation of bicyclo[2.1.1]hexene to benzene facilitates a very strong interaction between benzene and cations. Infrared (IR) frequencies and nuclear magnetic resonance (NMR) chemical shifts are shown to be valuable in characterizing cation-π interactions. The C-C bonds of the central six-membered rings are weakened due to metal ion binding. Based on the Quantum Theory of Atoms in Molecules (QTAIM), we have observed the presence of stabilizing H∙∙∙H interactions in two of the considered systems as opposed to the frequent description of these interactions as non-bonded repulsive interactions. Alkali metal ion binding with those two ligands slightly reduces the strength of such H∙∙∙H interactions.

Structural basis of ligand recognition in 5-HT3 receptors

PubMed Central

Kesters, Divya; Thompson, Andrew J; Brams, Marijke; van Elk, René; Spurny, Radovan; Geitmann, Matthis; Villalgordo, Jose M; Guskov, Albert; Helena Danielson, U; Lummis, Sarah C R; Smit, August B; Ulens, Chris

2013-01-01

The 5-HT3 receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT3 receptor. In the serotonin-bound structure, we observe hydrophilic interactions with loop E-binding site residues, which might enable transitions to channel opening. In the granisetron-bound structure, we observe a critical cation–π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5-HT3 receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high-affinity ligand binding in the human 5-HT3 receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti-emetics in the 5-HT3 receptor. PMID:23196367

Two zinc-binding domains in the transporter AdcA from Streptococcus pyogenes facilitate high-affinity binding and fast transport of zinc.

PubMed

Cao, Kun; Li, Nan; Wang, Hongcui; Cao, Xin; He, Jiaojiao; Zhang, Bing; He, Qing-Yu; Zhang, Gong; Sun, Xuesong

2018-04-20

Zinc is an essential metal in bacteria. One important bacterial zinc transporter is AdcA, and most bacteria possess AdcA homologs that are single-domain small proteins due to better efficiency of protein biogenesis. However, a double-domain AdcA with two zinc-binding sites is significantly overrepresented in Streptococcus species, many of which are major human pathogens. Using molecular simulation and experimental validations of AdcA from Streptococcus pyogenes , we found here that the two AdcA domains sequentially stabilize the structure upon zinc binding, indicating an organization required for both increased zinc affinity and transfer speed. This structural organization appears to endow Streptococcus species with distinct advantages in zinc-depleted environments, which would not be achieved by each single AdcA domain alone. This enhanced zinc transport mechanism sheds light on the significance of the evolution of the AdcA domain fusion, provides new insights into double-domain transporter proteins with two binding sites for the same ion, and indicates a potential target of antimicrobial drugs against pathogenic Streptococcus species. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Gating modifier toxins isolated from spider venom: modulation of voltage-gated sodium channels and the role of lipid membranes.

PubMed

Agwa, Akello J; Peigneur, Steve; Chow, Chun Yuen; Lawrence, Nicole; Craik, David J; Tytgat, Jan; King, Glenn F; Henriques, Sonia Troeira; Schroeder, Christina I

2018-04-27

Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures that modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here we examined whether there is a relationship among spider GMT amphipathicity, membrane binding and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Crystal structure of human glycine receptor-α3 bound to antagonist strychnine.

PubMed

Huang, Xin; Chen, Hao; Michelsen, Klaus; Schneider, Stephen; Shaffer, Paul L

2015-10-08

Neurotransmitter-gated ion channels of the Cys-loop receptor family are essential mediators of fast neurotransmission throughout the nervous system and are implicated in many neurological disorders. Available X-ray structures of prokaryotic and eukaryotic Cys-loop receptors provide tremendous insights into the binding of agonists, the subsequent opening of the ion channel, and the mechanism of channel activation. Yet the mechanism of inactivation by antagonists remains unknown. Here we present a 3.0 Å X-ray structure of the human glycine receptor-α3 homopentamer in complex with a high affinity, high-specificity antagonist, strychnine. Our structure allows us to explore in detail the molecular recognition of antagonists. Comparisons with previous structures reveal a mechanism for antagonist-induced inactivation of Cys-loop receptors, involving an expansion of the orthosteric binding site in the extracellular domain that is coupled to closure of the ion pore in the transmembrane domain.

Chemically synthesized glycosides of hydroxylated flavylium ions as suitable models of anthocyanins: binding to iron ions and human serum albumin, antioxidant activity in model gastric conditions.

PubMed

Al Bittar, Sheiraz; Mora, Nathalie; Loonis, Michèle; Dangles, Olivier

2014-12-11

Polyhydroxylated flavylium ions, such as 3',4',7-trihydroxyflavylium chloride (P1) and its more water-soluble 7-O-β-d-glucopyranoside (P2), are readily accessible by chemical synthesis and suitable models of natural anthocyanins in terms of color and species distribution in aqueous solution. Owing to their catechol B-ring, they rapidly bind FeIII, weakly interact with FeII and promote its autoxidation to FeIII. Both pigments inhibit heme-induced lipid peroxidation in mildly acidic conditions (a model of postprandial oxidative stress in the stomach), the colorless (chalcone) forms being more potent than the colored forms. Finally, P1 and P2 are moderate ligands of human serum albumin (HSA), their likely carrier in the blood circulation, with chalcones having a higher affinity for HSA than the corresponding colored forms.

Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

PubMed Central

Gajdosik, Martina Srajer; Clifton, James; Josic, Djuro

2012-01-01

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed. PMID:22520159

Binding of Divalent Cations to Polygalacturonate: A Mechanism Driven by the Hydration Water.

PubMed

Huynh, Uyen T D; Lerbret, Adrien; Neiers, Fabrice; Chambin, Odile; Assifaoui, Ali

2016-02-11

We have investigated the interactions between polygalacturonate (polyGal) and four divalent cations (M(2+) = Ba(2+), Ca(2+), Mg(2+), Zn(2+)) that differ in size and affinity for water. Our results evidence that M(2+)-polyGal interactions are intimately linked to the affinity of M(2+) for water. Mg(2+) interacts so strongly with water that it remains weakly bound to polyGal (polycondensation) by sharing water molecules from its first coordination shell with the carboxylate groups of polyGal. In contrast, the other cations form transient ionic pairs with polyGal by releasing preferentially one water molecule (for Zn(2+)) or two (for Ca(2+) and Ba(2+)), which corresponds to monodentate and bidentate binding modes with carboxylates, respectively. The mechanism for the binding of these three divalent cations to polyGal can be described by two steps: (i) monocomplexation and formation of point-like cross-links between polyGal chains (at low M(2+)/Gal molar ratios, R) and (ii) dimerization (at higher R). The threshold molar ratio, R*, between these two steps depends on the nature of divalent cations and is lower for calcium ions (R* < 0.1) than for zinc and barium ions (R* > 0.3). This difference may be explained by the intermediate affinity of Ca(2+) for water with respect to those of Zn(2+) and Ba(2+), which may induce the formation of cross-links of intermediate flexibility. By comparison, the lower and higher flexibilities of the cross-links formed by Zn(2+) and Ba(2+), respectively, may shift the formation of dimers to higher molar ratios (R*).

Synthetic water soluble di-/tritopic molecular receptors exhibiting Ca2+/Mg2+ exchange.

PubMed

Lavie-Cambot, Aurélie; Tron, Arnaud; Ducrot, Aurélien; Castet, Frédéric; Kauffmann, Brice; Beauté, Louis; Allouchi, Hassan; Pozzo, Jean-Luc; Bonnet, Célia S; McClenaghan, Nathan D

2017-05-23

Structural integration of two synthetic water soluble receptors for Ca 2+ and Mg 2+ , namely 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and o-aminophenol-N,N,O-triacetic acid (APTRA), respectively, gave novel di- and tritopic ionophores (1 and 2). As Mg 2+ and Ca 2+ cannot be simultaneously complexed by the receptors, allosteric control of complexation results. Potentiometric measurements established stepwise protonation constants and showed high affinity for Ca 2+ (log K = 6.08 and 8.70 for 1 and 2, respectively) and an excellent selectivity over Mg 2+ (log K = 3.70 and 5.60 for 1 and 2, respectively), which is compatible with magnesium-calcium ion exchange. While ion-exchange of a single Mg 2+ for a single Ca 2+ is possible in both 1 and 2, the simultaneous binding of two Mg 2+ by 2 appears prohibitive for replacement of these two ions by a single Ca 2+ . Ion-binding and exchange was further rationalized by DFT calculations.

Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

PubMed

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2017-04-01

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain*

PubMed Central

Yates, Luke A.; Lumb, Craig N.; Brahme, Nina N.; Zalyte, Ruta; Bird, Louise E.; De Colibus, Luigi; Owens, Raymond J.; Calderwood, David A.; Sansom, Mark S. P.; Gilbert, Robert J. C.

2012-01-01

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P3; KD ∼100 μm) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P3 as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P2 on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P3 is affected by the presence of PO4 ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species. PMID:23132860

Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64).

PubMed

Poznar, Monika; Hołubowicz, Rafał; Wojtas, Magdalena; Gapiński, Jacek; Banachowicz, Ewa; Patkowski, Adam; Ożyhar, Andrzej; Dobryszycki, Piotr

2017-11-01

Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Calcium-independent metal-ion catalytic mechanism of anthrax edema factor

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

Shen, Yuequan; Zhukovskaya, Natalia L.; Guo, Qing

2009-11-18

Edema factor (EF), a key anthrax exotoxin, has an anthrax protective antigen-binding domain (PABD) and a calmodulin (CaM)-activated adenylyl cyclase domain. Here, we report the crystal structures of CaM-bound EF, revealing the architecture of EF PABD. CaM has N- and C-terminal domains and each domain can bind two calcium ions. Calcium binding induces the conformational change of CaM from closed to open. Structures of the EF-CaM complex show how EF locks the N-terminal domain of CaM into a closed conformation regardless of its calcium-loading state. This represents a mechanism of how CaM effector alters the calcium affinity of CaM andmore » uncouples the conformational change of CaM from calcium loading. Furthermore, structures of EF-CaM complexed with nucleotides show that EF uses two-metal-ion catalysis, a prevalent mechanism in DNA and RNA polymerases. A histidine (H351) further facilitates the catalysis of EF by activating a water to deprotonate 3'OH of ATP. Mammalian adenylyl cyclases share no structural similarity with EF and they also use two-metal-ion catalysis, suggesting the catalytic mechanism-driven convergent evolution of two structurally diverse adenylyl cyclases.« less

Facile characterization of aptamer kinetic and equilibrium binding properties using surface plasmon resonance

PubMed Central

Chang, Andrew L.; McKeague, Maureen; Smolke, Christina D.

2015-01-01

Nucleic acid aptamers find widespread use as targeting and sensing agents in nature and biotechnology. Their ability to bind an extensive range of molecular targets, including small molecules, proteins, and ions, with high affinity and specificity enables their use in diverse diagnostic, therapeutic, imaging, and gene-regulatory applications. Here, we describe methods for characterizing aptamer kinetic and equilibrium binding properties using a surface plasmon resonance-based platform. This aptamer characterization platform is broadly useful for studying aptamer–ligand interactions, comparing aptamer properties, screening functional aptamers during in vitro selection processes, and prototyping aptamers for integration into nucleic acid devices. PMID:25432760

Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding

PubMed Central

Peng, Tao; Free, Paul; Fernig, David G.; Lim, Sierin; Tomczak, Nikodem

2016-01-01

Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages’ pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages’ core and low non-specific binding to the cages’ outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage’s core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently available approaches and provides a new route to design tailored and well-controlled hybrid nanoparticles. PMID:27622533

Native Electrospray Ionization Mass Spectrometry Reveals Multiple Facets of Aptamer-Ligand Interactions: From Mechanism to Binding Constants.

PubMed

Gülbakan, Basri; Barylyuk, Konstantin; Schneider, Petra; Pillong, Max; Schneider, Gisbert; Zenobi, Renato

2018-06-20

Aptamers are oligonucleotide receptors obtained through an iterative selection process from random-sequence libraries. Though many aptamers for a broad range of targets with high affinity and selectivity have been generated, a lack of high-resolution structural data and the limitations of currently available biophysical tools greatly impede understanding of the mechanisms of aptamer-ligand interactions. Here we demonstrate that an approach based on native electrospray ionization mass spectrometry (ESI-MS) can be successfully applied to characterize aptamer-ligand complexes in all details. We studied an adenosine-binding aptamer (ABA), a l-argininamide-binding aptamer (LABA), and a cocaine-binding aptamer (CBA) and their noncovalent interactions with ligands by native ESI-MS and complemented these measurements by ion mobility spectrometry (IMS), isothermal titration calorimetry (ITC), and circular dichroism (CD) spectroscopy. The ligand selectivity of the aptamers and the respective complex stoichiometry could be determined by the native ESI-MS approach. The ESI-MS data can also help refining the binding model for aptamer-ligand complexes and deliver accurate aptamer-ligand binding affinities for specific and nonspecific binding events. For specific ligands, we found K d1 = 69.7 μM and K d2 = 5.3 μM for ABA (two binding sites); K d1 = 22.04 μM for LABA; and K d1 = 8.5 μM for CBA.

SuperPain—a resource on pain-relieving compounds targeting ion channels

PubMed Central

Gohlke, Björn O.; Preissner, Robert; Preissner, Saskia

2014-01-01

Pain is more than an unpleasant sensory experience associated with actual or potential tissue damage: it is the most common reason for physician consultation and often dramatically affects quality of life. The management of pain is often difficult and new targets are required for more effective and specific treatment. SuperPain (http://bioinformatics.charite.de/superpain/) is freely available database for pain-stimulating and pain-relieving compounds, which bind or potentially bind to ion channels that are involved in the transmission of pain signals to the central nervous system, such as TRPV1, TRPM8, TRPA1, TREK1, TRESK, hERG, ASIC, P2X and voltage-gated sodium channels. The database consists of ∼8700 ligands, which are characterized by experimentally measured binding affinities. Additionally, 100 000 putative ligands are included. Moreover, the database provides 3D structures of receptors and predicted ligand-binding poses. These binding poses and a structural classification scheme provide hints for the design of new analgesic compounds. A user-friendly graphical interface allows similarity searching, visualization of ligands docked into the receptor, etc. PMID:24271391

SuperPain--a resource on pain-relieving compounds targeting ion channels.

PubMed

Gohlke, Björn O; Preissner, Robert; Preissner, Saskia

2014-01-01

Pain is more than an unpleasant sensory experience associated with actual or potential tissue damage: it is the most common reason for physician consultation and often dramatically affects quality of life. The management of pain is often difficult and new targets are required for more effective and specific treatment. SuperPain (http://bioinformatics.charite.de/superpain/) is freely available database for pain-stimulating and pain-relieving compounds, which bind or potentially bind to ion channels that are involved in the transmission of pain signals to the central nervous system, such as TRPV1, TRPM8, TRPA1, TREK1, TRESK, hERG, ASIC, P2X and voltage-gated sodium channels. The database consists of ∼8700 ligands, which are characterized by experimentally measured binding affinities. Additionally, 100 000 putative ligands are included. Moreover, the database provides 3D structures of receptors and predicted ligand-binding poses. These binding poses and a structural classification scheme provide hints for the design of new analgesic compounds. A user-friendly graphical interface allows similarity searching, visualization of ligands docked into the receptor, etc.

Location and ion-binding of membrane-associated valinomycin, a proton nuclear magnetic resonance study.

PubMed

Meers, P; Feigenson, G W

1988-03-03

Valinomycin, incorporated in small unilamellar vesicles of perdeuterated dimyristoylphosphatidylcholine, reveals several well-resolved 1H-NMR resonances. These resonances were used to examine the location, orientation and ion-binding of membrane-bound valinomycin. The order of affinity of membrane-bound valinomycin for cations is Rb+ greater than K+ greater than Cs+ greater than Ba2+, and binding is sensitive to surface change. The exchange between bound and free forms is fast on the NMR time scale. The intrinsic binding constants, extrapolated to zero anion concentration, are similar to those determined in aqueous solution. Rb+ and K+ show 1:1 binding to valinomycin, whereas the stoichiometry of Cs+ and Ba2+ is not certain. Paramagnetic chemical shift reagents and nitroxide spin label relaxation probes were used to study the location and orientation of valinomycin in the membrane. Despite relatively fast exchange of bound cations, the time average location of the cation-free form of valinomycin is deep within the bilayer under the conditions of these experiments. Upon complexation to K+, valinomycin moves closer to the interfacial region.

Multiple Metal Binding Domains Enhance the Zn(II) Selectivity of the Divalent Metal Ion Transporter AztA†

PubMed Central

Liu, Tong; Reyes-Caballero, Hermes; Li, Chenxi; Scott, Robert A.; Giedroc, David P.

2013-01-01

Transition metal-transporting P1B-type CPx ATPases play crucial roles in mediating metal homeostasis and resistance in all cells. The degree to which N-terminal metal binding domains (MBDs) confer metal specificity to the transporter is unclear. We show that the two MBDs of the Zn/Cd/Pb effluxing pump Anabaena AztA are functionally nonequivalent, but only with respect to zinc resistance. Inactivation of the a-MBD largely abrogates resistance to high intracellular Zn(II) levels, whereas inactivation of the b-MBD is not as deleterious. In contrast, inactivation of either the a- or b-MBD has little measurable impact on Cd(II) and Pb(II) resistance. The membrane proximal b-MBD binds Zn(II) with a higher affinity than the distal N-terminal a-MBD. Facile Zn(II)-specific intermolecular transfer from the a-MBD to the higher-affinity b-MBD is readily observed by 1H–15N HSQC spectroscopy. Unlike Zn(II), Cd(II) and Pb(II) form saturated 1:1 S4 or S3(O/N) complexes with AztAaHbH, where a single metal ion bridges the two MBDs. We propose that the tandem MBDs enhance Zn(II)-specific transport, while stabilizing a non-native inter-MBD Cd/Pb cross-linked structure that is a poor substrate and/or regulator for the transporter. PMID:17824670

Self-assembling cyclic tetrapeptide from alternating C-linked carbo-beta-amino acid [(S)-beta-Caa] and alpha-aminoxy acid [(R)-Ama]: a selective chloride ion receptor.

PubMed

Sharma, Gangavaram V M; Manohar, Vennampalli; Dutta, Samit Kumar; Sridhar, Bojja; Ramesh, Venna; Srinivas, Ragampeta; Kunwar, Ajit C

2010-02-19

A cyclic tetrapeptide is prepared from alternating (S)-beta-Caa (C-linked carbo-beta-amino acid) and (R)-Ama (alpha-aminoxy acid). Extensive NMR (in CDCl(3) solution) and mass spectral (MS) studies show its halide binding capacity, with a special affinity to the chloride ion. At higher concentration it was found to form molecular aggregates as evidenced from transmission electron microscopic and atomic force microscopic analysis, confirming the formation of nanorods.

Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger

PubMed Central

Lee, ChangKeun; Huang, Yihe; Faraldo-Gómez, José D.; Jiang, Youxing

2016-01-01

Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. PMID:27183196

Engineering an FMN-based iLOV protein for the detection of arsenic ions.

PubMed

Ravikumar, Yuvaraj; Nadarajan, Saravanan Prabhu; Lee, Chong-Soon; Yun, Hyungdon

2017-05-15

Over the past few decades, genetically encoded fluorescent proteins have been widely used as efficient probes to explore and investigate the roles of metal ions in biological processes. The discovery of small FMN-based fluorescent proteins, such as iLOV and FbFP, has enabled researchers to exploit these fluorescent reporter proteins for metal-sensing applications. In this study, we report the inherent binding properties of iLOV towards arsenic ions. The fluorescence quenching of iLOV was linearly related to the concentration of arsenic ions, and engineered proteins showed better sensitivity than the wild-type protein. Engineering key residues around the chromophore converted the iLOV protein into a highly sensitive sensor for As 3+ ions. iLOV N468S exhibited an improved binding affinity with a dissociation constant of 1.5 μM. Furthermore, the circular dichroism spectra indicated that the fluorescence quenching mechanism might be related to arsenic-protein complex formation. Thus, the reagentless sensing of arsenic can potentially be exploited to determine intracellular or environmental arsenic using a genetically encoded biosensing approach. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger

DOE PAGES

Liao, Jun; Marinelli, Fabrizio; Lee, Changkeun; ...

2016-05-16

Na +/Ca 2+ exchangers utilize the Na + electrochemical gradient across the plasma membrane to extrude intracellular Ca 2+, and play a central role in Ca 2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na +, Ca 2+ or Sr 2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3:1Na +/Ca 2+ exchange stoichiometry, and reveals the conformational changes at the onset ofmore » the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. Lastly, these calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na +/Ca 2+ antiport.« less

Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags.

PubMed

Mack, Laura; Brill, Boris; Delis, Natalia; Groner, Bernd

2014-12-01

The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Investigation of the binding of Salvianolic acid B to human serum albumin and the effect of metal ions on the binding

NASA Astrophysics Data System (ADS)

Chen, Tingting; Cao, Hui; Zhu, Shajun; Lu, Yapeng; Shang, Yanfang; Wang, Miao; Tang, Yanfeng; Zhu, Li

2011-10-01

The studies on the interaction between HSA and drugs have been an interesting research field in life science, chemistry and clinical medicine. There are also many metal ions present in blood plasma, thus the research about the effect of metal ions on the interaction between drugs and plasma proteins is crucial. In this study, the interaction of Salvianolic acid B (Sal B) with human serum albumin (HSA) was investigated by the steady-state, synchronous fluorescence and circular dichroism (CD) spectroscopies. The results showed that Sal B had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. Binding parameters calculated showed that Sal B was bound to HSA with the binding affinities of 10 5 L mol -1. The thermodynamic parameters studies revealed that the binding was characterized by positive enthalpy and positive entropy changes, and hydrophobic interactions were the predominant intermolecular forces to stabilize the complex. The specific binding distance r (2.93 nm) between donor (HSA) and acceptor (Sal B) was obtained according to Förster non-radiative resonance energy transfer theory. The synchronous fluorescence experiment revealed that Sal B cannot lead to the microenvironmental changes around the Tyr and Trp residues of HSA, and the binding site of Sal B on HSA is located in hydrophobic cavity of subdomain IIA. The CD spectroscopy indicated the secondary structure of HSA is not changed in the presence of Sal B. Furthermore, The effect of metal ions (e.g. Zn 2+, Cu 2+, Co 2+, Ni 2+, Fe 3+) on the binding constant of Sal B-HSA complex was also discussed.

Interactions of ligands with active and inactive conformations of the dopamine D2 receptor.

PubMed

Malmberg, A; Mohell, N; Backlund Höök, B; Johansson, A M; Hacksell, U; Nordvall, G

1998-04-10

The affinities of 19 pharmacologically diverse dopamine D2 receptor ligands were determined for the active and inactive conformations of cloned human dopamine D2 receptors expressed in Ltk cells. The agonist [3H]quinpirole was used to selectively label the guanine nucleotide-binding protein-coupled, active receptor conformation. The antagonist [3H]raclopride, in the presence of the non-hydrolysable GTP-analogue Gpp(NH)p and sodium ions and in the absence of magnesium ions, was used to label the free inactive receptor conformation. The intrinsic activities of the ligands were determined in a forskolin-stimulated cyclic AMP assay using the same cells. An excellent correlation was shown between the affinity ratios (KR/KRG) of the ligands for the two receptor conformations and their intrinsic activity (r=0.96). The ligands included eight structurally related and enantiopure 2-aminotetralin derivatives; the enantiomers of 5-hydroxy-2-(dipropylamino)tetralin, 5-methoxy-2-(dipropylamino)tetralin, 5-fluoro-2-(dipropylamino)tetralin and 2-(dipropylamino)tetralin. The (S)-enantiomers behaved as full agonists in the cyclic AMP assay and displayed a large KR/KRG ratio. The (R)-enantiomers were classified as partial agonists and had lower ratios. The structure-affinity relationships of these compounds at the active and the inactive receptor conformations were analysed separately, and used in conjunction with a homology based receptor model of the dopamine D2 receptor. This led to proposed binding modes for agonists, antagonists and partial agonists in the 2-aminotetralin series. The concepts used in this study should be of value in the design of ligands with predetermined affinity and intrinsic activity.

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions.

PubMed

Srikant, C B; Dahan, A; Craig, C

1990-02-04

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2+, Mn2+, Ca2+ and Co2+ augmented the binding of both T* SS-14 and LTT* SS-28, while higher than 4 mM Co2+ inhibited binding of both ligands. LTT* SS-28 binding was reduced in the presence of high concentrations of Ba2+ and Mn2+ also. Interestingly Ca2+ at higher than 10 mM preferentially inhibited LTT* SS-28 binding and increased the affinity of SS-14 but not SS-28 for LTT* SS-28 binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

PubMed

Finzi, Andrés; Cloutier, Jonathan; Cohen, Eric A

2003-07-01

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

Selectivity and permeation of alkali metal ions in K+-channels.

PubMed

Furini, Simone; Domene, Carmen

2011-06-24

Ion conduction in K(+)-channels is usually described in terms of concerted movements of K(+) progressing in a single file through a narrow pore. Permeation is driven by an incoming ion knocking on those ions already inside the protein. A fine-tuned balance between high-affinity binding and electrostatic repulsive forces between permeant ions is needed to achieve efficient conduction. While K(+)-channels are known to be highly selective for K(+) over Na(+), some K(+) channels conduct Na(+) in the absence of K(+). Other ions are known to permeate K(+)-channels with a more moderate preference and unusual conduction features. We describe an extensive computational study on ion conduction in K(+)-channels rendering free energy profiles for the translocation of three different alkali ions and some of their mixtures. The free energy maps for Rb(+) translocation show at atomic level why experimental Rb(+) conductance is slightly lower than that of K(+). In contrast to K(+) or Rb(+), external Na(+) block K(+) currents, and the sites where Na(+) transport is hindered are characterized. Translocation of K(+)/Na(+) mixtures is energetically unfavorable owing to the absence of equally spaced ion-binding sites for Na(+), excluding Na(+) from a channel already loaded with K(+). Copyright © 2011 Elsevier Ltd. All rights reserved.

Kinetic contribution to extracellular Na+/K+ selectivity in the Na+/K+ pump.

PubMed

Vleeskens, Elizabeth; Clarke, Ronald J

2018-05-01

The sodium potassium pump (Na + ,K + -ATPase) shows a high selectivity for K + over Na + binding from the extracellular medium. To understand the K + selectivity in the presence of a high concentration of competing Na + ions requires consideration of more than just ion binding affinities. Here, equilibrium-based calculations of the extracellular occupation of the Na + ,K + -ATPase transport sites by Na + and K + are compared to fluxes through Na + and K + transport pathways. The results show that, under physiological conditions, there is a 332-fold selectivity for pumping of K + from the extracellular medium into the cytoplasm relative to Na + , whereas equilibrium calculations alone predict only a 7.5-fold selectivity for K + . Thus, kinetic effects make a major contribution to the determination of extracellular K + selectivity.

Insight into the Interaction of Metal Ions with TroA from Streptococcus suis

PubMed Central

Zheng, Beiwen; Zhang, Qiangmin; Gao, Jia; Han, Huiming; Li, Ming; Zhang, Jingren; Qi, Jianxun; Yan, Jinghua; Gao, George F.

2011-01-01

Background The scavenging ability of sufficient divalent metal ions is pivotal for pathogenic bacteria to survive in the host. ATP-binding cassette (ABC)-type metal transporters provide a considerable amount of different transition metals for bacterial growth. TroA is a substrate binding protein for uptake of multiple metal ions. However, the function and structure of the TroA homologue from the epidemic Streptococcus suis isolates (SsTroA) have not been characterized. Methodology/Principal Findings Here we determined the crystal structure of SsTroA from a highly pathogenic streptococcal toxic shock syndrome (STSS)-causing Streptococcus suis in complex with zinc. Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that apo-SsTroA binds Zn2+ and Mn2+. Both metals bind to SsTroA with nanomolar affinity and stabilize the protein against thermal unfolding. Zn2+ and Mn2+ induce distinct conformational changes in SsTroA compared with the apo form as confirmed by both circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra. NMR data also revealed that Zn2+/Mn2+ bind to SsTroA in either the same site or an adjacent region. Finally, we found that the folding of the metal-bound protein is more compact than the corresponding apoprotein. Conclusions/Significance Our findings reveal a mechanism for uptake of metal ions in S. suis and this mechanism provides a reasonable explanation as to how SsTroA operates in metal transport. PMID:21611125

Cooperative Allosteric Ligand Binding in Calmodulin

NASA Astrophysics Data System (ADS)

Nandigrami, Prithviraj

Conformational dynamics is often essential for a protein's function. For example, proteins are able to communicate the effect of binding at one site to a distal region of the molecule through changes in its conformational dynamics. This so called allosteric coupling fine tunes the sensitivity of ligand binding to changes in concentration. A conformational change between a "closed" (apo) and an "open" (holo) conformation upon ligation often produces this coupling between binding sites. Enhanced sensitivity between the unbound and bound ensembles leads to a sharper binding curve. There are two basic conceptual frameworks that guide our visualization about ligand binding mechanisms. First, a ligand can stabilize the unstable "open" state from a dynamic ensemble of conformations within the unbound basin. This binding mechanism is called conformational selection. Second, a ligand can weakly bind to the low-affinity "closed" state followed by a conformational transition to the "open" state. In this dissertation, I focus on molecular dynamics simulations to understand microscopic origins of ligand binding cooperativity. A minimal model of allosteric binding transitions must include ligand binding/unbinding events, while capturing the transition mechanism between two distinct meta-stable free energy basins. Due in part to computational timescales limitations, work in this dissertation describes large-scale conformational transitions through a simplified, coarse-grained model based on the energy basins defined by the open and closed conformations of the protein Calmodulin (CaM). CaM is a ubiquitous calcium-binding protein consisting of two structurally similar globular domains connected by a flexible linker. The two domains of CaM, N-terminal domain (nCaM) and C-terminal domain (cCaM) consists of two helix-loop-helix motifs (the EF-hands) connected by a flexible linker. Each domain of CaM consists of two binding loops and binds 2 calcium ions each. The intact domain binds up to 4 calcium ions. The simulations use a coupled molecular dynamics/monte carlo scheme where the protein dynamics is simulated explicitly, while ligand binding/unbinding are treated implicitly. In the model, ligand binding/unbinding events coupled with a conformational change of the protein within the grand canonical ensemble. Here, ligand concentration is controlled through the chemical potential (micro). This allows us to use a simple thermodynamic model to analyze the simulated data and quantify binding cooperativity. Simulated binding titration curves are calculated through equilibrium simulations at different values of micro. First, I study domain opening transitions of isolated nCaM and cCaM in the absence of calcium. This work is motivated by results from a recent analytic variational model that predicts distinct domain opening transition mechanism for the domains of CaM. This is a surprising result because the domains have the same folded state topology. In the simulations, I find the two domains of CaM have distinct transition mechanism over a broad range of temperature, in harmony with the analytic predictions. In particular, the simulated transition mechanism of nCaM follows a two-state behavior, while domain opening in cCaM involves global unfolding and refolding of the tertiary structure. The unfolded intermediate also appears in the landscape of nCaM, but at a higher temperature than it appears in cCaM's energy landscape. This is consistent with nCaM's higher thermal stability. Under approximate physiological conditions, majority of the sampled transitions in cCaM involves unfolding and refolding during conformational change. Kinetically, the transient unfolding and refolding in cCaM significantly slows the domain opening and closing rates in cCaM. Second, I investigate the structural origins of binding affinity and allosteric cooperativity of binding 2 calcium-ions to each domain of CaM. In my work, I predict the order of binding strength of CaM's loops. I analyze simulated binding curves within the framework of the classic Monod-Wyman-Changeux (MWC) model of allostery to extract the binding free energies to the closed and open ensembles. The simulations predict that cCaM binds calcium with higher affinity and greater cooperativity than nCaM. Where it is possible to compare, these predictions are in good agreement with experimental results. The analysis of the simulations offers a rationale for why the two domains differ in cooperativity: the higher cooperativity of cCaM is due to larger difference in affinity of its binding loops. Third, I extend the work to investigate structural origins of binding cooperativity of 4 calcium-ions to intact CaM. I characterize the microscopic cooperativities of each ligation state and provide a kinetic description of the binding mechanism. Due to the heterogeneous nature of CaM's loops, as predicted in our simulations of isolated domains, I focus on investigating the influence of this heterogeneity on the kinetic flux of binding pathways as a function of concentration. The formalism developed for Network Models of protein folding kinetics, is used to evaluate the directed flux of all possible pathways between unligated and fully loaded CaM. (Abstract shortened by ProQuest.).

Halorhodopsin pumps Cl– and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

PubMed Central

Gunner, M. R.

2014-01-01

Key mutations differentiate the functions of homologous proteins. One example compares the inward ion pump halorhodopsin (HR) and the outward proton pump bacteriorhodopsin (BR). Of the nine essential buried ionizable residues in BR, six are conserved in HR. However, HR changes three BR acids, D85 in a central cluster of ionizable residues, D96, nearer the intracellular, and E204, nearer the extracellular side of the membrane to the small, neutral amino acids T111, V122, and T230, respectively. In BR, acidic amino acids are stationary anions whose proton affinity is modulated by conformational changes, establishing a sequence of directed binding and release of protons. Multiconformation continuum electrostatics calculations of chloride affinity and residue protonation show that, in reaction intermediates where an acid is ionized in BR, a Cl– is bound to HR in a position near the deleted acid. In the HR ground state, Cl– binds tightly to the central cluster T111 site and weakly to the extracellular T230 site, recovering the charges on ionized BR-D85 and neutral E204 in BR. Imposing key conformational changes from the BR M intermediate into the HR structure results in the loss of Cl– from the central T111 site and the tight binding of Cl– to the extracellular T230 site, mirroring the changes that protonate BR-D85 and ionize E204 in BR. The use of a mobile chloride in place of D85 and E204 makes HR more susceptible to the environmental pH and salt concentrations than BR. These studies shed light on how ion transfer mechanisms are controlled through the interplay of protein and ion electrostatics. PMID:25362051

Ion exchange materials, method of forming ion exchange materials, and methods of treating liquids

DOEpatents

Wertsching, Alan K.; Peterson, Eric S.; Wey, John E.

2007-12-25

The invention includes an ion affinity material having an organic component which is sulfonated and which is chemically bonded to an inorganic substrate component. The invention includes a method of forming a metal binding material. A solid support material comprising surface oxide groups is provided and an organic component having at least one alkyl halide is covalently linked to at least some of the surface oxide groups to form a modified support material. The at least one alkyl halide is subsequently converted into an alkyl sulfonate. The invention further includes a method and system for extracting ions from a liquid. An ion exchange material having a sulfonated alkyl silane component covalently bonded to a metal oxide support material is provided and a liquid is exposed to the ion exchange material.

Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

NASA Astrophysics Data System (ADS)

Batoulis, Helena; Schmidt, Thomas H.; Weber, Pascal; Schloetel, Jan-Gero; Kandt, Christian; Lang, Thorsten

2016-04-01

Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca2+ and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca2+ ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca2+ ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems.

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates. Conclusions Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches. PMID:22112852

[Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

PubMed

Skvortsov, A N; Zatulovskiĭ, E A; Puchkova, L V

2012-01-01

It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

Ion exchange selectivity for cross-linked polyacrylic acid

NASA Technical Reports Server (NTRS)

May, C. E.; Philipp, W. H.

1983-01-01

The ion separation factors for 21 common metal ions with cross-linked polyacrylic acid were determined as a function of pH and the percent of the cross-linked polyacrylic acid neutralized. The calcium ion was used as a reference. At a pH of 5 the decreasing order of affinity of the ions for the cross-linked polyacrylic acid was found to be: Hg++, Fe+++, Pb++, Cr+++, Cu++, Cd++, Al+++, Ag+, Zn++, Ni++, Mn++, Co++, Ca++, Sr++, Ba++, Mg++, K+, Rb+, Cs+, Na+, and Li+. Members of a chemical family exhibited similar selectivities. The Hg++ ion appeared to be about a million times more strongly bound than the alkali metal ions. The relative binding of most of the metal ions varied with pH; the very tightly and very weakly bound ions showed the largest variations with pH. The calcium ion-hydrogen ion equilibrium was perturbed very little by the presence of the other ions. The separation factors and selectivity coefficients are discussed in terms of equilibrium and thermodynamic significance.

α-Conotoxin dendrimers have enhanced potency and selectivity for homomeric nicotinic acetylcholine receptors.

PubMed

Wan, Jingjing; Huang, Johnny X; Vetter, Irina; Mobli, Mehdi; Lawson, Joshua; Tae, Han-Shen; Abraham, Nikita; Paul, Blessy; Cooper, Matthew A; Adams, David J; Lewis, Richard J; Alewood, Paul F

2015-03-11

Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3β2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels.

Structure of a highly acidic β-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs+-selective binding site

PubMed Central

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Matsumoto, Fumiko; Shibazaki, Chie; Shimizu, Rumi; Yamada, Mitsugu; Adachi, Motoyasu; Tamada, Taro; Kawamoto, Masahide; Tokunaga, Hiroko; Ishibashi, Matsujiro; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

2015-01-01

Environmentally friendly absorbents are needed for Sr2+ and Cs+, as the removal of the radioactive Sr2+ and Cs+ that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs+ or Sr2+. The crystal structure of a halophilic β-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space group P31 using X-ray crystallography. Moreover, the locations of bound Sr2+ and Cs+ ions were identified by anomalous X-ray diffraction. The location of one Cs+-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na+ (90 mM Na+/10 mM Cs+). From an activity assay using isothermal titration calorimetry, the bound Sr2+ and Cs+ ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs+-binding site provides important information that is useful for the design of artificial Cs+-binding sites that may be useful in the bioremediation of radioactive isotopes. PMID:25760604

Noncovalent Complexation of Monoamine Neurotransmitters and Related Ammonium Ions by Tetramethoxy Tetraglucosylcalix[4]arene

NASA Astrophysics Data System (ADS)

Torvinen, Mika; Kalenius, Elina; Sansone, Francesco; Casnati, Alessandro; Jänis, Janne

2012-02-01

The noncovalent complexation of monoamine neurotransmitters and related ammonium and quaternary ammonium ions by a conformationally flexible tetramethoxy glucosylcalix[4]arene was studied by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. The glucosylcalixarene exhibited highest binding affinity towards serotonin, norepinephrine, epinephrine, and dopamine. Structural properties of the guests, such as the number, location, and type of hydrogen bonding groups, length of the alkyl spacer between the ammonium head-group and the aromatic ring structure, and the degree of nitrogen substitution affected the complexation. Competition experiments and guest-exchange reactions indicated that the hydroxyl groups of guests participate in intermolecular hydrogen bonding with the glucocalixarene.

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTP gamma S to brain membranes.

PubMed

Moni, R W; Romero, F S; Daly, J W

1995-08-01

1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog, Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes. 2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 microM) for A1-adenosine receptors, 30% (100 microM) for A2a-adenosine receptors, 20% (2 microM) for alpha 2-adrenergic receptors, and 30% (10 microM) for 5HT1A receptors. High affinity agonist binding for A1-, alpha 2-, and 5HT1A-receptors was virtually abolished by GTP gamma S in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin. 3. The effect of adenoregulin on binding of N6-cyclohexyladenosine ([3H]CHA) to A1-receptors was relatively slow and was irreversible. Adenoregulin increased the Bmax value for [3H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [3H]CHA dissociation. Binding of the A1-selective antagonist, [3H]DPCPX, was maximally enhanced by only 13% at 2 microM adenoregulin. Basal and A1-adenosine receptor-stimulated binding of [35S]GTP gamma S were maximally enhanced 45% and 23%, respectively, by 50 microM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [3H]CHA and [3H]DPCPX were enhanced by adenoregulin. Binding of [3H]CHA to membranes from DDT1 MF-2 cells was maximally enhanced 17% at 20 microM adenoregulin. In intact DDT1 MF-2 cells, 20 microM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediated via the adenosine A1 receptor. 4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.

Mononuclear zinc(II) complexes of 2-((2-(piperazin-1-yl)ethylimino)methyl)-4-substituted phenols: Synthesis, structural characterization, DNA binding and cheminuclease activities

NASA Astrophysics Data System (ADS)

Ravichandran, J.; Gurumoorthy, P.; Karthick, C.; Kalilur Rahiman, A.

2014-03-01

Four new zinc(II) complexes [Zn(HL1-4)Cl2] (1-4), where HL1-4 = 2-((2-(piperazin-1-yl)ethylimino)methyl)-4-substituted phenols, have been isolated and fully characterized using various spectro-analytical techniques. The X-ray crystal structure of complex 4 shows the distorted trigonal-bipyramidal coordination geometry around zinc(II) ion. The crystal packing is stabilized by intermolecular NH⋯O hydrogen bonding interaction. The complexes display no d-d electronic band in the visible region due to d10 electronic configuration of zinc(II) ion. The electrochemical properties of the synthesized ligands and their complexes exhibit similar voltammogram at reduction potential due to electrochemically innocent Zn(II) ion, which evidenced that the electron transfer is due to the nature of the ligand. Binding interaction of complexes with calf thymus DNA was studied by UV-Vis absorption titration, viscometric titration and cyclic voltammetry. All complexes bind with CT DNA by intercalation, giving the binding affinity in the order of 2 > 1 ≫ 3 > 4. The prominent cheminuclease activity of complexes on plasmid DNA (pBR322 DNA) was observed in the absence and presence of H2O2. Oxidative pathway reveals that the underlying mechanism involves hydroxyl radical.

Locating the Binding Sites of Pb(II) Ion with Human and Bovine Serum Albumins

PubMed Central

Belatik, Ahmed; Hotchandani, Surat; Carpentier, Robert; Tajmir-Riahi, Heidar-Ali

2012-01-01

Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of KPb-HSA = 8.2 (±0.8)×104 M−1 and KPb-BSA = 7.5 (±0.7)×104 M−1. The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization. PMID:22574219

Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

PubMed Central

2012-01-01

Background Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1st and 2nd EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants. Results The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca2+ binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 105 and 5.0 × 102 M-1 with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1st non-canonical EF-hand is largely enhanced by the binding of Ca2+ to the 2nd canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca2+ binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg2+-binding in NCaBD. Conclusions We demonstrated that the 1st non-canonical EF-hand of NOX5 has very weak Ca2+ binding affinity compared with the 2nd canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca2+ binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca2+ specific. Graphical abstract PMID:22490336

Vanadium-Binding Ability of Nucleoside Diphosphate Kinase from the Vanadium-Rich Fan Worm, Pseudopotamilla occelata.

PubMed

Yamaguchi, Nobuo; Yoshinaga, Masafumi; Kamino, Kei; Ueki, Tatsuya

2016-06-01

Polychaete fan worms and ascidians accumulate high levels of vanadium ions. Several vanadiumbinding proteins, known as vanabins, have been found in ascidians. However, no vanadium-binding factors have been isolated from the fan worm. In the present study, we sought to identify vanadiumbinding proteins in the branchial crown of the fan worm using immobilized metal ion affinity chromatography. A nucleoside diphosphate kinase (NDK) homolog was isolated and determined to be a vanadium-binding protein. Kinase activity of the NDK homologue, PoNDK, was suppressed by the addition of V(IV), but was unaffected by V(V). The effect of V(IV) on PoNDK precedes its activation by Mg(II). This is the first report to describe the relationship between NDK and V(IV). PoNDK is located in the epidermis of the branchial crown, and its distribution is very similar to that of vanadium. These results suggest that PoNDK is associated with vanadium accumulation and metabolism in P. occelata.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

PubMed Central

2015-01-01

Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes. PMID:26681483

Copper removal by algal biomass: biosorbents characterization and equilibrium modelling.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Pinheiro, José P S; Domingos, Rute F; Boaventura, Rui A R

2009-04-30

The general principles of Cu(II) binding to algal waste from agar extraction, composite material and algae Gelidium, and different modelling approaches, are discussed. FTIR analyses provided a detailed description of the possible binding groups present in the biosorbents, as carboxylic groups (D-glucuronic and pyruvic acids), hydroxyl groups (cellulose, agar and floridean starch) and sulfonate groups (sulphated galactans). Potentiometric acid-base titrations showed a heterogeneous distribution of two major binding groups, carboxyl and hydroxyl, following the quasi-Gaussian affinity constant distribution suggested by Sips, which permitted to estimate the maximum amount of acid functional groups (0.36, 0.25 and 0.1 mmol g(-1)) and proton binding parameters (pK(H)=5.0, 5.3 and 4.4; m(H)=0.43, 0.37, 0.33), respectively for algae Gelidium, algal waste and composite material. A non-ideal, semi-empirical, thermodynamically consistent (NICCA) isotherm fitted better the experimental ion binding data for different pH values and copper concentrations, considering only the acid functional groups, than the discrete model. Values of pK(M) (3.2; 3.6 and 3.3), n(M) (0.98, 0.91, 1.0) and p (0.67, 0.53 and 0.43) were obtained, respectively for algae Gelidium, algal waste and composite material. NICCA model reflects the complex macromolecular systems that take part in biosorption considering the heterogeneity of the biosorbent, the competition between protons and metals ions to the binding sites and the stoichiometry for different ions.

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

PubMed Central

Senning, Eric N.; Aman, Teresa K.

2016-01-01

Biological membranes are complex assemblies of lipids and proteins that serve as platforms for cell signaling. We have developed a novel method for measuring the structure and dynamics of the membrane based on fluorescence resonance energy transfer (FRET). The method marries four technologies: (1) unroofing cells to isolate and access the cytoplasmic leaflet of the plasma membrane; (2) patch-clamp fluorometry (PCF) to measure currents and fluorescence simultaneously from a membrane patch; (3) a synthetic lipid with a metal-chelating head group to decorate the membrane with metal-binding sites; and (4) transition metal ion FRET (tmFRET) to measure short distances between a fluorescent probe and a transition metal ion on the membrane. We applied this method to measure the density and affinity of native and introduced metal-binding sites in the membrane. These experiments pave the way for measuring structural rearrangements of membrane proteins relative to the membrane. PMID:26755772

Thinking in cycles: MWC is a good model for acetylcholine receptor-channels

PubMed Central

Auerbach, Anthony

2012-01-01

Abstract Neuromuscular acetylcholine receptors have long been a model system for understanding the mechanisms of operation of ligand-gated ion channels and fast chemical synapses. These five subunit membrane proteins have two allosteric (transmitter) binding sites and a distant ion channel domain. Occupation of the binding sites by agonist molecules transiently increases the probability that the channel is ion-permeable. Recent experiments show that the Monod, Wyman and Changeux formalism for allosteric proteins, originally developed for haemoglobin, is an excellent model for acetylcholine receptors. By using mutations and single-channel electrophysiology, the gating equilibrium constants for receptors with zero, one or two bound agonist molecules, and the agonist association and dissociation rate constants from both the closed- and open-channel conformations, have been estimated experimentally. The change in affinity for each transmitter molecule between closed and open conformations provides ∼–5.1 kcal mol−1 towards the global gating isomerization of the protein. PMID:21807612

Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

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

Sine, Steven M.; Huang, Sun; Li, Shu-Xing

2013-09-01

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less

Peroxisome proliferator-binding protein: identification and partial characterization of nafenopin-, clofibric acid-, and ciprofibrate-binding proteins from rat liver.

PubMed Central

Lalwani, N D; Alvares, K; Reddy, M K; Reddy, M N; Parikh, I; Reddy, J K

1987-01-01

Peroxisome proliferators (PP) induce a highly predictable pleiotropic response in rat and mouse liver that is characterized by hepatomegaly, increase in peroxisome number in hepatocytes, and induction of certain peroxisomal enzymes. The PP-binding protein (PPbP) was purified from rat liver cytosol by a two-step procedure involving affinity chromatography and ion-exchange chromatography. Three PP, nafenopin and its structural analogs clofibric acid and ciprofibrate, were used as affinity ligands and eluting agents. This procedure yields a major protein with an apparent Mr of 70,000 on NaDodSO4/PAGE in the presence of reducing agent and Mr 140,000 (Mr 140,000-160,000) on gel filtration and polyacrylamide gradient gel electrophoresis under nondenaturing conditions, indicating that the active protein is a dimer. This protein has an acidic pI of 4.2 under nondenaturing conditions, which rises to 5.6 under denaturing conditions. The isolation of the same Mr 70,000 protein with three different, but structurally related, agents as affinity ligands and the immunological identity of the isolated proteins constitute strong evidence that this protein is the PPbP capable of recognizing PP that are structurally related to clofibrate. The PPbP probably plays an important role in the regulation of PP-induced pleiotropic response. Images PMID:3474650

Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT.

PubMed

Kowald, Gregory R; Stürzenbaum, Stephen R; Blindauer, Claudia A

2016-01-05

Earthworms express, as most animals, metallothioneins (MTs)-small, cysteine-rich proteins that bind d(10) metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, whilst a single Cd₇wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II), expressions in the presence of Zn(II) yielded mixtures. The average affinities of wMT-2 determined for either Cd(II) or Zn(II) are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by ¹H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo.

Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases.

PubMed

Soya, Naoto; Shoemaker, Glen K; Palcic, Monica M; Klassen, John S

2009-11-01

The first comparative thermodynamic study of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24 degrees C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn(2+), both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 10(4) M(-1) was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (approximately 1 x 10(4) M(-1)), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn(2+), both GTA and GTB recognize their respective trisaccharide products but with a low affinity approximately 10(3) M(-1); the presence of UDP and Mn(2+) has no effect on A trisaccharide binding but precludes B-trisaccharide binding.

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins.

PubMed

Suzuki, N; Mihashi, K

1991-01-01

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

How Structure Defines Affinity in Protein-Protein Interactions

PubMed Central

Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

2014-01-01

Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

ESI-MS measurements for the equilibrium constants of copper(II)-insulin complexes.

PubMed

Gülfen, Mustafa; Özdemir, Abdil; Lin, Jung-Lee; Chen, Chung-Hsuan

2018-06-01

Trace elements regulate many biological reactions in the body. Copper(II) is known as one of trace elements and capable of binding to proteins. Insulin is a blood glucose-lowering peptide hormone and it is secreted by the pancreatic β-cells. In this study, Cu(II)-insulin complexes were investigated by using ESI-MS method. Insulin molecule gives ESI-MS peaks at +4, +5, +6 and +7 charged states. Cu(II)-insulin complexes can be monitored and quantified on the ESI-MS spectra as the shifted peaks according to insulin peaks. The solutions of Cu(II)-insulin complexes at different pHs and mole ratios of Cu(II) ions to insulin molecule were measured on the ESI-MS. The highest complex formation ratio for Cu(II)-insulin were found at pH 7. The multiple bindings of Cu(II) ions to insulin molecule was observed. The formation equilibrium constants of Cu(II)-insulin complexes were calculated as Kf 1 : 3.34 × 10 4 , Kf 2 : 2.99 × 10 4 , Kf 3 : 7.00 × 10 3 and Kf 4 :2.86 × 10 3 . The specific binding property of Cu(II) ions was controlled by using different spray ion sources including electrospray and nano-electrospray. The binding property of Cu(II) also investigated by MS/MS fragmentation. It was concluded from the ESI-MS measurements that Cu(II) ion has a high affinity to insulin molecules to form stable complexes. Copyright © 2018 Elsevier B.V. All rights reserved.

Protonation states of histidine and other key residues in deoxy normal human adult hemoglobin by neutron protein crystallography

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

Kovalevsky, Andrey, E-mail: ayk@lanl.gov; Chatake, Toshiyuki; Shibayama, Naoya

2010-11-01

Using neutron diffraction analysis, the protonation states of 35 of 38 histidine residues were determined for the deoxy form of normal human adult hemoglobin. Distal and buried histidines may contribute to the increased affinity of the deoxy state for hydrogen ions and its decreased affinity for oxygen compared with the oxygenated form. The protonation states of the histidine residues key to the function of deoxy (T-state) human hemoglobin have been investigated using neutron protein crystallography. These residues can reversibly bind protons, thereby regulating the oxygen affinity of hemoglobin. By examining the OMIT F{sub o} − F{sub c} and 2F{sub o}more » − F{sub c} neutron scattering maps, the protonation states of 35 of the 38 His residues were directly determined. The remaining three residues were found to be disordered. Surprisingly, seven pairs of His residues from equivalent α or β chains, αHis20, αHis50, αHis58, αHis89, βHis63, βHis143 and βHis146, have different protonation states. The protonation of distal His residues in the α{sub 1}β{sub 1} heterodimer and the protonation of αHis103 in both subunits demonstrates that these residues may participate in buffering hydrogen ions and may influence the oxygen binding. The observed protonation states of His residues are compared with their ΔpK{sub a} between the deoxy and oxy states. Examination of inter-subunit interfaces provided evidence for interactions that are essential for the stability of the deoxy tertiary structure.« less

Assigning crystallographic electron densities with free energy calculations—The case of the fluoride channel Fluc

PubMed Central

2018-01-01

Approximately 90% of the structures in the Protein Data Bank (PDB) were obtained by X-ray crystallography or electron microscopy. Whereas the overall quality of structure is considered high, thanks to a wide range of tools for structure validation, uncertainties may arise from density maps of small molecules, such as organic ligands, ions or water, which are non-covalently bound to the biomolecules. Even with some experience and chemical intuition, the assignment of such disconnected electron densities is often far from obvious. In this study, we suggest the use of molecular dynamics (MD) simulations and free energy calculations, which are well-established computational methods, to aid in the assignment of ambiguous disconnected electron densities. Specifically, estimates of (i) relative binding affinities, for instance between an ion and water, (ii) absolute binding free energies, i.e., free energies for transferring a solute from bulk solvent to a binding site, and (iii) stability assessments during equilibrium simulations may reveal the most plausible assignments. We illustrate this strategy using the crystal structure of the fluoride specific channel (Fluc), which contains five disconnected electron densities previously interpreted as four fluoride and one sodium ion. The simulations support the assignment of the sodium ion. In contrast, calculations of relative and absolute binding free energies as well as stability assessments during free MD simulations suggest that four of the densities represent water molecules instead of fluoride. The assignment of water is compatible with the loss of these densities in the non-conductive F82I/F85I mutant of Fluc. We critically discuss the role of the ion force fields for the calculations presented here. Overall, these findings indicate that MD simulations and free energy calculations are helpful tools for modeling water and ions into crystallographic density maps. PMID:29771936

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

NASA Astrophysics Data System (ADS)

Cubrilovic, Dragana; Biela, Adam; Sielaff, Frank; Steinmetzer, Torsten; Klebe, Gerhard; Zenobi, Renato

2012-10-01

NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (KD) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in KD and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the KD values ranging from 2.9 to 17.6 μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (KD) measured via nanoESI-MS were compared with kinetic inhibition constants (Ki) in solution.

Molecular basis for subtype-specificity and high-affinity zinc inhibition in the GluN1-GluN2A NMDA receptor amino terminal domain

PubMed Central

Romero-Hernandez, Annabel; Simorowski, Noriko; Karakas, Erkan

2016-01-01

Summary Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors containing a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concentration of zinc that binds to an extracellular domain called an amino terminal domain (ATD). Despite physiological importance, the molecular mechanism underlying the high-affinity zinc inhibition has been incomplete due to lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc binding site and reveals distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability supporting the general model where the bi-lobe motion in ATD regulates the channel activity in NMDA receptors. PMID:27916457

Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro.

PubMed

Jonsson, Andreas; Wållberg, Helena; Herne, Nina; Ståhl, Stefan; Frejd, Fredrik Y

2009-08-17

Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For ZTNF-alpha:185, subnanomolar affinity (KD=0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the ZTNF-alpha:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

Nanostructured Block Polymer Membranes as High Capacity Adsorbers for the Capture of Metal Ions from Water

NASA Astrophysics Data System (ADS)

Boudouris, Bryan; Weidman, Jacob; Mulvenna, Ryan; Phillip, William

The efficient removal of metal ions from aqueous streams is of significant import in applications ranging from industrial waste treatment to the purification of drinking water. An emerging paradigm associated with this separation is one that utilizes membrane adsorbers as a means by which to bind metal salt contaminants. Here, we demonstrate that the casting of an A-B-C triblock polymer using the self-assembly and non-solvent induced phase separation (SNIPS) methodology results in a nanoporous membrane geometry. The nature of the triblock polymer affords an extremely high density of binding sites within the membrane. As such, we demonstrate that the membranes with binding capacities equal to that of state-of-the-art packed bed columns. Moreover, because the affinity of the C moiety can be tuned, highly selective binding events can occur based solely on the chemistry of the block polymer and the metal ions in solution (i.e., in a manner that is independent of the size of the metal ions). Due to these combined facts, these membranes efficiently remove heavy metal (e.g., lead- and cadmium-based) salts from contaminated water streams with greater than 95% efficiency. Finally, we show that the membranes can be regenerated through a simple treatment in order to provide long-lasting adsorber systems as well. Thus, it is anticipated that these nanostructured triblock polymer membranes are a platform by which to obtain next-generation water purification processes.

Nanobio interfaces: charge control of enzyme/inorganic interfaces for advanced biocatalysis.

PubMed

Deshapriya, Inoka K; Kumar, Challa V

2013-11-19

Specific approaches to the rational design of nanobio interfaces for enzyme and protein binding to nanomaterials are vital for engineering advanced, functional nanobiomaterials for biocatalysis, sensing, and biomedical applications. This feature article presents an overview of our recent discoveries on structural, functional, and mechanistic details of how enzymes interact with inorganic nanomaterials and how they can be controlled in a systematic manner using α-Zr(IV)phosphate (α-ZrP) as a model system. The interactions of a number of enzymes having a wide array of surface charges, sizes, and functional groups are investigated. Interactions are carefully controlled to screen unfavorable repulsions and enhance favorable interactions for high affinity, structure retention, and activity preservation. In specific cases, catalytic activities and substrate selectivities are improved over those of the pristine enzymes, and two examples of high activity near the boiling point of water have been demonstrated. Isothermal titration calorimetric studies indicated that enzyme binding is coupled to ion sequestration or release to or from the nanobio interface, and binding is controlled in a rational manner. We learned that (1) bound enzyme stabilities are improved by lowering the entropy of the denatured state; (2) maximal loadings are obtained by matching charge footprints of the enzyme and the nanomaterial surface; (3) binding affinities are improved by ion sequestration at the nanobio interface; and (4) maximal enzyme structure retention is obtained by biophilizing the nanobio interface with protein glues. The chemical and physical manipulations of the nanobio interface are significant not only for understanding the complex behaviors of enzymes at biological interfaces but also for desiging better functional nanobiomaterials for a wide variety of practical applications.

Quantifying protein microstructure and electrostatic effects on the change in Gibbs free energy of binding in immobilized metal affinity chromatography.

PubMed

Pathange, Lakshmi P; Bevan, David R; Zhang, Chenming

2008-03-01

Electrostatic forces play a major role in maintaining both structural and functional properties of proteins. A major component of protein electrostatics is the interactions between the charged or titratable amino acid residues (e.g., Glu, Lys, and His), whose pK(a) (or the change of the pK(a)) value could be used to study protein electrostatics. Here, we report the study of electrostatic forces through experiments using a well-controlled model protein (T4 lysozyme) and its variants. We generated 10 T4 lysozyme variants, in which the electrostatic environment of the histidine residue was perturbed by altering charged and neutral amino acid residues at various distances from the histidine (probe) residue. The electrostatic perturbations were theoretically quantified by calculating the change in free energy (DeltaDeltaG(E)) using Coulomb's law. On the other hand, immobilized metal affinity chromatography (IMAC) was used to quantify these perturbations in terms of protein binding strength or change in free energy of binding (DeltaDeltaG(B)), which varies from -0.53 to 0.99 kcal/mol. For most of the variants, there is a good correlation (R(2) = 0.97) between the theoretical DeltaDeltaG(E) and experimental DeltaDeltaG(B) values. However, there are three deviant variants, whose histidine residue was found to be involved in site-specific interactions (e.g., ion pair and steric hindrance), which were further investigated by molecular dynamics simulation. This report demonstrates that the electrostatic (DeltaDeltaG(Elec)) and microstructural effects (DeltaDeltaG(Micro)) in a protein can be quantified by IMAC through surface histidine mediated protein-metal ion interaction and that the unique microstructure around a histidine residue can be identified by identifying the abnormal binding behaviors during IMAC.

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent.

PubMed

Howlett, Robert M; Hughes, Bethan M; Hitchcock, Andrew; Kelly, David J

2012-06-01

Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c-cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K(d) value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wild-type strain and a decreased rate of uptake of (63)NiCl(2). The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system.

Is There Consistency between the Binding Affinity and Inhibitory Potential of Natural Polyphenols as α-amylase Inhibitors?

PubMed

Xu, Wei; Shao, Rong; Xiao, Jianbo

2016-07-26

The inhibitory potential of natural polyphenols for α-amylases has attracted great interests among researchers. The structure-affinity properties of natural polyphenols binding to α-amylase and the structure-activity relationship of dietary polyphenols inhibiting α-amylase were deeply investigated. There is a lack of consistency between the structure-affinity relationship and the structure-activity relationship of natural polyphenols as α-amylase inhibitors. Is it consistent between the binding affinity and inhibitory potential of natural polyphenols as with α-amylase inhibitors? It was found that the consistency between the binding affinity and inhibitory potential of natural polyphenols as with α-amylase inhibitors is not equivocal. For example, there is no consistency between the binding affinity and the inhibitory potential of quercetin and its glycosides as α-amylase inhibitors. However, catechins with higher α-amylase inhibitory potential exhibited higher affinity with α-amylase.

Screening Carbohydrate Libraries for Protein Interactions Using the Direct ESI-MS Assay. Applications to Libraries of Unknown Concentration

NASA Astrophysics Data System (ADS)

Kitova, Elena N.; El-Hawiet, Amr; Klassen, John S.

2014-08-01

A semiquantitative electrospray ionization mass spectrometry (ESI-MS) binding assay suitable for analyzing mixtures of oligosaccharides, at unknown concentrations, for interactions with target proteins is described. The assay relies on the differences in the ratio of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS at two or more initial protein concentrations to distinguish low affinity (≤103 M-1) ligands from moderate and high affinity (>105 M-1) ligands present in the library and to rank their affinities. Control experiments were performed on solutions of a single chain antibody and a mixture of synthetic oligosaccharides, with known affinities, in the absence and presence of a 40-component carbohydrate library to demonstrate the implementation and reliability of the assay. The application of the assay for screening natural libraries of carbohydrates against proteins is also demonstrated using mixtures of human milk oligosaccharides, isolated from breast milk, and fragments of a bacterial toxin and human galectin 3.

Metal chelate affinity precipitation of RNA and purification of plasmid DNA

NASA Technical Reports Server (NTRS)

Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

2003-01-01

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

Highly Preorganized Ligand 1,10-Phenanthroline-2,9-dicarboxylic Acid for the Selective Recovery of Uranium from Seawater in the Presence of Competing Vanadium Species

DOE PAGES

Lashley, Mark A.; Ivanov, Alexander S.; Bryantsev, Vyacheslav S.; ...

2016-09-30

Studies of the complexation of new promising ligands with uranyl (UO 2 2+) and other seawater cations can aid the development of more efficient, selective, and robust sorbents for the recovery of uranium from seawater. Here, we propose that the ligand design principles based on structural preorganization can be successfully applied to obtain a dramatic enhancement in UO 2 2+ ion binding affinity and selectivity. This concept is exemplified through the investigation of the com-plexes of UO 2 2+, VO 2+, and VO 2+ with the highly preorganized ligand PDA (1,10-phenanthroline-2,9-dicarboxylic acid) using a combination of fluores-cence and absorbance techniques,more » along with den-sity functional theory (DFT) calculations. Moreover, the measured stability constant value, log K1, of 16.5 for the UO 2 2+/PDA complex is very high compared to uranyl complexes with other dicarboxylic ligands. Moreover, PDA exhibits strong selectivity for uranyl over vanadium ions, since the determined sta-bility constant values of the PDA complexes of the vanadium ions are quite low (V(IV) log K1 = 7.4, V(V) = 7.3). Finally, the structures of the corresponding UO 2 2+, VO 2+, and VO 2+ complexes with PDA were identified by systematic DFT calculations, and helped to interpret the stronger binding affinity for uranium over the vanadium ions. Due to its high chemical stability, selectivity, and structural preor-ganization for UO 2 2+ complexation, PDA is a very promising candidate that can be potentially used in the development of novel adsorbent materials for the selective extraction of uranium from sea-water.« less

Highly Preorganized Ligand 1,10-Phenanthroline-2,9-dicarboxylic Acid for the Selective Recovery of Uranium from Seawater in the Presence of Competing Vanadium Species

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

Lashley, Mark A.; Ivanov, Alexander S.; Bryantsev, Vyacheslav S.

Studies of the complexation of new promising ligands with uranyl (UO 2 2+) and other seawater cations can aid the development of more efficient, selective, and robust sorbents for the recovery of uranium from seawater. Here, we propose that the ligand design principles based on structural preorganization can be successfully applied to obtain a dramatic enhancement in UO 2 2+ ion binding affinity and selectivity. This concept is exemplified through the investigation of the com-plexes of UO 2 2+, VO 2+, and VO 2+ with the highly preorganized ligand PDA (1,10-phenanthroline-2,9-dicarboxylic acid) using a combination of fluores-cence and absorbance techniques,more » along with den-sity functional theory (DFT) calculations. Moreover, the measured stability constant value, log K1, of 16.5 for the UO 2 2+/PDA complex is very high compared to uranyl complexes with other dicarboxylic ligands. Moreover, PDA exhibits strong selectivity for uranyl over vanadium ions, since the determined sta-bility constant values of the PDA complexes of the vanadium ions are quite low (V(IV) log K1 = 7.4, V(V) = 7.3). Finally, the structures of the corresponding UO 2 2+, VO 2+, and VO 2+ complexes with PDA were identified by systematic DFT calculations, and helped to interpret the stronger binding affinity for uranium over the vanadium ions. Due to its high chemical stability, selectivity, and structural preor-ganization for UO 2 2+ complexation, PDA is a very promising candidate that can be potentially used in the development of novel adsorbent materials for the selective extraction of uranium from sea-water.« less

Opposing intermolecular tuning of Ca2+ affinity for Calmodulin by its target peptides

NASA Astrophysics Data System (ADS)

Cheung, Margaret

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2+) by integrating coarse-grained models and all-atomistic simulations with non-equilibrium physics. We focused on binding between CaM and two specific targets, Ca2+/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca2+ signaling pathways in neurons. It was shown experimentally that Ca2+/CaM binds to the CaMKII peptide with higher affinity than the Ng peptide. The binding of CaMKII peptide to CaM in return increases the Ca2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide, which binds to Ca2+-free CaM or Ca2+/CaM with similar binding affinity. Unlike CaM-CaMKII peptide that allowed structure determination by crystallography, the structural description of CaM-Ng peptide is unknown due to low binding affinity, therefore, we computationally generated an ensemble of CaM-Ng peptide structures by matching the changes in the chemical shifts of CaM upon Ng peptide binding from nuclear magnetic resonance experiments. We computed the changes in Ca2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca2+ for CaM in the presence of Ng by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca2+ binding loops particularly at C-domain of CaM, enabling Ca2+release. In contrast, CaMKII increases Ca2+ affinity for the C-domain of CaM by stabilizing the two Ca2+ binding loops.

Existence of three subtypes of bradykinin B2 receptors in guinea pig.

PubMed

Seguin, L; Widdowson, P S; Giesen-Crouse, E

1992-12-01

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of binding affinity of CJ-023,423 for human prostanoid EP4 receptor.

PubMed

Murase, Akio; Nakao, Kazunari; Takada, Junji

2008-01-01

In order to characterize the receptor binding pharmacology of CJ-023,423, a potent and selective EP4 antagonist, we performed a radioligand receptor binding assay under various assay conditions. An acidic (pH 6) and hypotonic buffer is a conventional, well-known buffer for prostaglandin E2 receptor binding assays. CJ-023,423 showed moderate binding affinity for human EP4 receptor under conventional buffer conditions. However, its binding affinity was greatly increased under neutral (pH 7.4) and isotonic buffer conditions. In this report, the binding mechanism between CJ-023,423 and human EP4 receptor is discussed based on the binding affinities determined under various assay conditions. Copyright 2008 S. Karger AG, Basel.

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma.

PubMed

Ikonomopoulou, M P; Bradley, A J; Whittier, J M; Ibrahim, K

2006-11-01

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4 degrees C with high affinity (K (a) = 1.49 +/- 0.09 x 10(9) M(-1); 0.17 +/- 0.02 x 10(7) M(-1)) and low binding capacity (B (max) = 3.24 +/- 0.84 x 10(-5) M; 0.33 +/- 0.06 x 10(-4) M). The binding affinity and capacity of testosterone at 23 and 36 degrees C, respectively were similar to those determined at 4 degrees C. However, oestradiol showed no binding activity at 36 degrees C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36 degrees C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.

Cis→Trans Isomerization of Pro7 in Oxytocin Regulates Zn2+ Binding

NASA Astrophysics Data System (ADS)

Fuller, Daniel R.; Glover, Matthew S.; Pierson, Nicholas A.; Kim, DoYong; Russell, David H.; Clemmer, David E.

2016-08-01

Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn2+ to the nine-residue peptide hormone oxytocin (OT, Cys1-Tyr2-Ile3-Gln4-Asn5-Cys6-Pro7-Leu8-Gly9-NH2, having a disulfide bond between Cys1 and Cys6 residues). Zn2+ binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899-3906 (1979)]. In the absence of Zn2+, we find evidence for two primary OT conformations, which arise because the Cys6-Pro7 peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn2+, we determine binding constants in water of KA = 1.43 ± 0.24 and 0.42 ± 0.12 μM-1, for the trans- and cis-configured populations, respectively. The Zn2+ bound form of OT, having a cross section of Ω = 235 Å2, has Pro7 in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993-6000 (2008)], in which it was proposed that Zn2+ binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro7-Leu8-Gly9-NH2, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn2+. In this way, the proline residue regulates Zn2+ binding to OT and, hence, is important in receptor binding.

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

PubMed

Krall, Jacob; Jensen, Claus Hatt; Bavo, Francesco; Falk-Petersen, Christina Birkedahl; Haugaard, Anne Stæhr; Vogensen, Stine Byskov; Tian, Yongsong; Nittegaard-Nielsen, Mia; Sigurdardóttir, Sara Björk; Kehler, Jan; Kongstad, Kenneth Thermann; Gloriam, David E; Clausen, Rasmus Prætorius; Harpsøe, Kasper; Wellendorph, Petrine; Frølund, Bente

2017-11-09

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar K i ) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

Use of magnetic circular dichroism to study dinuclear metallohydrolases and the corresponding biomimetics.

PubMed

Larrabee, James A; Schenk, Gerhard; Mitić, Nataša; Riley, Mark J

2015-09-01

Magnetic circular dichroism (MCD) is a convenient technique for providing structural and mechanistic insight into enzymatic systems in solution. The focus of this review is on aspects of geometric and electronic structure that can be determined by MCD, and how this method can further our understanding of enzymatic mechanisms. Dinuclear Co(II) systems that catalyse hydrolytic reactions were selected to illustrate the approach. These systems all contain active sites with similar structures consisting of two Co(II) ions bridged by one or two carboxylates and a water or hydroxide. In most of these active sites one Co(II) is five-coordinate and one is six-coordinate, with differing binding affinities. It is shown how MCD can be used to determine which binding site--five or six-coordinate--has the greater affinity. Importantly, zero-field-splitting data and magnetic exchange coupling constants may be determined from the temperature and field dependence of MCD data. The relevance of these data to the function of the enzymatic systems is discussed.

An Enhanced Platform to Analyse Low-Affinity Amyloid β Protein by Integration of Electrical Detection and Preconcentrator.

PubMed

Yoo, Yong Kyoung; Yoon, Dae Sung; Kim, Gangeun; Kim, Jinsik; Han, Sung Il; Lee, Junwoo; Chae, Myung-Sic; Lee, Sang-Myung; Lee, Kyu Hyoung; Hwang, Kyo Seon; Lee, Jeong Hoon

2017-10-30

Sensitivity and limit of detection (LOD) enhancement are essential criteria for the development of ultrasensitive molecular sensors. Although various sensor types have been investigated to enhance sensitivity and LOD, analyte detection and its quantification are still challenging, particularly for protein-protein interactions with low association constants. To solve this problem, here, we used ion concentration polarization (ICP)-based preconcentration to increase the local concentration of analytes in a microfluidic platform for LOD improvement. This was the first demonstration of a microfluidic device with an integrated ICP preconcentrator and interdigitated microelectrode (IME) sensor to detect small changes in surface binding between antigens and antibodies. We detected the amyloid beta (Aβ) protein, an Alzheimer's disease marker, with low binding affinity to its antibodies by adopting ICP preconcentration phenomena. We demonstrated that a combination of ICP preconcentrator and IME sensor increased the LOD by 13.8-fold to femtomolar level (8.15 fM), which corresponds to a significant advance for clinical applications.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

PubMed Central

Itakura, Yoko; Nakamura-Tsuruta, Sachiko; Kominami, Junko; Tateno, Hiroaki; Hirabayashi, Jun

2017-01-01

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations. PMID:28556796

Interaction between phloretin and the red blood cell membrane

PubMed Central

1976-01-01

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane. PMID:5575

Influence of ionic complexation on release rate profiles from multiple water-in-oil-in-water (W/O/W) emulsions.

PubMed

Bonnet, Marie; Cansell, Maud; Placin, Frédéric; David-Briand, Elisabeth; Anton, Marc; Leal-Calderon, Fernando

2010-07-14

Water-in-oil-in-water (W/O/W) double emulsions were prepared, and the kinetics of release of magnesium ions from the internal to the external water phase was followed. Different chelating agents (phosvitin and gluconate) were used to bind magnesium within the prospect of improving the ion retention in the internal aqueous droplets. Magnesium release was monitored for 1 month of storage, for each formulation, with and without chelation, at two storage temperatures (4 and 25 degrees C). Leakage occurred without film rupturing (coalescence) and was mainly due to entropically driven diffusion/permeation phenomena. The experimental results revealed a clear correlation between the effectiveness of chelating agents to delay the delivery and their binding capacity characterized by the equilibrium affinity constant. The kinetic data (percent released versus time curves) were interpreted within the framework of a kinetic model based on diffusion and taking into account magnesium chelation.

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification.

PubMed

Shin, Min Jae; Tan, Lihan; Jeong, Min Ho; Kim, Ji-Heung; Choe, Woo-Seok

2011-08-05

Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl(2)-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification. Copyright © 2011 Elsevier B.V. All rights reserved.

Binding affinities of cationic dyes in the presence of activated charcoal and anionic surfactant in the premicellar region

NASA Astrophysics Data System (ADS)

Ali, Farman; Ibrahim, Muhammad; Khan, Fawad; Bibi, Iram; Shah, Syed W. H.

2018-03-01

Binding preferences of cationic dyes malachite green and methylene blue in a mixed charcoal-sodium dodecyl sulfate system have been investigated using UV-visible absorption spectroscopy. The dye adsorption shows surfactant-dependent patterns, indicating diverse modes of interactions. At low surfactant concentration, a direct binding to charcoal is preferred. Comparatively greater quantities of surfactant lead to attachment of dye-surfactant complex to charcoal through hydrophobic interactions. A simple model was employed for determination of equilibrium constant K eq and concentration of dye-surfactant ion pair N DS for both dyes. The values of binding parameters revealed that malachite green was directly adsorbed onto charcoal, whereas methylene blue was bound through surfactant monomers. The model is valid for low surfactant concentrations in the premicellar region. These findings have significance for material and environmental sciences.

Microfluidic free-flow electrophoresis for the discovery and characterisation of calmodulin binding partners

NASA Astrophysics Data System (ADS)

Herling, Therese; Linse, Sara; Knowles, Tuomas

2015-03-01

Non-covalent and transient protein-ligand interactions are integral to cellular function and malfunction. Key steps in signalling and regulatory pathways rely on reversible non-covalent protein-protein binding or ion chelation. Here we present a microfluidic free-flow electrophoresis method for detecting and characterising protein-ligand interactions in solution. We apply this method to probe the binding equilibria of calmodulin, a central protein to calcium signalling pathways. In this study we characterise the specific binding of calmodulin to phosphorylase kinase, a known target, and creatine kinase, which we identify as a putative binding partner through a protein array screen and surface plasmon resonance experiments. We verify the interaction between calmodulin and creatine kinase in solution using free-flow electrophoresis and investigate the effect of calcium and sodium chloride on the calmodulin-ligand binding affinity in free solution without the presence of a potentially interfering surface. Our results demonstrate the general applicability of quantitative microfluidic electrophoresis to characterise binding equilibria between biomolecules in solution.

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

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

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

Zinc binding groups for histone deacetylase inhibitors.

PubMed

Zhang, Lei; Zhang, Jian; Jiang, Qixiao; Zhang, Li; Song, Weiguo

2018-12-01

Zinc binding groups (ZBGs) play a crucial role in targeting histone deacetylase inhibitors (HDACIs) to the active site of histone deacetylases (HDACs), thus determining the potency of HDACIs. Due to the high affinity to the zinc ion, hydroxamic acid is the most commonly used ZBG in the structure of HDACs. An alternative ZBG is benzamide group, which features excellent inhibitory selectivity for class I HDACs. Various ZBGs have been designed and tested to improve the activity and selectivity of HDACIs, and to overcome the pharmacokinetic limitations of current HDACIs. Herein, different kinds of ZBGs are reviewed and their features have been discussed for further design of HDACIs.

Effects of the central potassium ions on the G-quadruplex and stabilizer binding.

PubMed

Wang, Zhiguo; Liu, Jun-Ping

2017-03-01

Human telomeres undertake the structure of intra-molecular parallel G-quadruplex in the presence of K + in eukaryotic cell. Stabilization of the telomere G-quadruplex represents a potential strategy to prevent telomere lengthening by telomerase in cancer therapy. Current work demonstrates that the binding of central K + with the parallel G-quadruplex is a coordinated water directed step-wise process. The K + above the top G-tetrad is prone to leak into environment and the 5'-adenine quickly flips over the top G-tetrad, leading to the bottom gate of G-tetrads as the only viable pathway of K + binding. Present molecular dynamics studies on the two most potent stabilizers RHPS4 and BRACO-19 reveal that the central K + has little influence on the binding conformations of the bound stabilizers. But without the central K + , either RHPS4 or BRACO-19 cannot stabilize the structure of G-quadruplex. The binding strength of stabilizers evaluated by the MM-PBSA method follows the order of BRACO-19> RHPS4, which agrees with the experimental results. The difference in binding affinities between RHPS4 and BRACO-19 is probably related to the ability to form intramolecular hydrogen bonds and favorable van del Waals interactions with G-quadruplex. In the models that have one central K + located at the upper/lower binding site, the corresponding top/bottom stacked stabilizers show more favorable binding affinities, indicating the apparent promoting effect of central K + on the stabilizer binding. Our findings provide further insights into the regulatory effect of K + on the G-quadruplex targeted binding, which is meaningful to the development of G-quadruplex stabilizers. Copyright © 2017 Elsevier Inc. All rights reserved.

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors.

PubMed

Jadey, Snehal; Auerbach, Anthony

2012-07-01

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes ("catch" and "hold") that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement ("capping"). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation.

[3H]MK-801 binding sites in post-mortem human frontal cortex.

PubMed

Kornhuber, J; Mack-Burkhardt, F; Kornhuber, M E; Riederer, P

1989-03-29

The binding of [3H]MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate) was investigated in extensively washed homogenates of post-mortem human frontal cortex. The association of [3H]MK-801 proceeded slowly (t1/2 = 553 min) and reached equilibrium only after a prolonged incubation (greater than 24 h). The dissociation of [3H]MK-801 from the binding site was also slow (t1/2 = 244 min). Glutamate, glycine and magnesium markedly increased the rate of association (t1/2 = 14.8 min) and dissociation (t1/2 = 36.5 min). At equilibrium, the binding was not altered by these substances. Specific binding was linear with protein concentration, was saturable, reversible, stereoselective, heat-labile and was nearly absent in the white matter. Scatchard analysis of the saturation curves obtained at equilibrium indicated that there was a high-affinity (Kd1 1.39 +/- 0.21 nM, Bmax1 0.483 +/- 0.084 pmol/mg protein) and a low-affinity (Kd2 116.25 +/- 50.79 nM, Bmax2 3.251 +/- 0.991 pmol/mg protein) binding site. All competition curves obtained with (+)-MK-801, (-)-MK-801, phencyclidine and ketamine had Hill coefficients of less than unity and were best explained by a two-site model. Thus, our results demonstrate the presence of binding sites for MK-801 in post-mortem human brains and provide evidence for binding site heterogeneity. Furthermore, glutamate, glycine and magnesium accelerate the association and dissociation of [3H]MK-801 to and from its binding sites. The results add support to the hypothesis that MK-801, glutamate, glycine and magnesium all bind to different sites on the NMDA receptor-ion channel complex.

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors

PubMed Central

Jadey, Snehal

2012-01-01

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes (“catch” and “hold”) that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement (“capping”). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation. PMID:22732309

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.

PubMed

Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-05-03

Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors

PubMed Central

Koshimizu, Taka-aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-01-01

Reducing Na+ in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na+-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na+ sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na+ increased cell surface [3H]AVP binding and decreased receptor internalization. Substitution of Na+ by Cs+ or NH4+ inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na+ over Cs+. Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations. PMID:27138239

Synthesis and characterization of Ag+-decorated poly(glycidyl methacrylate) microparticle design for the adsorption of nucleic acids.

PubMed

Erol, Kadir; Uzunoglu, Aytekin; Köse, Kazım; Sarıca, Büşra; Avcı, Emre; Köse, Dursun A

2018-04-01

In this study, we report on the adsorption of RNA and DNA molecules by exploiting the high binding affinity of these nucleic acids to Ag + ions anchored on magnetic poly(glycidyl methacrylate) (PGMA) microparticles. PGMA microparticles were synthesized and modified with nicotinamide which enabled to anchor Ag + ions on the surface. The successful preparation of PGMA was confirmed by the presence of characteristic FTIR peaks. The ESR results showed that the incorporation of FeNi salt to the polymeric structure provided a magnetic property to the microparticles. The amount of nicotinamide and Ag + ions used to modify the surface of the particles were found to be 1.79 wt% and 52.6 mg Ag/g microparticle, respectively. The high affinity of nucleic acids to Ag + ions were exploited for the adsorption studies. At the optimum working conditions, the adsorption capacity of microparticles was found to be 40.1 and 11.48 mg nucleic acid/g microparticle for RNA and DNA, respectively. Our study indicated that the use of novel Ag + -decorated magnetic PGMA particles can be successfully employed as adsorbents for fast, easy, and cost-friendly adsorption of nucleic acids with high purity as well as high in quantity. Copyright © 2018 Elsevier B.V. All rights reserved.

Synthesis and binding affinity of neuropeptide Y at opiate receptors.

PubMed

Kiddle, James J; McCreery, Heather J; Soles, Sonia

2003-03-24

Neuropeptide Y and several metabolic fragments were synthesized and evaluated for binding affinity at non-selective opiate receptors. Neuropeptide Y and several C-terminal fragments were shown to bind to non-selective opiate receptors with an affinity similar to that of Leu-enkephalin.

[Interaction of human factor X with thromboplastin].

PubMed

Kiselev, S V; Zubairov, D M; Timarbaev, V N

2003-01-01

The binding of 125I-labeled human factor X to native and papaine-treated tissue tromboplastin in the presence of CaCl2 or EDTA was studied. The Scatchard analysis suggests the existence of high (Kd=l,8 x10(-9) M) and low affinity binding sites on the thromboplastin surface. The removal of Ca2+ reduced affinity of factor X to the high affinity sites. This was accompanied by some increase of their number. Proteolysis by papaine decreased affinity of high affinity sites and caused the increase of their number in the presence of Ca2+. In the absence of Ca2+ the affinity remained unchanged, but the number of sites decreased. At low concentrations of factor X positive cooperativity for high affinity binding sites was observed. It did not depend on the presence of Ca2+. The results indirectly confirm the role of hydrophobic interactons in Ca2+ dependent binding of factor X to thromboplastin and the fact that heterogeneity of this binding is determined by mesophase structure of the thromboplastin phospholipids.

Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein.

PubMed

Balogh, Ria K; Gyurcsik, Béla; Hunyadi-Gulyás, Éva; Christensen, Hans E M; Jancsó, Attila

2016-07-01

Metal ion regulation is essential for living organisms. In prokaryotes metal ion dependent transcriptional factors, the so-called metalloregulatory proteins play a fundamental role in controlling the concentration of metal ions. These proteins recognize metal ions with an outstanding selectivity. A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes the expression and purification of such proteins challenging. Here we describe a method for the purification of the copper-regulatory CueR protein under optimized conditions. In order to avoid protein precipitation and/or eventual aggregation and to get rid of the co-purifying Escherichia coli elongation factor, our procedure consisted of four steps supplemented by DNA digestion. Subsequent anion exchange on Sepharose FF Q 16/10, affinity chromatography on Heparin FF 16/10, second anion exchange on Source 30 Q 16/13 and gel filtration on Superdex 75 26/60 resulted in large amounts of pure CueR protein without any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Strittmatter, S.M.; Snyder, S.H.

We demonstrate that (3H)captopril selectively labels angiotensin converting enzyme (EC 3.14.15.1) (ACE) and employ this technique to probe enzyme-inhibitor interactions. (3H)Captopril binding sites copurify with ACE activity from rat lung or rat brain. At each stage of the purification the Vmax/Bmax ratio, or kcat is 17,000 min-1 with hippuryl-L-histidyl-L-leucine as substrate. The specificity of (3H)captopril binding is apparent in the similar pharmacologic profile of inhibition in crude and pure enzyme preparations. Furthermore, binding sites and enzyme activity comigrate in gel filtration and sucrose gradient sedimentation experiments. Equilibrium analysis of (3H)captopril binding to purified ACE reveals a Bmax of 6 nmol/mgmore » of protein (KD = 2 nM), demonstrating the presence of one inhibitor binding site per polypeptide chain. The kinetics of (3H)captopril binding are characterized by monophasic association and dissociation rate constants of 0.026 nM-1 min-1 and 0.034 min-1, respectively. The affinity of ACE for both (3H) captopril and enalaprilat is greater at 37 degrees than at 0 degree, demonstrating that these interactions are entropically driven, perhaps by an isomerization of the enzyme molecule. The ionic requirements for (3H)captopril binding and substrate catalysis differ. Chloride and bromide ion, but not fluoride, are about 100-fold more potent stimulators of binding than catalysis. When the active site Zn2+ ion is replaced by Co2+, catalysis was stimulated 2-fold, whereas binding activity was decreased by 70%.« less

The example of calix[4]pyrrole derivative containing Bodipy unit: fluorometric and colorimetric sensor for F- ion.

PubMed

Taner, Bilge; Kursunlu, Ahmed Nuri; Güler, Ersin

2014-01-24

A novel chemosensor based on calix[4]pyrrole derivative modified by Bodipy unit has been synthesized, and its complexes with various anions were investigated. The results show that the receptors can selectively recognize biologically important fluoride ions. The binding affinity for fluoride ions was investigated by naked-eye color change, absorption, emission, proton nuclear magnetic resonance spectroscopy. The addition of fluoride ions to an acetonitrile solution of chemosensor can result in an obvious color change (brownish yellow color to straw yellow). The stoichiometries between the receptor and fluoride were determined from the molar ratio plots using the UV-visible spectra, which showed evident 1:1. The proton nuclear magnetic resonance spectral data supported the fluoride anion recognition with the disappearance of the amino proton peaks. Published by Elsevier B.V.

Computational Design of Metal Ion Sequestering Agents

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

Hay, Benjamin P.; Rapko, Brian M.

Organic ligands that exhibit a high degree of metal ion recognition are essential precursors for developing separation processes and sensors for metal ions. Since the beginning of the nuclear era, much research has focused on discovering ligands that target specific radionuclides. Members of the Group 1A and 2A cations (e.g., Cs, Sr, Ra) and the f-block metals (actinides and lanthanides) are of primary concern to DOE. Although there has been some success in identifying ligand architectures that exhibit a degree of metal ion recognition, the ability to control binding affinity and selectivity remains a significant challenge. The traditional approach formore » discovering such ligands has involved lengthy programs of organic synthesis and testing that, in the absence of reliable methods for screening compounds before synthesis, have resulted in much wasted research effort.« less

Like-charged protein-polyelectrolyte complexation driven by charge patches

NASA Astrophysics Data System (ADS)

Yigit, Cemil; Heyda, Jan; Ballauff, Matthias; Dzubiella, Joachim

2015-08-01

We study the pair complexation of a single, highly charged polyelectrolyte (PE) chain (of 25 or 50 monomers) with like-charged patchy protein models (CPPMs) by means of implicit-solvent, explicit-salt Langevin dynamics computer simulations. Our previously introduced set of CPPMs embraces well-defined zero-, one-, and two-patched spherical globules each of the same net charge and (nanometer) size with mono- and multipole moments comparable to those of globular proteins with similar size. We observe large binding affinities between the CPPM and the like-charged PE in the tens of the thermal energy, kBT, that are favored by decreasing salt concentration and increasing charge of the patch(es). Our systematic analysis shows a clear correlation between the distance-resolved potentials of mean force, the number of ions released from the PE, and CPPM orientation effects. In particular, we find a novel two-site binding behavior for PEs in the case of two-patched CPPMs, where intermediate metastable complex structures are formed. In order to describe the salt-dependence of the binding affinity for mainly dipolar (one-patched) CPPMs, we introduce a combined counterion-release/Debye-Hückel model that quantitatively captures the essential physics of electrostatic complexation in our systems.

Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule

PubMed Central

Nakanishi, Tsuyoshi; Ando, Eiji; Furuta, Masaru; Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru; Tsunasawa, Susumu; Nishimura, Osamu

2007-01-01

We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3. PMID:18166671

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis.

PubMed

Duggin, Iain G; Matthews, Jacqueline M; Dixon, Nicholas E; Wake, R Gerry; Mackay, Joel P

2005-04-01

Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA.

Temporal Hierarchy of Gene Expression Mediated by Transcription Factor Binding Affinity and Activation Dynamics

PubMed Central

Gao, Rong

2015-01-01

ABSTRACT Understanding cellular responses to environmental stimuli requires not only the knowledge of specific regulatory components but also the quantitative characterization of the magnitude and timing of regulatory events. The two-component system is one of the major prokaryotic signaling schemes and is the focus of extensive interest in quantitative modeling and investigation of signaling dynamics. Here we report how the binding affinity of the PhoB two-component response regulator (RR) to target promoters impacts the level and timing of expression of PhoB-regulated genes. Information content has often been used to assess the degree of conservation for transcription factor (TF)-binding sites. We show that increasing the information content of PhoB-binding sites in designed phoA promoters increased the binding affinity and that the binding affinity and concentration of phosphorylated PhoB (PhoB~P) together dictate the level and timing of expression of phoA promoter variants. For various PhoB-regulated promoters with distinct promoter architectures, expression levels appear not to be correlated with TF-binding affinities, in contrast to the intuitive and oversimplified assumption that promoters with higher affinity for a TF tend to have higher expression levels. However, the expression timing of the core set of PhoB-regulated genes correlates well with the binding affinity of PhoB~P to individual promoters and the temporal hierarchy of gene expression appears to be related to the function of gene products during the phosphate starvation response. Modulation of the information content and binding affinity of TF-binding sites may be a common strategy for temporal programming of the expression profile of RR-regulated genes. PMID:26015501

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

PubMed Central

Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

2011-01-01

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

Facile synthesis of Fe3O4@PDA core-shell microspheres functionalized with various metal ions: A systematic comparison of commonly-used metal ions for IMAC enrichment.

PubMed

Jiang, Jiebing; Sun, Xueni; Li, Yan; Deng, Chunhui; Duan, Gengli

2018-02-01

Metal ions differed greatly in affinity towards phosphopeptides, and thus it is essential to systematically compare the phosphopeptides enrichment ability of different metal ions usually used in the IMAC techniques. In this work, for the first time, eight metal ions, including Nb 5+ , Ti 4+ , Zr 4+ , Ga 3+ , Y 3+ , In 3+ , Ce 4+ , Fe 3+ , were immobilized on the polydopamine (PDA)-coated Fe 3 O 4 (denoted as Fe 3 O 4 @PDA-M n+ ), and systematically compared by the real biosamples, in addition to standard phosphopeptides. Fe 3 O 4 microspheres were synthesized via the solvothermal reaction, followed by self-polymerization of dopamine on the surface. Then through taking advantage of the hydroxyl and amino group of PDA, the eight metal ions were easily adhered to the surface of Fe 3 O 4 @PDA. After characterization, the resultant Fe 3 O 4 @PDA-M n+ microspheres were applied to phosphopeptides enrichment based on the binding affinity between metal ions and phosphopeptides. According to the results, different metal ions presented diverse phosphopeptides enrichment efficiency in terms of selectivity, sensitivity and the enrichment ability from real complex samples, and Fe 3 O 4 @PDA-Nb 5+ and Fe 3 O 4 @PDA-Ti 4+ showed obvious advantages of the phosphopeptides enrichment effect after the comparison. This systematic comparison may provide certain reference for the use and development of IMAC materials in the future. Copyright © 2017 Elsevier B.V. All rights reserved.

Functionalized Fullerene Targeting Human Voltage-Gated Sodium Channel, hNav1.7.

PubMed

Hilder, Tamsyn A; Robinson, Anna; Chung, Shin-Ho

2017-08-16

Mutations of hNa v 1.7 that cause its activities to be enhanced contribute to severe neuropathic pain. Only a small number of hNa v 1.7 specific inhibitors have been identified, most of which interact with the voltage-sensing domain of the voltage-activated sodium ion channel. In our previous computational study, we demonstrated that a [Lys 6 ]-C 84 fullerene binds tightly (affinity of 46 nM) to Na v Ab, the voltage-gated sodium channel from the bacterium Arcobacter butzleri. Here, we extend this work and, using molecular dynamics simulations, demonstrate that the same [Lys 6 ]-C 84 fullerene binds strongly (2.7 nM) to the pore of a modeled human sodium ion channel hNa v 1.7. In contrast, the fullerene binds only weakly to a mutated model of hNa v 1.7 (I1399D) (14.5 mM) and a model of the skeletal muscle hNa v 1.4 (3.7 mM). Comparison of one representative sequence from each of the nine human sodium channel isoforms shows that only hNa v 1.7 possesses residues that are critical for binding the fullerene derivative and blocking the channel pore.

Selective Capture of Cesium and Thallium from Natural Waters and Simulated Wastes with Copper Ferrocyanide Functionalized Mesoporous Silica

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

Sangvanich, Thanapon; Sukwarotwat, Vichaya; Wiacek, Robert J.

2010-10-15

Copper(II) ferrocyanide immobilized inside mesoporous silica MCM-41 supports (Cu-FC-EDA-SAMMSTM) has been evaluated against iron(III) hexacyanoferrate(II) (insoluble Prussian blue) for the sorption of cesium (Cs+) and thallium (Tl+) from natural waters and simulated wastes. The affinities (in term of distribution coefficients, Kd) of both sorbents for Cs and Tl were measured as a function of solution pH, competing cations, and matrices. For the entire pH studied (pH 0.1 to 7.3), Cu-FC-EDA-SAMMS had higher affinities for Cs and Tl (one to two orders of magnitude higher Kd) than Prussian blue and was less negatively impacted by the solution pH, competing cations, andmore » matrices. The adsorption isotherms and kinetics of the two sorbents for Cs and/or Tl were also determined in seawater and simulated acid and alkaline wastes. SAMMS outperformed Prussian blue in terms of maximum adsorption capacity (e.g., 21.7 versus 2.6 mg Cs/g in acid waste stimulant, pH 1.1), and rate (e.g., over 95 wt% of Cs was removed after 2 minutes with SAMMS, while only 75 wt% was removed with Prussian blue). The lower affinity, capacity, and rate of Cs and Tl sorption on Prussian blue than those on Cu-FC-EDA-SAMMS were attributed to the molecular pore sizes, which restrict mass transport, and the insoluble Cs abducts of the Prussian blue, which restrict the ability of neighboring binding sites to further bind Cs ions. On the other hand, the large pores of SAMMS not only enable faster diffusion and faster binding chemistry, but they also allow isolation of binding sites so that one Cs binding event does not impact further Cs binding. In addition, iron (Fe) dissolved from insoluble Prussian blue over 10-fold of that from Cu-FC-EDA-SAMMS after 24 hours of contact time, indicating poorer material stability of Prussian blue.« less

Chemistry of carcinogenic metals.

PubMed Central

Martell, A E

1981-01-01

The periodic distribution of known and suspected carcinogenic metal ions is described, and the chemical behavior of various types of metal ions is explained in terms of the general theory of hard and soft acids and bases. The chelate effect is elucidated, and the relatively high stability of metal chelates in very dilute solutions is discussed. The concepts employed for the chelate effect are extended to explain the high stabilities of macrocyclic and cryptate complexes. Procedures for the use of equilibrium data to determine the speciation of metal ions and complexes under varying solution conditions are described. Methods for assessing the interferences by hydrogen ion, competing metal ions, hydrolysis, and precipitation are explained, and are applied to systems containing iron(III) chelates of fourteen chelating agents designed for effective binding of the ferric ion. The donor groups available for the building up of multidentate ligands are presented, and the ways in which they may be combined to achieve high affinity and selectivity for certain types of metal ions are explained. PMID:6791915

Structure of a highly acidic β-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs{sup +}-selective binding site

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

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo

2015-03-01

The tertiary structure of a β-lactamase derived from the halobacterium Chromohalobacter sp. 560 (HaBLA) was determined by X-ray crystallography. Three unique Sr{sup 2+}-binding sites and one Cs{sup +}-binding site were discovered in the HaBLA molecule. Environmentally friendly absorbents are needed for Sr{sup 2+} and Cs{sup +}, as the removal of the radioactive Sr{sup 2+} and Cs{sup +} that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs{sup +}more » or Sr{sup 2+}. The crystal structure of a halophilic β-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space group P3{sub 1} using X-ray crystallography. Moreover, the locations of bound Sr{sup 2+} and Cs{sup +} ions were identified by anomalous X-ray diffraction. The location of one Cs{sup +}-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na{sup +} (90 mM Na{sup +}/10 mM Cs{sup +}). From an activity assay using isothermal titration calorimetry, the bound Sr{sup 2+} and Cs{sup +} ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs{sup +}-binding site provides important information that is useful for the design of artificial Cs{sup +}-binding sites that may be useful in the bioremediation of radioactive isotopes.« less

PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: MODEL II

EPA Science Inventory

The training set used to derive a common reactivity pattern (COREPA) model for estrogen receptor (ER) binding affinity in Model I (see Abstract I in this series) was extended to include 47 rat estrogen receptor (rER) relative binding affinity (RBA) measurements in addition to the...

PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: A COMPARISON OF THREE MODELS

EPA Science Inventory

A comparative analysis of how three COREPA models for ER binding affinity performed when used to predict potential estrogen receptor (ER) ligands is presented. Models I and II were developed based on training sets of 232 and 279 rat ER binding affinity measurements, respectively....

Binding of benzocaine in batrachotoxin-modified Na+ channels. State- dependent interactions

PubMed Central

1994-01-01

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX- modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use- dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed. PMID:8195785

Hyaluronate-binding proteins of murine brain.

PubMed

Marks, M S; Chi-Rosso, G; Toole, B P

1990-01-01

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.

Characterization of nicotine binding to the rat brain P/sub 2/ preparation: the identification of multiple binding sites which include specific up-regulatory site(s)

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

Sloan, J.W.

1984-01-01

These studies show that nicotine binds to the rat brain P/sub 2/ preparation by saturable and reversible processes. Multiple binding sites were revealed by the configuration of saturation, kinetic and Scatchard plots. A least squares best fit of Scatchard data using nonlinear curve fitting programs confirmed the presence of a very high affinity site, an up-regulatory site, a high affinity site and one or two low affinity sites. Stereospecificity was demonstrated for the up-regulatory site where (+)-nicotine was more effective and for the high affinity site where (-)-nicotine had a higher affinity. Drugs which selectively up-regulate nicotine binding site(s) havemore » been identified. Further, separate very high and high affinity sites were identified for (-)- and (+)-(/sup 3/H)nicotine, based on evidence that the site density for the (-)-isomer is 10 times greater than that for the (+)-isomer at these sites. Enhanced nicotine binding has been shown to be a statistically significant phenomenon which appears to be a consequence of drugs binding to specific site(s) which up-regulate binding at other site(s). Although Scatchard and Hill plots indicate positive cooperatively, up-regulation more adequately describes the function of these site(s). A separate up-regulatory site is suggested by the following: (1) Drugs vary markedly in their ability to up-regulate binding. (2) Both the affinity and the degree of up-regulation can be altered by structural changes in ligands. (3) Drugs with specificity for up-regulation have been identified. (4) Some drugs enhance binding in a dose-related manner. (5) Competition studies employing cold (-)- and (+)-nicotine against (-)- and (+)-(/sup 3/H)nicotine show that the isomers bind to separate sites which up-regulate binding at the (-)- and (+)-nicotine high affinity sites and in this regard (+)-nicotine is more specific and efficacious than (-)-nicotine.« less

Probing Selenium-Ion Distributions and Changes in Redox-State at Biofilm/Mineral Interfaces by Coupling Long-period X-ray Standing Wave and XANES Measurements

NASA Astrophysics Data System (ADS)

Templeton, A. S.; Trainor, T. P.; Spormann, A. M.; Brown, G. E.

2002-12-01

Metal sorption and precipitation reactions at biological as well as mineral surfaces are important controls on metal speciation and bioavailability in natural environments. When highly hydrated biofilms form on mineral surfaces, numerous competitive and synergistic effects are predicted to occur. Experimentally, it is challenging to determine where the sorbed metal ions are localized, the relative affinity of the biological vs. mineral surface sites, or to monitor biomineralization reactions or changes in metal speciation that may also occur. A large part of the difficulty is due to the low concentrations of sorbed ions, the small length-scale of the biofilm-mineral interface, and the complex interplay between microbially-catalayzed redox transformations vs. sorption and/or transport processes. Long-period x-ray standing wave (XSW) techniques are well-suited to determining the vertical distribution of metal(oid) species within biofilms overlying mineral surfaces. We will discuss experiments where Se fluorescence yield profiles are used to compare the affinity of Burkholderia cepacia biofilms for binding Se(IV) and Se(VI) species relative to underlying alpha-Al2O3 substrates over three orders of magnitude in [Se]. In addition, we will discuss how coupling the XSW experiments to grazing-incidence, spatially-resolved Se K-edge XANES spectroscopy can be used to differentiate between the oxidation state of the Se complexes localized within the biofilm vs. the mineral surface. This approach is used to monitor changes in the relative distributions of Se(VI), Se(IV) and Se(0) species as a function of time and proximity to the mineral surface. The long-period XSW data show that selenite preferentially binds to the oxide surfaces, particularly at low [Se]. When B. cepacia is metabolically active, B. cepacia rapidly reduces a fraction of the Se(IV) to the red elemental Se form. In contrast, selenate is preferentially partitioned into the B. cepacia biofilms at all [Se] tested due to a lower affinity for binding to the mineral surface. XANES spectra show that rapid reduction of selenate by B. cepacia to Se(IV) and Se(0) species subsequently results in a vertical segregation of Se species at the B. cepacia/alpha-Al2O3 interface. Elemental Se accumulates within the biofilm with the Se(VI), whereas selenite intermediates preferentially sorb to the underlying oxide surface.

The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1), Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo

PubMed Central

Sankova, Tatiana P.; Orlov, Iurii A.; Saveliev, Andrey N.; Kirilenko, Demid A.; Babich, Polina S.; Brunkov, Pavel N.; Puchkova, Ludmila V.

2017-01-01

There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST) and the N-terminal domain (ectodomain) of human high affinity copper transporter CTR1 (hNdCTR1), which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell’s copper metabolism and its chelating properties are discussed. PMID:29099786

The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1), Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo.

PubMed

Sankova, Tatiana P; Orlov, Iurii A; Saveliev, Andrey N; Kirilenko, Demid A; Babich, Polina S; Brunkov, Pavel N; Puchkova, Ludmila V

2017-11-03

There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST) and the N-terminal domain (ectodomain) of human high affinity copper transporter CTR1 (hNdCTR1), which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell's copper metabolism and its chelating properties are discussed.

HD Exchange and PLIMSTEX Determine the Affinities and Order of Binding of Ca2+ with Troponin C

PubMed Central

Huang, Richard Y-C.; Rempel, Don L.; Gross, Michael L.

2011-01-01

Troponin C (TnC), present in all striated muscle, is the Ca2+-activated trigger that initiates myocyte contraction. The binding of Ca2+ to TnC initiates a cascade of conformational changes involving the constituent proteins of the thin filament. The functional properties of TnC and its ability to bind Ca2+ have significant regulatory influence on the contractile reaction of muscle. Changes in TnC may also correlate with cardiac and various other muscle-related diseases. We report here the implementation of the PLIMSTEX strategy (Protein Ligand Interaction by Mass Spectrometry, Titration and H/D Exchange) to elucidate the binding affinity of TnC with Ca2+ and, more importantly, to determine the order of Ca2+ binding of the four EF hands of the protein. The four equilibrium constants, K1 = (5 ± 5) × 10 M-1, K2 = (1.8 ± 0.8) × 107 M-1, K3 = (4.2 ± 0.9) × 106 M-1, and K4 = (1.6 ± 0.6) × 106 M-1, agree well with determinations by other methods and serve to increase our confidence in the PLIMSTEX approach. We determined the order of binding to the four EF hands to be III, IV, II, and I by extracting from the H/DX results the deuterium patterns for each EF hand for each state of the protein (apo through fully Ca2+ bound). This approach, demonstrated for the first time, may be general for determining binding orders of metal ions and other ligands to proteins. PMID:21574565

Binding of naringin and naringenin with hen egg white lysozyme: A spectroscopic investigation and molecular docking study

NASA Astrophysics Data System (ADS)

Das, Sourav; Ghosh, Pooja; Koley, Sudipta; Singha Roy, Atanu

2018-03-01

The interactions of naringenin (NG) and naringin (NR) with Hen Egg White Lysozyme (HEWL) in aqueous medium have been investigated using UV-vis spectroscopy, steady-state fluorescence, circular dichroism (CD), Fourier Transform infrared spectroscopy (FT-IR) and molecular docking analyses. Both NG and NR can quench the intrinsic fluorescence of HEWL via static quenching mechanism. At 300 K, the value of binding constant (Kb) of HEWL-NG complex (5.596 ± 0.063 × 104 M- 1) was found to be greater than that of HEWL-NR complex (3.404 ± 0.407 × 104 M- 1). The negative ΔG° values in cases of both the complexes specify the spontaneous binding. The binding distance between the donor (HEWL) and acceptor (NG/NR) was estimated using the Försters theory and the possibility of non-radiative energy transfer from HEWL to NG/NR was observed. The presence of metal ions (Ca2 +, Cu2 + and Fe2 +) decreased the binding affinity of NG/NR towards HEWL. Synchronous fluorescence studies indicate the change in Trp micro-environment due to the incorporation of NG/NR into HEWL. CD and FT-IR studies indicated that the α-helicity of the HEWL was slightly enhanced due to ligand binding. NG and NR inhibited the enzymatic activity of HEWL and exhibited their affinity for the active site of HEWL. Molecular docking studies revealed that both NG and NR bind in the close vicinity of Trp 62 and Trp 63 residues which is vital for the catalytic activity.

FecB, a periplasmic ferric-citrate transporter from E. coli, can bind different forms of ferric-citrate as well as a wide variety of metal-free and metal-loaded tricarboxylic acids.

PubMed

Banerjee, Sambuddha; Paul, Subrata; Nguyen, Leonard T; Chu, Byron C H; Vogel, Hans J

2016-01-01

The Escherichia coli Fec system, consisting of an outer membrane receptor (FecA), a periplasmic substrate binding protein (FecB) and an inner membrane permease-ATPase type transporter (FecC/D), plays an important role in the uptake and transport of Fe(3+)-citrate. Although several FecB sequences from various organisms have been reported, there are no biophysical or structural data available for this protein to date. In this work, using isothermal titration calorimetry (ITC), we report for the first time the ability of FecB to bind different species of Fe(3+)-citrate as well as other citrate complexes with trivalent (Ga(3+), Al(3+), Sc(3+) and In(3+)) and a representative divalent metal ion (Mg(2+)) with low μM affinity. Interestingly, ITC experiments with various iron-free di- and tricarboxylic acids show that FecB can bind tricarboxylates with μM affinity but not biologically relevant dicarboxylates. The ability of FecB to bind with metal-free citrate is also observed in (1)H,(15)N HSQC-NMR titration experiments reported here at two different pH values. Further, differential scanning calorimetry (DSC) experiments indicate that the ligand-bound form of FecB has greater thermal stability than ligand-free FecB under all pH and ligand conditions tested, which is consistent with the idea of domain closure subsequent to ligand binding for this type of periplasmic binding proteins.

Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC

PubMed Central

Chen, Qiang; Cheng, Mary Hongying; Xu, Yan; Tang, Pei

2010-01-01

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (KD) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins. PMID:20858424

Use of terbium as a probe of tRNA tertiary structure and folding.

PubMed Central

Hargittai, M R; Musier-Forsyth, K

2000-01-01

Lanthanide metals such as terbium have previously been shown to be useful for mapping metal-binding sites in RNA. Terbium binds to the same sites on RNA as magnesium, however, with a much higher affinity. Thus, low concentrations of terbium ions can easily displace magnesium and promote phosphodiester backbone scission. At higher concentrations, terbium cleaves RNA in a sequence-independent manner, with a preference for single-stranded, non-Watson-Crick base-paired regions. Here, we show that terbium is a sensitive probe of human tRNALys,3 tertiary structure and folding. When 1 microM tRNA is used, the optimal terbium ion concentration for detecting Mg2+-induced tertiary structural changes is 50-60 microM. Using these concentrations of RNA and terbium, a magnesium-dependent folding transition with a midpoint (KMg) of 2.6 mM is observed for unmodified human tRNALys,3. At lower Tb3+ concentrations, cleavage is restricted to nucleotides that constitute specific metal-binding pockets. This small chemical probe should also be useful for detecting protein induced structural changes in RNA. PMID:11105765

Two-sided block of a dual-topology F- channel.

PubMed

Turman, Daniel L; Nathanson, Jacob T; Stockbridge, Randy B; Street, Timothy O; Miller, Christopher

2015-05-05

The Fluc family is a set of small membrane proteins forming F(-)-specific electrodiffusive ion channels that rescue microorganisms from F(-) toxicity during exposure to weakly acidic environments. The functional channel is built as a dual-topology homodimer with twofold symmetry parallel to the membrane plane. Fluc channels are blocked by nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries. The unusual symmetrical antiparallel dimeric architecture of Flucs demands that the two chemically equivalent monobody-binding epitopes reside on opposite ends of the channel, a double-sided blocking situation that has never before presented itself in ion channel biophysics. However, it is not known if both sites can be simultaneously occupied, and if so, whether monobodies bind independently or cooperatively to their transmembrane epitopes. Here, we use direct monobody-binding assays and single-channel recordings of a Fluc channel homolog to reveal a novel trimolecular blocking behavior that reveals a doubly occupied blocked state. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity between the two blocking sites.

Solvation of actinide salts in water using a polarizable continuum model.

PubMed

Kumar, Narendra; Seminario, Jorge M

2015-01-29

In order to determine how actinide atoms are dressed when solvated in water, density functional theory calculations have been carried out to study the equilibrium structure of uranium plutonium and thorium salts (UO2(2+), PuO2(2+), Pu(4+), and Th(4+)) both in vacuum as well as in solution represented by a conductor-like polarizable continuum model. This information is of paramount importance for the development of sensitive nanosensors. Both UO2(2+) and PuO2(2+) ions show coordination number of 4-5 with counterions replacing one or two water molecules from the first coordination shell. On the other hand, Pu(4+), has a coordination number of 8 both when completely solvated and also in the presence of chloride and nitrate ions with counterions replacing water molecules in the first shell. Nitrates were found to bind more strongly to Pu(IV) than chloride anions. In the case of the Th(IV) ion, the coordination number was found to be 9 or 10 in the presence of chlorides. Moreover, the Pu(IV) ion shows greater affinity for chlorides than the Th(IV) ion. Adding dispersion and ZPE corrections to the binding energy does not alter the trends in relative stability of several conformers because of error cancelations. All structures and energetics of these complexes are reported.

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva.

PubMed

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui; Xiao, Yi

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

Divers models of divalent cation interaction to calcium-binding proteins: techniques and anthology.

PubMed

Cox, Jos A

2013-01-01

Intracellular Ca(2+)-binding proteins (CaBPs) are sensors of the calcium signal and several of them even shape the signal. Most of them are equipped with at least two EF-hand motifs designed to bind Ca(2+). Their affinities are very variable, can display cooperative effects, and can be modulated by physiological Mg(2+) concentrations. These binding phenomena are monitored by four major techniques: equilibrium dialysis, fluorimetry with fluorescent Ca(2+) indicators, flow dialysis, and isothermal titration calorimetry. In the last quarter of the twentieth century reports on the ion-binding characteristics of several abundant wild-type CaBPs were published. With the advent of recombinant CaBPs it became possible to determine these properties on previously inaccessible proteins. Here I report on studies by our group carried out in the last decade on eight families of recombinant CaBPs, their mutants, or truncated domains. Moreover this chapter deals with the currently used methods for quantifying the binding of Ca(2+) and Mg(2+) to CaBPs.

Opposing Intermolecular Tuning of Ca2+ Affinity for Calmodulin by Neurogranin and CaMKII Peptides.

PubMed

Zhang, Pengzhi; Tripathi, Swarnendu; Trinh, Hoa; Cheung, Margaret S

2017-03-28

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca 2+ ) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca 2+ /CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca 2+ signaling pathways in neurons. It was shown experimentally that Ca 2+ /CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca 2+ -free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca 2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng 13-49 ), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng 13-49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng 13-49 structures by matching the changes in the chemical shifts of CaM upon Ng 13-49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca 2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca 2+ for CaM in the presence of Ng 13-49 by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca 2+ binding loops particularly at C-domain of CaM, enabling Ca 2+ release. In contrast, CaMKII peptide increases Ca 2+ affinity for the C-domain of CaM by stabilizing the two Ca 2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng 13-49 and holoCaM-CaMKII delineates the importance of CaM's progressive mechanism of target binding on its Ca 2+ binding affinities. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Modulation of DNA binding by gene-specific transcription factors.

PubMed

Schleif, Robert F

2013-10-01

The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

Method for lake restoration

DOEpatents

Dawson, Gaynor W.; Mercer, Basil W.

1979-01-01

A process for removing pollutants or minerals from lake, river or ocean sediments or from mine tailings is disclosed. Magnetically attractable collection units containing an ion exchange or sorbent media with an affinity for a chosen target substance are distributed in the sediments or tailings. After a period of time has passed sufficient for the particles to bind up the target substances, a magnet drawn through the sediments or across the tailings retrieves the units along with the target substance.

Acid-base properties of humic and fulvic acids formed during composting.

PubMed

Plaza, César; Senesi, Nicola; Polo, Alfredo; Brunetti, Gennaro

2005-09-15

The soil acid-base buffering capacity and the biological availability, mobilization, and transport of macro- and micronutrients, toxic metal ions, and xenobiotic organic cations in soil are strongly influenced by the acid-base properties of humic substances, of which humic and fulvic acids are the major fractions. For these reasons, the proton binding behavior of the humic acid-like (HA) and fulvic acid-like (FA) fractions contained in a compost are believed to be instrumental in its successful performance in soil. In this work, the acid-base properties of the HAs and FAs isolated from a mixture of the sludge residue obtained from olive oil mill wastewater (OMW) evaporated in an open-air pond and tree cuttings (TC) at different stages of composting were investigated by a current potentiometric titration method and the nonideal competitive adsorption (NICA)-Donnan model. The NICA-Donnan model provided an excellent description of the acid-base titration data, and pointed out substantial differences in site density and proton-binding affinity between the HAs and FAs examined. With respect to FAs, HAs were characterized by a smaller content of carboxylic- and phenolic-type groups and their larger affinities for proton binding. Further, HAs featured a greater heterogeneity in carboxylic-type groups than FAs. The composting process increased the content and decreased the proton affinity of carboxylic- and phenolic-type groups of HAs and FAs, and increased the heterogeneity of phenolic-type groups of HAs. As a whole, these effects indicated that the composting process could produce HA and FA fractions with greater cation binding capacities. These results suggest that composting of organic materials improves their agronomic and environmental value by increasing their potential to retain and exchange macro- and micronutrients, and to reduce the bioavailability of organic and inorganic pollutants.

Temporal changes in elemental composition in decomposing filamentous algae (Cladophora glomerata and Pilayella littoralis) determined with PIXE and PIGE.

PubMed

Lill, J-O; Salovius-Laurén, S; Harju, L; Rajander, J; Saarela, K-E; Lindroos, A; Heselius, S-J

2012-01-01

Particle-induced X-ray emission and particle-induced gamma-ray emission spectrometry were successfully applied in a study of the elemental composition of decomposing filamentous algae. Fresh brown (Pilayella littoralis) and green (Cladophora glomerata) algal materials were placed in cages at 4m depth in a water column of 8m in the Archipelago Sea, northern Baltic Sea. Every second week decaying algae were sampled from the cages to allow measurements of changes in the elemental compositions. In the study of the elemental losses the concentrations were compensated for the mass reduction. The results show that sulphur, chlorine and partly potassium were lost during decomposition of P. littoralis and C. glomerata. Most of the other elements studied were recovered in the remaining algal mass. Special attention was paid to sorption and desorption of elements, including metal binding capacity, in the decaying algal materials. The affinity order of different cations to the two algal species was established by calculation of conditional distribution coefficients, D'(M). For instance for P. littoralis the following series of binding strength (affinity) of cations were obtained: Al>Ti>Fe > Mn>Ni, Cu>Ba, Cr, Zn>Rb>K, Sr>Pb>Ca>Na>Mg. Notably is that the binding strength of strontium was more than 10 times higher for P. littoralis than for C. glomerata. Due to their high binding capacity and good affinity and selectivity for heavy metal ions these algae have great potential as biological sorbents. Large variations in elemental content during decomposition complicate the use of algae for environmental monitoring. Copyright © 2011 Elsevier B.V. All rights reserved.

Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

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

Zhang, Lianying; College of Life Science, Dezhou University, Dezhou 253023; Ren, Xiao-Min

2014-09-15

Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group.more » For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.« less

Structural and Thermodynamic Consequences of the Replacement of Zinc with Environmental Metals on ERα-DNA Interactions

PubMed Central

Deegan, Brian J.; Bona, Anna M.; Bhat, Vikas; Mikles, David C.; McDonald, Caleb B.; Seldeen, Kenneth L.; Farooq, Amjad

2011-01-01

Estrogen receptor α (ERα) acts as a transcription factor by virtue of the ability of its DNA-binding (DB) domain, comprised of a tandem pair of zinc fingers, to recognize the estrogen response element (ERE) within the promoters of target genes. Herein, using an array of biophysical methods, we probe structural consequences of the replacement of zinc within the DB domain of ERα with various environmental metals and their effects on the thermodynamics of binding to DNA. Our data reveal that while the DB domain reconstituted with divalent ions of zinc, cadmium, mercury and cobalt binds to DNA with affinities in the nanomolar range, divalent ions of barium, copper, iron, lead, manganese, nickel and tin are unable to regenerate DB domain with DNA-binding potential though they can compete with zinc for coordinating the cysteine ligands within the zinc fingers. We also show that the metal-free DB domain is a homodimer in solution and that the binding of various metals only results in subtle secondary and tertiary structural changes, implying that metal-coordination may only be essential for DNA-binding. Collectively, our findings provide mechanistic insights into how environmental metals may modulate the physiological function of a key nuclear receptor involved in mediating a plethora of cellular functions central to human health and disease. PMID:22038807

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel.

PubMed

Yang, Fan; Vu, Simon; Yarov-Yarovoy, Vladimir; Zheng, Jie

2016-06-28

Vanilloids activation of TRPV1 represents an excellent model system of ligand-gated ion channels. Recent studies using cryo-electron microcopy (cryo-EM), computational analysis, and functional quantification revealed the location of capsaicin-binding site and critical residues mediating ligand-binding and channel activation. Based on these new findings, here we have successfully introduced high-affinity binding of capsaicin and resiniferatoxin to the vanilloid-insensitive TRPV2 channel, using a rationally designed minimal set of four point mutations (F467S-S498F-L505T-Q525E, termed TRPV2_Quad). We found that binding of resiniferatoxin activates TRPV2_Quad but the ligand-induced open state is relatively unstable, whereas binding of capsaicin to TRPV2_Quad antagonizes resiniferatoxin-induced activation likely through competition for the same binding sites. Using Rosetta-based molecular docking, we observed a common structural mechanism underlying vanilloids activation of TRPV1 and TRPV2_Quad, where the ligand serves as molecular "glue" that bridges the S4-S5 linker to the S1-S4 domain to open these channels. Our analysis revealed that capsaicin failed to activate TRPV2_Quad likely due to structural constraints preventing such bridge formation. These results not only validate our current working model for capsaicin activation of TRPV1 but also should help guide the design of drug candidate compounds for this important pain sensor.

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.

Specificity and Affinity Quantification of Flexible Recognition from Underlying Energy Landscape Topography

PubMed Central

Chu, Xiakun; Wang, Jin

2014-01-01

Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less) flexibility leads to weaker (stronger) coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition. PMID:25144525

Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

PubMed Central

Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

2012-01-01

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

Specificity and affinity quantification of flexible recognition from underlying energy landscape topography.

PubMed

Chu, Xiakun; Wang, Jin

2014-08-01

Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less) flexibility leads to weaker (stronger) coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition.

How Much Binding Affinity Can be Gained by Filling a Cavity?

PubMed Central

Kawasaki, Yuko; Chufan, Eduardo E.; Lafont, Virginie; Hidaka, Koushi; Kiso, Yoshiaki; Amzel, L. Mario; Freire, Ernesto

2011-01-01

Binding affinity optimization is critical during drug development. Here we evaluate the thermodynamic consequences of filling a binding cavity with functionalities of increasing van der Waals radii (-H, -F, -Cl and CH3) that improve the geometric fit without participating in hydrogen bonding or other specific interactions. We observe a binding affinity increase of two orders of magnitude. There appears to be three phases in the process. The first phase is associated with the formation of stable van der Waals interactions. This phase is characterized by a gain in binding enthalpy and a loss in binding entropy, attributed to a loss of conformational degrees of freedom. For the specific case presented in this paper, the enthalpy gain amounts to −1.5 kcal/mol while the entropic losses amount to +0.9 kcal/mol resulting in a net 3.5-fold affinity gain. The second phase is characterized by simultaneous enthalpic and entropic gains. This phase improves the binding affinity 25-fold. The third phase represents the collapse of the trend and is triggered by the introduction of chemical functionalities larger than the binding cavity itself (CH(CH3)2). It is characterized by large enthalpy and affinity losses. The thermodynamic signatures associated with each phase provide guidelines for lead optimization. PMID:20028396

Parameterization of Ca+2-protein interactions for molecular dynamics simulations.

PubMed

Project, Elad; Nachliel, Esther; Gutman, Menachem

2008-05-01

Molecular dynamics simulations of Ca+2 ions near protein were performed with three force fields: GROMOS96, OPLS-AA, and CHARMM22. The simulations reveal major, force-field dependent, inconsistencies in the interaction between the Ca+2 ions with the protein. The variations are attributed to the nonbonded parameterizations of the Ca+2-carboxylates interactions. The simulations results were compared to experimental data, using the Ca+2-HCOO- equilibrium as a model. The OPLS-AA force field grossly overestimates the binding affinity of the Ca+2 ions to the carboxylate whereas the GROMOS96 and CHARMM22 force fields underestimate the stability of the complex. Optimization of the Lennard-Jones parameters for the Ca+2-carboxylate interactions were carried out, yielding new parameters which reproduce experimental data. Copyright 2007 Wiley Periodicals, Inc.

Ammonium boranes for the selective complexation of cyanide or fluoride ions in water.

PubMed

Hudnall, Todd W; Gabbaï, François P

2007-10-03

With the recognition of aqueous fluoride and cyanide ions as an objective, we have investigated the anion binding properties of two isomeric ammonium boranes, namely [p-(Mes2B)C6H4(NMe3)]+ ([1]+) and [o-(Mes2B)C6H4(NMe3)]+ ([2]+). These cationic boranes, which could be obtained by reaction of the known 4- and 2-dimesitylboryl-N,N-dimethylaniline with MeOTf, have been investigated both experimentally and computationally. They both react with fluoride and cyanide ions in organic solvents to afford the corresponding fluoroborate/ or cyanoborate/ammonium zwitterions 1F, 1CN, 2F, and 2CN. In aqueous solution, however, these cationic boranes behave as remarkably selective receptors. Indeed, [1]+ only complexes cyanide ions while [2]+ only complexes fluoride ions. In H2O/DMSO 60:40 vol (HEPES 6 mM, pH 7), the cyanide binding constant of [1]+ and the fluoride binding constant of [2]+ are respectively equal to 3.9 (+/-0.1) x 108 and 910 (+/-50) M-1. Structural and computational studies indicate that both steric and electronic effects contribute to the unusual selectivity displayed by these cationic boranes. Owing to favorable Coulombic effects, the para-derivative [1]+ has a very high affinity for cyanide; yet these effects are not sufficiently intense to allow complexation of the more efficiently hydrated and less basic fluoride anion. In the case of the ortho-derivative [2]+, the proximity of the ammonium moiety leads to an increase in the Lewis acidity of the boron center thus making fluoride binding possible. However, steric effects prevent cyanide coordination to the boron center of [2]+. Finally, cation [1]+ and [2]+ bind their dedicated anions reversibly and show a negligible response in the presence of other common anions including Cl-, Br-, I-, NO3-, OAc-, H2PO4-, and HSO4-.

Biosorption of Microelements by Spirulina: Towards Technology of Mineral Feed Supplements

PubMed Central

Chojnacka, Katarzyna

2014-01-01

Surface characterization and metal ion adsorption properties of Spirulina sp. and Spirulina maxima were verified by various instrumental techniques. FTIR spectroscopy and potentiometric titration were used for qualitative and quantitative determination of metal ion-binding groups. Comparative FTIR spectra of natural and Cu(II)-treated biomass proved involvement of both phosphoryl and sulfone groups in metal ions sorption. The potentiometric titration data analysis provided the best fit with the model assuming the presence of three types of surface functional groups and the carboxyl group as the major binding site. The mechanism of metal ions biosorption was investigated by comparing the results from multielemental analyses by ICP-OES and SEM-EDX. Biosorption of Cu(II), Mn(II), Zn(II), and Co(II) ions by lyophilized Spirulina sp. was performed to determine the metal affinity relationships for single- and multicomponent systems. Obtained results showed the replacement of naturally bound ions: Na(I), K(I), or Ca(II) with sorbed metal ions in a descending order of Mn(II) > Cu(II) > Zn(II) > Co(II) for single- and Cu(II) > Mn(II) > Co(II) > Zn(II) for multicomponent systems, respectively. Surface elemental composition of natural and metal-loaded material was determined both by ICP-OES and SEM-EDX analysis, showing relatively high value of correlation coefficient between the concentration of Na(I) ions in algal biomass. PMID:25386594

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*

PubMed Central

Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

2015-01-01

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

PubMed Central

Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

2015-01-01

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes. PMID:26053890

In silico assessment of the conduction mechanism of the Ryanodine Receptor 1 reveals previously unknown exit pathways.

PubMed

Heinz, Leonard P; Kopec, Wojciech; de Groot, Bert L; Fink, Rainer H A

2018-05-02

The ryanodine receptor 1 is a large calcium ion channel found in mammalian skeletal muscle. The ion channel gained a lot of attention recently, after multiple independent authors published near-atomic cryo electron microscopy data. Taking advantage of the unprecedented quality of structural data, we performed molecular dynamics simulations on the entire ion channel as well as on a reduced model. We calculated potentials of mean force for Ba 2+ , Ca 2+ , Mg 2+ , K + , Na + and Cl - ions using umbrella sampling to identify the key residues involved in ion permeation. We found two main binding sites for the cations, whereas the channel is strongly repulsive for chloride ions. Furthermore, the data is consistent with the model that the receptor achieves its ion selectivity by over-affinity for divalent cations in a calcium-block-like fashion. We reproduced the experimental conductance for potassium ions in permeation simulations with applied voltage. The analysis of the permeation paths shows that ions exit the pore via multiple pathways, which we suggest to be related to the experimental observation of different subconducting states.

Evolution reversed: the ability to bind iron restored to the N-lobe of the murine inhibitor of carbonic anhydrase by strategic mutagenesis.

PubMed

Mason, Anne B; Judson, Gregory L; Bravo, Maria Cristina; Edelstein, Andrew; Byrne, Shaina L; James, Nicholas G; Roush, Eric D; Fierke, Carol A; Bobst, Cedric E; Kaltashov, Igor A; Daughtery, Margaret A

2008-09-16

The murine inhibitor of carbonic anhydrase (mICA) is a member of the superfamily related to the bilobal iron transport protein transferrin (TF), which binds a ferric ion within a cleft in each lobe. Although the gene encoding ICA in humans is classified as a pseudogene, an apparently functional ICA gene has been annotated in mice, rats, cows, pigs, and dogs. All ICAs lack one (or more) of the amino acid ligands in each lobe essential for high-affinity coordination of iron and the requisite synergistic anion, carbonate. The reason why ICA family members have lost the ability to bind iron is potentially related to acquiring a new function(s), one of which is inhibition of certain carbonic anhydrase (CA) isoforms. A recombinant mutant of the mICA (W124R/S188Y) was created with the goal of restoring the ligands required for both anion (Arg124) and iron (Tyr188) binding in the N-lobe. Absorption and fluorescence spectra definitively show that the mutant binds ferric iron in the N-lobe. Electrospray ionization mass spectrometry confirms the presence of both ferric iron and carbonate. At the putative endosomal pH of 5.6, iron is released by two slow processes indicative of high-affinity coordination. Induction of specific iron binding implies that (1) the structure of mICA resembles those of other TF family members and (2) the N-lobe can adopt a conformation in which the cleft closes when iron binds. Because the conformational change in the N-lobe indicated by metal binding does not impact the inhibitory activity of mICA, inhibition of CA was tentatively assigned to the C-lobe. Proof of this assignment is provided by limited trypsin proteolysis of porcine ICA.

Molecular docking and dynamics simulation study on the influence of Zn2+ on the binding modes of aggrecanase with its inhibitors.

PubMed

Suganya, Panneer S R; Kalva, Sukesh; Saleena, Lilly M

2014-01-01

Zinc plays a vital role in structural organization, regulation of function and stabilization of the folded protein, which ultimately activates or inactivates the binding sites of the protein. Its transition makes a major change in the protein and its binding affinity. The ligand binding aggrecanases can be influenced by Zn2+ ions; therefore the study focuses on checking the binding mode in the presence and absence of zinc using Docking and Molecular dynamics simulation. The crystal structure with zinc was considered as wild type (ADAMTS-4-1Zn2+, ADAMTS-5-1Zn2+) and the crystal structure without zinc was considered as the mutant type (ADAMTS-4-0Zn2+, ADAMTS-5-0Zn2+). Mutations were made manually by deleting the zinc atom. ADAMTS-4-1Zn2+ had the best Glide score of -12.66 kcal·mol−1, whereas ADAMTS-4-0Zn2+ had -11.69 kcal·mol−1. ADAMTS-4-1Zn2+ had the best glide energy of -72.29 kcal·mol−1, whereas ADAMTS-4-0Zn2+ had-68.44 kcal·mol−1. ADAMTS-4-1Zn2+ had the best glide e-model of -116.34, whereas ADAMTS-4-0Zn2+ had -104.264. The RMSD value for ADAMTS-4-1Zn2+ and ADAMTS-4-0Zn2+ was 1.9. These results suggested that the absence of zinc decreases the binding affinity of ADAMTS-4 with its inhibitor. ADAMTS-5-1Zn2+ had the best Glide score of -8.32 kcal·mol−1, whereas ADAMTS-5-0Zn2+ had -6.62 kcal·mol−1. ADAMTS-5-1Zn2+ had the best glide energy of -70.28 kcal·mol−1, whereas ADAMTS-5-0Zn2+ had -66.02 kcal·mol−1. ADAMTS-5-1Zn2+ had the best glide e-model of-108.484, whereas ADAMTS-5-0Zn2+ had -93.81. The RMSD value for ADAMTS-5-1Zn2+ and ADAMTS-5-0Zn2+ was 0.48Å. These results confirmed that the absence of zinc decreased the binding affinity of ADAMTS-5 with its inhibitor whereas the presence extended the docking energy range and strengthened the binding affinity. Per-residue interaction study, MM-GBSA and Molecular Dynamics showed that all the four complexes underwent extensive structural changes whereas the complex with zinc was stable throughout the simulation period.

Batrachotoxin Changes the Properties of the Muscarinic Receptor in Rat Brain and Heart: Possible Interaction(s) between Muscarinic Receptors and Sodium Channels

NASA Astrophysics Data System (ADS)

Cohen-Armon, Malca; Kloog, Yoel; Henis, Yoav I.; Sokolovsky, Mordechai

1985-05-01

The effects of Na+-channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors in homogenates of rat brain and heart were studied. BTX enhanced the affinity for the binding of the agonists carbamoylcholine and acetylcholine to the muscarinic receptors in brainstem and ventricle, but not in the cerebral cortex. Analysis of the data according to a two-site model for agonist binding indicated that the effect of BTX was to increase the affinity of the agonists to the high-affinity site. Guanyl nucleotides, known to induce interconversion of high-affinity agonist binding sites to the low-affinity state, canceled the effect of BTX on carbamoylcholine and acetylcholine binding. BTX had no effect on the binding of the agonist oxotremorine or on the binding of the antagonist [3H]-N-methyl-4-piperidyl benzilate. The local anesthetics dibucaine and tetracaine antagonized the effect of BTX on the binding of muscarinic agonists at concentrations known to inhibit the activation of Na+ channels by BTX. On the basis of these findings, we propose that in specific tissues the muscarinic receptors may interact with the BTX binding site (Na+ channels).

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody.

PubMed

Serpa, Gisele; Augusto, Elisabeth Fátima Pires; Tamashiro, Wirla Maria Silva Cunha; Ribeiro, Mariana Borçoe; Miranda, Everson Alves; Bueno, Sônia Maria Alves

2005-02-25

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me(2+)-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn(2+) complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose.

Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

NASA Astrophysics Data System (ADS)

Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

2016-11-01

Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

Purification of L-( sup 3 H) Nicotine eliminates low affinity binding

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

Romm, E.; Marks, M.J.; Collins, A.C.

1990-01-01

Some studies of L-({sup 3}H) nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicated that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-({sup 3}H)nicotine eliminates the low affinity site.

Highly selectively monitoring heavy and transition metal ions by a fluorescent sensor based on dipeptide.

PubMed

Neupane, Lok Nath; Thirupathi, Ponnaboina; Jang, Sujung; Jang, Min Jung; Kim, Jung Hwa; Lee, Keun-Hyeung

2011-09-15

Fluorescent sensor (DMH) based on dipeptide was efficiently synthesized in solid phase synthesis. The dipeptide sensor shows sensitive response to Ag(I), Hg(II), and Cu(II) among 14 metal ions in 100% aqueous solution. The fluorescent sensor differentiates three heavy metal ions by response type; turn on response to Ag(I), ratiometric response to Hg(II), and turn off detection of Cu(II). The detection limits of the sensor for Ag(I) and Cu(II) were much lower than the EPA's drinking water maximum contaminant levels (MCL). Specially, DMH penetrated live cells and detected intracellular Ag(+) by turn on response. We described the fluorescent change, binding affinity, detection limit for the metal ions. The study of a heavy metal-responsive sensor based on dipeptide demonstrates its potential utility in the environment field. Copyright © 2011 Elsevier B.V. All rights reserved.

Functional antagonistic properties of clozapine at the 5-HT3 receptor.

PubMed

Hermann, B; Wetzel, C H; Pestel, E; Zieglgänsberger, W; Holsboer, F; Rupprecht, R

1996-08-23

The atypical neuroleptic clozapine is thought to exert its psychopharmacological actions through a variety of neurotransmitter receptors. It binds preferentially to D4 and 5-HT2 receptors; however, little is known on it's interaction with the 5-HT3 receptor. Using a cell line stably expressing the 5-HT3 receptor, whole-cell voltage-clamp analysis revealed functional antagonistic properties of clozapine at low nanomolar concentrations in view of a binding affinity in the upper nanomolar range. Because the concentration of clozapine required for an interaction with the 5-HT3 receptor can be achieved with therapeutical doses, functional antagonistic properties at this ligand-gated ion channel may contribute to its unique psychopharmacological profile.

The FOXP2 forkhead domain binds to a variety of DNA sequences with different rates and affinities.

PubMed

Webb, Helen; Steeb, Olga; Blane, Ashleigh; Rotherham, Lia; Aron, Shaun; Machanick, Philip; Dirr, Heini; Fanucchi, Sylvia

2017-07-01

FOXP2 is a member of the P subfamily of FOX transcription factors, the DNA-binding domain of which is the winged helix forkhead domain (FHD). In this work we show that the FOXP2 FHD is able to bind to various DNA sequences, including a novel sequence identified in this work, with different affinities and rates as detected using surface plasmon resonance. Combining the experimental work with molecular docking, we show that high-affinity sequences remain bound to the protein for longer, form a greater number of interactions with the protein and induce a greater structural change in the protein than low-affinity sequences. We propose a binding model for the FOXP2 FHD that involves three types of binding sequence: low affinity sites which allow for rapid scanning of the genome by the protein in a partially unstructured state; moderate affinity sites which serve to locate the protein near target sites and high-affinity sites which secure the protein to the DNA and induce a conformational change necessary for functional binding and the possible initiation of downstream transcriptional events. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

PubMed Central

Orcutt, Kelly Davis; Slusarczyk, Adrian L; Cieslewicz, Maryelise; Ruiz-Yi, Benjamin; Bhushan, Kumar R; Frangioni, John V; Wittrup, K Dane

2014-01-01

Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals. PMID:21315278

DNA Mismatch Binding and Antiproliferative Activity of Rhodium Metalloinsertors

PubMed Central

Ernst, Russell J.; Song, Hang; Barton, Jacqueline K.

2009-01-01

Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M−1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. PMID:19175313

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

PubMed

Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

2008-11-21

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.

LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

PubMed

Marshall, J C; Shakespear, R A; Odell, W D

1976-11-01

Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

Analysis of solute-protein interactions and solute-solute competition by zonal elution affinity chromatography.

PubMed

Tao, Pingyang; Poddar, Saumen; Sun, Zuchen; Hage, David S; Chen, Jianzhong

2018-02-02

Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets. Copyright © 2018 Elsevier Inc. All rights reserved.

Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

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

Poat, J.A.; Cripps, H.E.; Iversen, L.L.

1988-05-01

Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no furthermore » increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.« less

Exopolysaccharides regulate calcium flow in cariogenic biofilms

PubMed Central

Varenganayil, Muth M.; Decho, Alan W.

2017-01-01

Caries-associated biofilms induce loss of calcium from tooth surfaces in the presence of dietary carbohydrates. Exopolysaccharides (EPS) provide a matrix scaffold and an abundance of primary binding sites within biofilms. The role of EPS in binding calcium in cariogenic biofilms is only partially understood. Thus, the aim of the present study is to investigate the relationship between the calcium dissolution rates and calcium tolerance of caries-associated bacteria and yeast as well as to examine the properties of EPS to quantify its binding affinity for dissolved calcium. Calcium dissolution was measured by dissolution zones on Pikovskaya’s agar. Calcium tolerance was assessed by isothermal microcalorimetry (IMC) by adding CaCl2 to the bacterial cultures. Acid-base titration and Fourier transform infrared (FTIR) spectroscopy were used to identify possible functional groups responsible for calcium binding, which was assessed by isothermal titration calorimetry (ITC). Lactobacillus spp. and mutans streptococci demonstrated calcium dissolution in the presence of different carbohydrates. All strains that demonstrated high dissolution rates also revealed higher rates of calcium tolerance by IMC. In addition, acidic functional groups were predominantly identified as possible binding sites for calcium ions by acid-base titration and FTIR. Finally, ITC revealed EPS to have a higher binding affinity for calcium compared, for example, to lactic acid. In conclusion, this study illustrates the role of EPS in terms of the calcium tolerance of cariogenic microbiota by determining the ability of EPS to control free calcium concentrations within the biofilms as a self-regulating mode of action in the pathogenesis of dental caries. PMID:29023506

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes.

PubMed

Woll, Kellie A; Murlidaran, Sruthi; Pinch, Benika J; Hénin, Jérôme; Wang, Xiaoshi; Salari, Reza; Covarrubias, Manuel; Dailey, William P; Brannigan, Grace; Garcia, Benjamin A; Eckenhoff, Roderic G

2016-09-23

Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes*

PubMed Central

Woll, Kellie A.; Murlidaran, Sruthi; Pinch, Benika J.; Hénin, Jérôme; Wang, Xiaoshi; Salari, Reza; Covarrubias, Manuel; Dailey, William P.; Brannigan, Grace; Garcia, Benjamin A.; Eckenhoff, Roderic G.

2016-01-01

Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity. PMID:27462076

Selective retention of basic compounds by metal aquo-ion affinity chromatography.

PubMed

Asakawa, Yoshiki; Yamamoto, Eiichi; Asakawa, Naoki

2014-10-01

A novel metal aquo-ion affinity chromatography has been developed for the analysis of basic compounds using heat-treated silica gel containing hydrated metal cations (metal aquo-ions) as the packing material. The packing materials of the metal aquo-ion affinity chromatography were prepared by the immobilization of a single metal component such as Fe(III), Al(III), Ag(I), and Ni(II) on silica gel followed by extensive heat treatment. The immobilized metals form aquo-ions to present cation-exchange ability for basic analytes and the cation-exchange ability for basic analytes depends on pKa of the immobilized metal species. In the present study, to evaluate the retention characteristics of metal aquo-ion affinity chromatography, the on-line solid-phase extraction of drugs was investigated. Obtained data clearly evidence the selective retention capability of metal aquo-ion affinity chromatography for basic analytes with sufficient capacity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Calcium ion binding properties of Medicago truncatula calcium/calmodulin-dependent protein kinase.

PubMed

Swainsbury, David J K; Zhou, Liang; Oldroyd, Giles E D; Bornemann, Stephen

2012-09-04

A calcium/calmodulin-dependent protein kinase (CCaMK) is essential in the interpretation of calcium oscillations in plant root cells for the establishment of symbiotic relationships with rhizobia and mycorrhizal fungi. Some of its properties have been studied in detail, but its calcium ion binding properties and subsequent conformational change have not. A biophysical approach was taken with constructs comprising either the visinin-like domain of Medicago truncatula CCaMK, which contains EF-hand motifs, or this domain together with the autoinhibitory domain. The visinin-like domain binds three calcium ions, leading to a conformational change involving the exposure of hydrophobic surfaces and a change in tertiary but not net secondary or quaternary structure. The affinity for calcium ions of visinin-like domain EF-hands 1 and 2 (K(d) = 200 ± 50 nM) was appropriate for the interpretation of calcium oscillations (~125-850 nM), while that of EF-hand 3 (K(d) ≤ 20 nM) implied occupancy at basal calcium ion levels. Calcium dissociation rate constants were determined for the visinin-like domain of CCaMK, M. truncatula calmodulin 1, and the complex between these two proteins (the slowest of which was 0.123 ± 0.002 s(-1)), suggesting the corresponding calcium association rate constants were at or near the diffusion-limited rate. In addition, the dissociation of calmodulin from the protein complex was shown to be on the same time scale as the dissociation of calcium ions. These observations suggest that the formation and dissociation of the complex between calmodulin and CCaMK would substantially mirror calcium oscillations, which typically have a 90 s periodicity.

Multi-shell model of ion-induced nucleic acid condensation

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

Tolokh, Igor S.; Drozdetski, Aleksander V.; Pollack, Lois

2016-04-21

We present a semi-quantitative model of condensation of short nucleic acid (NA) duplexes in- duced by tri-valent cobalt hexammine (CoHex) ions. The model is based on partitioning of bound counterion distribution around single NA duplex into “external” and “internal” ion binding shells distinguished by the proximity to duplex helical axis. The duplex aggregation free energy is de- composed into attraction and repulsion components represented by simple analytic expressions. The source of the short-range attraction between NA duplexes in the aggregated phase is the in- teraction of CoHex ions in the overlapping regions of the “external” shells with the oppositely chargedmore » duplexes. The attraction depends on CoHex binding affinity to the “external” shell of nearly neutralized duplex and the number of ions in the shell overlapping volume. For a given NA duplex sequence and structure, these parameters are estimated from molecular dynamics simula- tion. The attraction is opposed by the residual repulsion of nearly neutralized duplexes as well as duplex configurational entropy loss upon aggregation. The estimates of the aggregation free energy are consistent with the experimental range of NA duplex condensation propensities, including the unusually poor condensation of RNA structures and subtle sequence effects upon DNA conden- sation. The model predicts that, in contrast to DNA, RNA duplexes may condense into tighter packed aggregates with a higher degree of duplex neutralization. The model also predicts that longer NA fragments will condense easier than shorter ones. The ability of this model to explain experimentally observed trends in NA condensation, lends support to proposed NA condensation picture based on the multivalent “ion binding shells”.« less

HIGH-AFFINITY T CELL RECEPTOR DIFFERENTIATES COGNATE PEPTIDE-MHC AND ALTERED PEPTIDE LIGANDS WITH DISTINCT KINETICS AND THERMODYNAMICS

PubMed Central

Persaud, Stephen P.; Donermeyer, David L.; Weber, K. Scott; Kranz, David M.; Allen, Paul M.

2010-01-01

Interactions between the T cell receptor and cognate peptide-MHC are crucial initiating events in the adaptive immune response. These binding events are highly specific yet occur with micromolar affinity. Even weaker interactions between TCR and self-pMHC complexes play critical regulatory roles in T cell development, maintenance and coagonist activity. Due to their low affinity, the kinetics and thermodynamics of such weak interactions are difficult to study. In this work, we used M15, a high-affinity TCR engineered from the 3.L2 TCR system, to study the binding properties, thermodynamics, and specificity of two altered peptide ligands (APLs). Our affinity measurements of the high-affinity TCR support the view that the wild type TCR binds these APLs in the millimolar affinity range, and hence very low affinities can still elicit biological functions. Finally, single methylene differences among the APLs gave rise to strikingly different binding thermodynamics. These minor changes in the pMHC antigen were associated with significant and unpredictable changes in both the entropy and enthalpy of the reaction. As the identical TCR was analyzed with several structurally similar ligands, the distinct thermodynamic binding profiles provide a mechanistic perspective on how exquisite antigen specificity is achieved by the T cell receptor. PMID:20334923

Production of a Purified Marine Neurotoxin and Demonstration of its Binding Affinity to Ion Channel Receptors

DTIC Science & Technology

1989-06-10

the ciguatera Implicated toxins, maitotoxin, does not displace brevetoxin from its unique receptor and therefore must produce its toxic 49octs with a...James R. Balthrop, John A. Babinchak, Penny B. Travis, Teresa L. Herring and Pam Y. Brown-Eyo Ciguatera is a tropical fish-borne disease in which both a...synaptosome bound toxin from free toxin following in vitro bindina. we have demonstruted that one of the ciguatera implicated toxins, maitotoxin

Pharmacological characterization of the cloned kappa opioid receptor as a kappa 1b subtype.

PubMed

Lai, J; Ma, S W; Zhu, R H; Rothman, R B; Lentes, K U; Porreca, F

1994-10-27

Substantial pharmacological evidence in vitro and in vivo has suggested the existence of subtypes of the kappa opioid receptor. Quantitative radioligand binding techniques resolved the presence of two high affinity binding sites for the kappa 1 ligand [3H]U69,593 in mouse brain membranes, termed kappa 1a and kappa 1b, respectively. Whereas the kappa 1a site has high affinity for fedotozine and oxymorphindole and low affinity for bremazocine and alpha-neoendorphin, site kappa 1b has high affinity for bremazocine and alpha-neoendorphin and low affinity for fedotozine and oxymorphindole. CI-977 and U69,593 bind equally well at both sites. To determine the relationship between these kappa 1 receptor subtypes and the recently cloned mouse kappa 1 receptor (KOR), we examined [3H]U69,593 binding to the KOR in stably transfected cells (KORCHN-8). Competition of [3H]U69,593 binding to the KOR by bremazocine, alpha-neoendorphin, fedotozine and oxymorphindole resolved a single class of binding sites at which these agents had binding affinities similar to that of the kappa 1b site present in mouse brain. These results suggest that the cloned KOR corresponds to the kappa 1 site in mouse brain defined as kappa 1b.

Calculation of Host-Guest Binding Affinities Using a Quantum-Mechanical Energy Model.

PubMed

Muddana, Hari S; Gilson, Michael K

2012-06-12

The prediction of protein-ligand binding affinities is of central interest in computer-aided drug discovery, but it is still difficult to achieve a high degree of accuracy. Recent studies suggesting that available force fields may be a key source of error motivate the present study, which reports the first mining minima (M2) binding affinity calculations based on a quantum mechanical energy model, rather than an empirical force field. We apply a semi-empirical quantum-mechanical energy function, PM6-DH+, coupled with the COSMO solvation model, to 29 host-guest systems with a wide range of measured binding affinities. After correction for a systematic error, which appears to derive from the treatment of polar solvation, the computed absolute binding affinities agree well with experimental measurements, with a mean error 1.6 kcal/mol and a correlation coefficient of 0.91. These calculations also delineate the contributions of various energy components, including solute energy, configurational entropy, and solvation free energy, to the binding free energies of these host-guest complexes. Comparison with our previous calculations, which used empirical force fields, point to significant differences in both the energetic and entropic components of the binding free energy. The present study demonstrates successful combination of a quantum mechanical Hamiltonian with the M2 affinity method.

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

PubMed Central

Tilley, Drew C.; Eum, Kenneth S.; Fletcher-Taylor, Sebastian; Austin, Daniel C.; Dupré, Christophe; Patrón, Lilian A.; Garcia, Rita L.; Lam, Kit; Yarov-Yarovoy, Vladimir; Cohen, Bruce E.; Sack, Jon T.

2014-01-01

Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX–fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling. PMID:25331865

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

PubMed

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

2010-07-01

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

An evaluation of ferrihydrite- and Metsorb™-DGT techniques for measuring oxyanion species (As, Se, V, P): effective capacity, competition and diffusion coefficients.

PubMed

Price, Helen L; Teasdale, Peter R; Jolley, Dianne F

2013-11-25

This study investigated several knowledge gaps with respect to the diffusive gradients in thin films (DGT) technique for measurement of oxyanions (As(III), As(V), Se(IV), Se(VI), PO4(3-), and V(V)) using the ferrihydrite and Metsorb™ binding layers. Elution efficiencies for each binding layer were higher with 1:20 dilutions, as analytical interferences for ICP-MS were minimised. Diffusion coefficients measured by diffusion cell and by DGT time-series experiments were found to agree well and generally agreed with previously reported values, although a range of diffusion coefficients have been reported for inorganic As and Se species. The relative binding affinity for both ferrihydrite and Metsorb™ was PO4(3-) ≈ As(V)>V(V) ≈ As(III)>Se(IV) > Se(VI) and effective binding capacities were measured in single ion solutions, and spiked synthetic freshwater and seawater, advising practical decisions about DGT monitoring. Under the conditions tested the performance of both ferrihydrite and Metsorb™ binding layers was directly comparable for As(V), As(III) Se(IV), V(V) and PO4(3-) over a deployment spanning ≤ 2 days for both freshwater and seawater. In order to return quantitative data for several analytes we recommend that the DGT method using either ferrihydrite or Metsorb™ be deployed for a maximum of 2 days in marine waters likely to contain high levels of the most strongly adsorbing oxyanions contaminants. The high pH, the competitive ions present in seawater and the identity of co-adsorbing ions affect the capacity of each binding layer for the analytes of interest. In freshwaters, longer deployment times can be considered but the concentration and identity of co-adsorbing ions may impact on quantitative uptake of Se(IV). This study found ferrihydrite-DGT outperformed Metsorb-DGT while previous studies have found the opposite, with variation in binding materials masses used being a likely reason. Clearly, preparation of both binding layers should always be optimised to produce the highest capacity possible, especially for seawater deployments. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

PubMed Central

Hay, Iain D.; Du, Jinping; Burr, Natalie

2014-01-01

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. PMID:25344238

Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms

NASA Astrophysics Data System (ADS)

McQuaid, Jeffrey B.; Kustka, Adam B.; Oborník, Miroslav; Horák, Aleš; McCrow, John P.; Karas, Bogumil J.; Zheng, Hong; Kindeberg, Theodor; Andersson, Andreas J.; Barbeau, Katherine A.; Allen, Andrew E.

2018-03-01

In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton. Although most dissolved iron in the marine environment is complexed with organic molecules, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts, including the ferric iron-concentrating protein ISIP2A, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution and are abundant in marine environmental genomic datasets, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

Direct Measurement of Equilibrium Constants for High-Affinity Hemoglobins

PubMed Central

Kundu, Suman; Premer, Scott A.; Hoy, Julie A.; Trent, James T.; Hargrove, Mark S.

2003-01-01

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (KD ≪ 1 μM) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and ∼100 μM−1 respectively, indicate that they are not capable of facilitating oxygen transport. PMID:12770899

Degenerate Pax2 and Senseless binding motifs improve detection of low-affinity sites required for enhancer specificity

PubMed Central

Zandvakili, Arya; Campbell, Ian; Weirauch, Matthew T.

2018-01-01

Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF binding sites (TFBSs) from background sequences represents a significant challenge. Here, we show that a Drosophila regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, we developed a method that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, we found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs. PMID:29617378

Spectroscopic and TDDFT investigation on highly selective fluorogenic chemosensor and construction of molecular logic gates.

PubMed

Basheer, Sabeel M; Kumar, Saravana Loganathan Ashok; Kumar, Moorthy Saravana; Sreekanth, Anandaram

2017-03-01

1,5-Bis(2-fluorene)thiocarbohydrazone (FBTC) was designed and synthesized for selective sensing of fluoride and copper ions. The binding constants of FBTC towards fluoride and copper ions have been calculated using the Benesi-Hildebrand equation, and FBTC has more binding affinity towards copper ion than fluoride ion. The 1 H NMR and 13 C NMR titration studies strongly support the deprotonation was taken from the N-H protons followed by the formation of hydrogen bond via N-H … F. To understand the fluoride ion sensing mechanism, theoretical investigation had been carried out using the density functional theory and time-dependent density functional theory. The theoretical data well reproduced the experimental results. The deprotonation process has a moderate transition barrier (481.55kcal/mol). The calculated ΔE and ΔG values (-253.92 and -192.41kcal/mol respectively) suggest the feasibility of sensing process. The potential energy curves give the optimized structures of FBTC-F complex in the ground state and excited state, which states the proton transition occurs at the excited state. The excited state proton transition mechanism was further confirmed with natural bond orbital analysis. The reversibility of the sensor was monitored by the alternate addition of F - and Cu 2+ ions, which was explained with "Read-Erase-Write-Read" behaviour. The multi-ion detection of sensor used to construct the molecular logic gate, such as AND, OR, NOR and INHIBITION logic gates. Copyright © 2016 Elsevier B.V. All rights reserved.

Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

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

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

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

Mechanisms of iron and copper-frataxin interactions.

PubMed

Han, T H L; Camadro, J M; Santos, R; Lesuisse, E; El Hage Chahine, J M; Ha-Duong, N T

2017-08-16

Frataxin is a mitochondrial protein whose deficiency is the cause of Friedreich's ataxia, a hereditary neurodegenerative disease. This protein plays a role in iron-sulfur cluster biosynthesis, protection against oxidative stress and iron metabolism. In an attempt to provide a better understanding of the role played by metals in its metabolic functions, the mechanisms of mitochondrial metal binding to frataxin in vitro have been investigated. A purified recombinant yeast frataxin homolog Yfh1 binds two Cu(ii) ions with a K d1 (Cu II ) of 1.3 × 10 -7 M and a K d2 (Cu II ) of 3.1 × 10 -4 M and a single Cu(i) ion with a higher affinity than for Cu(ii) (K d (Cu I ) = 3.2 × 10 -8 M). Mn(ii) forms two complexes with Yfh1 (K d1 (Mn II ) = 4.0 × 10 -8 M; K d2 (Mn II ) = 4.0 × 10 -7 M). Cu and Mn bind Yfh1 with higher affinities than Fe(ii). It is established for the first time that the mechanisms of the interaction of iron and copper with frataxin are comparable and involve three kinetic steps. The first step occurs in the 50-500 ms range and corresponds to a first metal uptake. This is followed by two other kinetic processes that are related to a second metal uptake and/or to a change in the conformation leading to thermodynamic equilibrium. Frataxin deficient Δyfh1 yeast cells exhibited a marked growth defect in the presence of exogenous Cu or Mn. Mitochondria from Δyfh1 strains also accumulated higher amounts of copper, suggesting a functional role of frataxin in vivo in copper homeostasis.

Analyzing the components of the free-energy landscape in a calcium selective ion channel by Widom's particle insertion method

NASA Astrophysics Data System (ADS)

Boda, Dezső; Giri, Janhavi; Henderson, Douglas; Eisenberg, Bob; Gillespie, Dirk

2011-02-01

The selectivity filter of the L-type calcium channel works as a Ca2 + binding site with a very large affinity for Ca2 + versus Na+. Ca2 + replaces half of the Na+ ions in the filter even when these ions are present in 1 μM and 30 mM concentrations in the bath, respectively. The energetics of this strong selectivity is analyzed in this paper. We use Widom's particle insertion method to compute the space-dependent profiles of excess chemical potential in our grand canonical Monte Carlo simulations. These profiles define the free-energy landscape for the various ions. Following Gillespie [Biophys. J. 94, 1169 (2008)], the difference of the excess chemical potentials for the two competing ions defines the advantage that one of the ions has over the other in the competition for space in the crowded selectivity filter. These advantages depend on ionic bath concentrations: the ion that is present in the bath in larger quantity (Na+) has the "number" advantage which is balanced by the free-energy advantage of the other ion (Ca2 +). The excess chemical potentials are decomposed into hard sphere exclusion and electrostatic components. The electrostatic terms correspond to interactions with the mean electric field produced by ions and induced charges as well to ionic correlations beyond the mean field description. Dielectrics are needed to produce micromolar Ca2 + versus Na+ selectivity in the L-type channel. We study the behavior of these terms with changes in bath concentrations of ions, charges, and diameters of ions, as well as geometrical parameters such as radius of the pore and the dielectric constant of the protein. Ion selectivity in calcium binding proteins probably has a similar mechanism.

Analyzing the components of the free-energy landscape in a calcium selective ion channel by Widom's particle insertion method.

PubMed

Boda, Dezso; Giri, Janhavi; Henderson, Douglas; Eisenberg, Bob; Gillespie, Dirk

2011-02-07

The selectivity filter of the L-type calcium channel works as a Ca(2+) binding site with a very large affinity for Ca(2+) versus Na(+). Ca(2+) replaces half of the Na(+) ions in the filter even when these ions are present in 1 μM and 30 mM concentrations in the bath, respectively. The energetics of this strong selectivity is analyzed in this paper. We use Widom's particle insertion method to compute the space-dependent profiles of excess chemical potential in our grand canonical Monte Carlo simulations. These profiles define the free-energy landscape for the various ions. Following Gillespie [Biophys. J. 94, 1169 (2008)], the difference of the excess chemical potentials for the two competing ions defines the advantage that one of the ions has over the other in the competition for space in the crowded selectivity filter. These advantages depend on ionic bath concentrations: the ion that is present in the bath in larger quantity (Na(+)) has the "number" advantage which is balanced by the free-energy advantage of the other ion (Ca(2+)). The excess chemical potentials are decomposed into hard sphere exclusion and electrostatic components. The electrostatic terms correspond to interactions with the mean electric field produced by ions and induced charges as well to ionic correlations beyond the mean field description. Dielectrics are needed to produce micromolar Ca(2+) versus Na(+) selectivity in the L-type channel. We study the behavior of these terms with changes in bath concentrations of ions, charges, and diameters of ions, as well as geometrical parameters such as radius of the pore and the dielectric constant of the protein. Ion selectivity in calcium binding proteins probably has a similar mechanism.

Increasing the affinity of selective bZIP-binding peptides through surface residue redesign.

PubMed

Kaplan, Jenifer B; Reinke, Aaron W; Keating, Amy E

2014-07-01

The coiled-coil dimer is a prevalent protein interaction motif that is important for many cellular processes. The basic leucine-zipper (bZIP) transcription factors are one family of proteins for which coiled-coil mediated dimerization is essential for function, and misregulation of bZIPs can lead to disease states including cancer. This makes coiled coils attractive protein-protein interaction targets to disrupt using engineered molecules. Previous work designing peptides to compete with native coiled-coil interactions focused primarily on designing the core residues of the interface to achieve affinity and specificity. However, folding studies on the model bZIP GCN4 show that coiled-coil surface residues also contribute to binding affinity. Here we extend a prior study in which peptides were designed to bind tightly and specifically to representative members of each of 20 human bZIP families. These "anti-bZIP" peptides were designed with an emphasis on target-binding specificity, with contributions to design-target specificity and affinity engineered considering only the coiled-coil core residues. High-throughput testing using peptide arrays indicated many successes. We have now measured the binding affinities and specificities of anti-bZIPs that bind to FOS, XBP1, ATF6, and CREBZF in solution and tested whether redesigning the surface residues can increase design-target affinity. Incorporating residues that favor helix formation into the designs increased binding affinities in all cases, providing low-nanomolar binders of each target. However, changes in surface electrostatic interactions sometimes changed the binding specificity of the designed peptides. © 2014 The Protein Society.

Structure and mechanism of Zn2+-transporting P-type ATPases

PubMed Central

Wang, Kaituo; Sitsel, Oleg; Meloni, Gabriele; Autzen, Henriette Elisabeth; Andersson, Magnus; Klymchuk, Tetyana; Nielsen, Anna Marie; Rees, Douglas C.; Nissen, Poul; Gourdon, Pontus

2014-01-01

Zinc is an essential micronutrient for all living organisms, required for signaling and proper function of a range of proteins involved in e.g. DNA-binding and enzymatic catalysis1. In prokaryotes and photosynthetic eukaryotes Zn2+-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn2+ and related elements2,3. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2.Pi) of ZntA from Shigella sonnei, determined at 3.2 and 2.7 Å resolution, respectively. The structures reveal a similar fold as the Cu+-ATPases with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn2+ ions. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including Cys392, Cys394 and Asp714. The pathway closes in the E2.Pi state where Asp714 interacts with the conserved Lys693, which possibly stimulates Zn2+ release as a built-in counter-ion, as also proposed for H+-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter-transport. These findings suggest a mechanistic link between PIB-type Zn2+-ATPases and PIII-type H+-ATPases, and show at the same time structural features of the extracellular release pathway that resemble the PII-type ATPases such as the sarco(endo)plasmic reticulum Ca2+-ATPase4,5 (SERCA) and Na+,K+-ATPase6. PMID:25132545

Comparative study of glycated hemoglobin by ion exchange chromatography and affinity binding nycocard reader in type 2 diabetes mellitus.

PubMed

Gautam, N; Dubey, R K; Jayan, A; Nepaune, Y; Padmavathi, P; Chaudhary, S; Jha, S K; Sinha, A K

2014-12-01

The aim of this study was to compare the level of glycated hemoglobin (HbA1c) in type 2 Diabetes Mellitus (DM) patients by two different methods namely Ion Exchange Chromatography and Affinity Binding Nycocard Reader. This is a cross-sectional study conducted on confirmed type 2 diabetes mellitus patients (n = 100) who visited Out Patients Department of the Universal College of Medical Sciences Teaching hospital, Bhairahawa, Nepal from November 2012 to March 2013. The diagnosis of diabetes mellitus was done on the basis of their fasting (164.46 ± 45.33 mg/dl) and random (187.93 ± 78.02 mg/dl) serum glucose level along with clinical history highly suggestive of type 2 DM. The HbA1c values of (7.8 ± 1.9%) and (8.0 ± 2.2%) were found in DM patients as estimated by those two different methods respectively. The highest frequency was observed in HbA1c > 8.0% indicating maximum cases were under very poor glycemic control. However, there were no significant differences observed in HbA1c value showing both methods are comparable in nature and can be used in lab for ease of estimation. The significant raised in HbA1c indicates complications associated with DM and monitoring of therapy become hard for those patients. Despite having standard reference method for HbA1c determination, the availability of report at the time of the patient visit can be made easy by using Nycocard Reader and Ion Exchange Chromatography techniques without any delay in communicating glycemic control, clinical decision-making and changes in treatment regimen.

Quantitative characterization of galectin-3-C affinity mass spectrometry measurements: Comprehensive data analysis, obstacles, shortcuts and robustness.

PubMed

Haramija, Marko; Peter-Katalinić, Jasna

2017-10-30

Affinity mass spectrometry (AMS) is an emerging tool in the field of the study of protein•carbohydrate complexes. However, experimental obstacles and data analysis are preventing faster integration of AMS methods into the glycoscience field. Here we show how analysis of direct electrospray ionization mass spectrometry (ESI-MS) AMS data can be simplified for screening purposes, even for complex AMS spectra. A direct ESI-MS assay was tested in this study and binding data for the galectin-3C•lactose complex were analyzed using a comprehensive and simplified data analysis approach. In the comprehensive data analysis approach, noise, all protein charge states, alkali ion adducts and signal overlap were taken into account. In a simplified approach, only the intensities of the fully protonated free protein and the protein•carbohydrate complex for the main protein charge state were taken into account. In our study, for high intensity signals, noise was negligible, sodiated protein and sodiated complex signals cancelled each other out when calculating the K d value, and signal overlap influenced the Kd value only to a minor extent. Influence of these parameters on low intensity signals was much higher. However, low intensity protein charge states should be avoided in quantitative AMS analyses due to poor ion statistics. The results indicate that noise, alkali ion adducts, signal overlap, as well as low intensity protein charge states, can be neglected for preliminary experiments, as well as in screening assays. One comprehensive data analysis performed as a control should be sufficient to validate this hypothesis for other binding systems as well. Copyright © 2017 John Wiley & Sons, Ltd.

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

Predicting MHC-II binding affinity using multiple instance regression

PubMed Central

EL-Manzalawy, Yasser; Dobbs, Drena; Honavar, Vasant

2011-01-01

Reliably predicting the ability of antigen peptides to bind to major histocompatibility complex class II (MHC-II) molecules is an essential step in developing new vaccines. Uncovering the amino acid sequence correlates of the binding affinity of MHC-II binding peptides is important for understanding pathogenesis and immune response. The task of predicting MHC-II binding peptides is complicated by the significant variability in their length. Most existing computational methods for predicting MHC-II binding peptides focus on identifying a nine amino acids core region in each binding peptide. We formulate the problems of qualitatively and quantitatively predicting flexible length MHC-II peptides as multiple instance learning and multiple instance regression problems, respectively. Based on this formulation, we introduce MHCMIR, a novel method for predicting MHC-II binding affinity using multiple instance regression. We present results of experiments using several benchmark datasets that show that MHCMIR is competitive with the state-of-the-art methods for predicting MHC-II binding peptides. An online web server that implements the MHCMIR method for MHC-II binding affinity prediction is freely accessible at http://ailab.cs.iastate.edu/mhcmir. PMID:20855923

Mechanisms of the inhibition of enzymatic hydrolysis of waste pulp fibers by calcium carbonate and the influence of nonionic surfactant for mitigation.

PubMed

Min, Byeong Cheol; Ramarao, Bandaru V

2017-06-01

Recycled paper mills produce large quantities of fibrous rejects and fines which are usually sent to landfills as solid waste. These cellulosic materials can be enzymatically hydrolyzed into sugars for the production of biofuels and biomaterials. Paper mill wastes also contain large amounts of calcium carbonate which inhibits cellulase activity. The calcium carbonate (30%, w/w) decreased 40-60% of sugar yield of unbleached softwood kraft pulp. The prime mechanisms for this are by pH variation, competitive and non-productive binding, and aggregation effect. Addition of acetic acid (pH adjustment) increased the sugar production from 19 to 22 g/L of paper mill waste fibers. Strong affinity of enzyme-calcium carbonate decreased free enzyme in solution and hindered sugar production. Electrostatic and hydrogen bond interactions are mainly possible mechanism of enzyme-calcium carbonate adsorption. The application of the nonionic surfactant Tween 80 alleviated the non-productive binding of enzyme with the higher affinity on calcium carbonate. Dissociated calcium ion also inhibited the hydrolysis by aggregation of enzyme.

High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies

PubMed Central

Anthony, Kelsey C.; You, Changjiang; Piehler, Jacob; Pomeranz Krummel, Daniel A.

2014-01-01

SUMMARY There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle (AuNPtris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of AuNPtris-NTA labeled proteins by electron microscopy is further ensured by the reagent’s short conformationally restricted linker. We have employed AuNPtris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. AuNPtris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our new labeling reagent should find broad application in non-covalent site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies. PMID:24560806

Natural hidden antibodies reacting with DNA or cardiolipin bind to thymocytes and evoke their death.

PubMed

Zamulaeva, I A; Lekakh, I V; Kiseleva, V I; Gabai, V L; Saenko, A S; Shevchenko, A S; Poverenny, A M

1997-08-18

Both free and hidden natural antibodies to DNA or cardiolipin were obtained from immunoglobulins of a normal donor. The free antibodies reacting with DNA or cardiolipin were isolated by means of affinity chromatography. Antibodies occurring in an hidden state were disengaged from the depleted immunoglobulins by ion-exchange chromatography and were then affinity-isolated on DNA or cardiolipin sorbents. We used flow cytometry to study the ability of free and hidden antibodies to bind to rat thymocytes. Simultaneously, plasma membrane integrity was tested by propidium iodide (PI) exclusion. The hidden antibodies reacted with 65.2 +/- 10.9% of the thymocytes and caused a fast plasma membrane disruption. Cells (28.7 +/- 7.1%) were stained with PI after incubation with the hidden antibodies for 1 h. The free antibodies bound to a very small fraction of the thymocytes and did not evoke death as compared to control without antibodies. The possible reason for the observed effects is difference in reactivity of the free and hidden antibodies to phospholipids. While free antibodies reacted preferentially with phosphotidylcholine, hidden antibodies reacted with cardiolipin and phosphotidylserine.

[Relations between location of elements in periodic table and affinity for the kidneys (author's transl)].

PubMed

Ando, A; Hisada, K; Ando, I

1977-10-01

The distribution of many inorganic compounds in rats was investigated in order to evaluate kidney affinity of inorganic compounds. In these experiments, 30%, 10-20% and 4-10% of administered dose was localized in the kidneys in 203Hg-acetate and 203 Bi-acetate, in H198AuCl4, 103PdCl2, 201TlCl, 210Pd(NO3)2 and H2(127M)TeO3, and in Na2(51)CrO4, 54MnCl2, (114m)InCl3 and 7BeCl2, respectively. Some bipositive ions and anions was hardly taken up into the kidneys. And in many hard acids according to classification of Lewis acids, the uptake rate into the kidneys was usually small. On the other hand, Hg, Au and Bi, which have strong binding power to the protein, showed high uptake rate in the kidneys. As Hg++, Au+ and Bi+++ was soft acids according to classification of Lewis acids, it was thought that these elements would bind strongly to soft base (RSH, RS-) present in the kidney.

Conformational Preference of ‘CαNN’ Short Peptide Motif towards Recognition of Anions

PubMed Central

Banerjee, Raja

2013-01-01

Among several ‘anion binding motifs’, the recently described ‘CαNN’ motif occurring in the loop regions preceding a helix, is conserved through evolution both in sequence and its conformation. To establish the significance of the conserved sequence and their intrinsic affinity for anions, a series of peptides containing the naturally occurring ‘CαNN’ motif at the N-terminus of a designed helix, have been modeled and studied in a context free system using computational techniques. Appearance of a single interacting site with negative binding free-energy for both the sulfate and phosphate ions, as evidenced in docking experiments, establishes that the ‘CαNN’ segment has an intrinsic affinity for anions. Molecular Dynamics (MD) simulation studies reveal that interaction with anion triggers a conformational switch from non-helical to helical state at the ‘CαNN’ segment, which extends the length of the anchoring-helix by one turn at the N-terminus. Computational experiments substantiate the significance of sequence/structural context and justify the conserved nature of the ‘CαNN’ sequence for anion recognition through “local” interaction. PMID:23516403

Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly destabilizing epileptogenic mutation.

PubMed

Alberdi, Araitz; Gomis-Perez, Carolina; Bernardo-Seisdedos, Ganeko; Alaimo, Alessandro; Malo, Covadonga; Aldaregia, Juncal; Lopez-Robles, Carlos; Areso, Pilar; Butz, Elisabeth; Wahl-Schott, Christian; Villarroel, Alvaro

2015-11-01

We show that the combination of an intracellular bi-partite calmodulin (CaM)-binding site and a distant assembly region affect how an ion channel is regulated by a membrane lipid. Our data reveal that regulation by phosphatidylinositol(4,5)bisphosphate (PIP2) and stabilization of assembled Kv7.2 subunits by intracellular coiled-coil regions far from the membrane are coupled molecular processes. Live-cell fluorescence energy transfer measurements and direct binding studies indicate that remote coiled-coil formation creates conditions for different CaM interaction modes, each conferring different PIP2 dependency to Kv7.2 channels. Disruption of coiled-coil formation by epilepsy-causing mutation decreases apparent CaM-binding affinity and interrupts CaM influence on PIP2 sensitivity. © 2015. Published by The Company of Biologists Ltd.

Separation of an associated 90K heat shock protein from the glucocorticoid receptor complex

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

Miller-Diener, A.; Kirsch, T.; Grove, B.

1986-05-01

A 90K heat shock protein(HSP), observed to copurify with the glucocorticoid receptor(GR), can be separated from the complex by 2 methods, allowing investigation of the role of HSP on kinase activity that was previously reported to be inherent to purified activated GR. Na/sub 2/MoO/sub 4/ stabilized unactivated rat hepatic GR complexes have been purified to >10,000-fold using a purification scheme that involves batchwise treatment of cytosol with phosphocellulose/DNAcellulose, elution from an affinity resin, gel filtration and ion exchange chromatography. Samples were subjected to 10-20% gradient SDS-PAGE. Proteins were transferred to nitrocellulose and blotted against monoclonal antibodies to GR(3A6), HSP ormore » nonspecific IgM/G. Immunoblots indicated that HSP was separated from unactivated GR complexes at the affinity step prior to elution of GR with active steroid. GR eluted from the resin with /sup 3/H Triamcinolone acetonide or /sup 3/H Dexamethasone mesylate had an apparent M/sub r/ = 94-96,000 for the steroid binding subunit and is recognized by 3A6. Purification of GR minus the affinity step resulted in copurification of HSP throughout the procedure. However, after Sephadex G75 filtration and subsequent incubation at 25/sup 0/C, 30 min., HSP was separated from activated (DNA binding) GR on DEAE cellulose-52. HSP did not enhance or inhibit /sup 32/P incorporation of the 94K steroid binding subunit nor did it affect phosphorylation of histones by GR.« less

Accurate and sensitive quantification of protein-DNA binding affinity.

PubMed

Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

2018-04-17

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

Accurate and sensitive quantification of protein-DNA binding affinity

PubMed Central

Rastogi, Chaitanya; Rube, H. Tomas; Kribelbauer, Judith F.; Crocker, Justin; Loker, Ryan E.; Martini, Gabriella D.; Laptenko, Oleg; Freed-Pastor, William A.; Prives, Carol; Stern, David L.; Mann, Richard S.; Bussemaker, Harmen J.

2018-01-01

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. PMID:29610332

Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom.

PubMed

Kochibe, N; Matta, K L

1989-01-05

A lectin in the fruiting bodies of Psathyrella velutina was purified by affinity chromatography on a chitin column and subsequent ion-exchange chromatography. P. velutina lectin (PVL) tends to aggregate irreversibly in buffered saline, but the addition of glycerol (10%, v/v) to lectin solutions was found to prevent aggregate formation. PVL is assumed to occur as a monomer of a polypeptide of Mr = 40,000 as determined by gel filtration and by gel electrophoresis in the presence of sodium dodecyl sulfate. PVL is specific for N-acetylglucosamine (GlcNAc). It was determined by equilibrium dialysis to have four binding sites/polypeptide molecule showing an average intrinsic association constant of K0 = 6.4 x 10(3) M-1 toward this sugar. The binding specificity of the lectin was studied by hemagglutination inhibition assays and by avidin-biotin-mediated enzyme immunoassays using various GlcNAc-containing saccharides. The results indicate that methyl N-acetyl beta-glucosaminide was a slightly better inhibitor than the corresponding alpha-anomer. PVL binds well to oligosaccharides bearing nonreducing terminal beta-GlcNAc linked 1----6 or 1----3 but poorly to those having a 1----4 linkage, such as N-acetylated chito-oligosaccharides. It also binds to the subterminal GlcNAc moiety when it is substituted at the C-6 position but does not interact with the moiety when substituted either at C-3 or C-4. Thus, these results show that PVL is quite different in its binding specificity from other GlcNAc-binding lectins of higher plants since they bind preferentially to beta-GlcNAc in 1----4 linkage and they have a high affinity for chitin oligosaccharides.

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

PubMed Central

Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

2013-01-01

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

Solubilization and purification of melatonin receptors from lizard brain.

PubMed

Rivkees, S A; Conron, R W; Reppert, S M

1990-09-01

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

PubMed Central

Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

2016-01-01

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

Development of acetophenone ligands as potential neuroimaging agents for cholinesterases.

PubMed

Jollymore-Hughes, Courtney T; Pottie, Ian R; Martin, Earl; Rosenberry, Terrone L; Darvesh, Sultan

2016-11-01

Association of cholinesterase with β-amyloid plaques and tau neurofibrillary tangles in Alzheimer's disease offers an opportunity to detect disease pathology during life. Achieving this requires development of radiolabelled cholinesterase ligands with high enzyme affinity. Various fluorinated acetophenone derivatives bind to acetylcholinesterase with high affinity, including 2,2,2-trifluoro-1-(3-dimethylaminophenyl)ethanone (1) and 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (2). Such compounds also offer potential for incorporation of radioactive fluorine ( 18 F) for Positron Emission Tomography (PET) imaging of cholinesterases in association with Alzheimer's disease pathology in the living brain. Here we describe the synthesis of two meta-substituted chlorodifluoroacetophenones using a Weinreb amide strategy and their rapid conversion to the corresponding trifluoro derivatives through nucleophilic substitution by fluoride ion, in a reaction amenable to incorporating 18 F for PET imaging. In vitro kinetic analysis indicates tight binding of the trifluoro derivatives to cholinesterases. Compound 1 has a K i value of 7nM for acetylcholinesterase and 1300nM for butyrylcholinesterase while for compound 2 these values are 0.4nM and 26nM, respectively. Tight binding of these compounds to cholinesterase encourages their development for PET imaging detection of cholinesterase associated with Alzheimer's disease pathology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

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

Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent

2007-10-01

Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosismore » mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.« less

A Unitary Anesthetic Binding Site at High Resolution

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

Vedula, L. Sangeetha; Brannigan, Grace; Economou, Nicoleta J.

2009-10-21

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA{sub A} receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA{sub A} receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show thatmore » apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA{sub A} receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.« less

A Unitary Anesthetic Binding Site at High Resolution

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

L Vedula; G Brannigan; N Economou

2011-12-31

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA{sub A} receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA{sub A} receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show thatmore » apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA{sub A} receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.« less

A Unitary Anesthetic-Binding Site at High Resolution

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

Vedula, L.; Brannigan, G; Economou, N

2009-01-01

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABAA receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA{sub A} receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritinmore » also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA{sub A} receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.« less

Identification of a unique Ca2+-binding site in rat acid-sensing ion channel 3.

PubMed

Zuo, Zhicheng; Smith, Rachel N; Chen, Zhenglan; Agharkar, Amruta S; Snell, Heather D; Huang, Renqi; Liu, Jin; Gonzales, Eric B

2018-05-25

Acid-sensing ion channels (ASICs) evolved to sense changes in extracellular acidity with the divalent cation calcium (Ca 2+ ) as an allosteric modulator and channel blocker. The channel-blocking activity is most apparent in ASIC3, as removing Ca 2+ results in channel opening, with the site's location remaining unresolved. Here we show that a ring of rat ASIC3 (rASIC3) glutamates (Glu435), located above the channel gate, modulates proton sensitivity and contributes to the formation of the elusive Ca 2+ block site. Mutation of this residue to glycine, the equivalent residue in chicken ASIC1, diminished the rASIC3 Ca 2+ block effect. Atomistic molecular dynamic simulations corroborate the involvement of this acidic residue in forming a high-affinity Ca 2+ site atop the channel pore. Furthermore, the reported observations provide clarity for past controversies regarding ASIC channel gating. Our findings enhance understanding of ASIC gating mechanisms and provide structural and energetic insights into this unique calcium-binding site.

Application of the principle of linked functions to ATP-driven ion pumps: kinetics of activation by ATP.

PubMed Central

Reynolds, J A; Johnson, E A; Tanford, C

1985-01-01

If a ligand binds with unequal affinity to two distinct states of a protein, then the equilibrium between the two states becomes a function of the concentration of the ligand. A necessary consequence is that the ligand must also affect the forward and/or reverse rate constants for transition between the two states. For an enzyme or transport protein with such a transition as a slow step in the catalytic cycle, the overall rate also becomes a function of ligand concentration. These conclusions are independent of whether or not the ligand is a direct participant in the reaction. If it is a direct participant, then the kinetic effect arising from the principle of linked functions is distinct from the direct catalytic effect. These principles suffice to account for the biphasic response of the hydrolytic activity of ATP-driven ion pumps to the concentration of ATP, without the need to invoke more than one ATP binding site per catalytic center. PMID:2987939

Let's get specific: the relationship between specificity and affinity.

PubMed

Eaton, B E; Gold, L; Zichi, D A

1995-10-01

The factors that lead to high-affinity binding are a good fit between the surfaces of the two molecules in their ground state and charge complementarity. Exactly the same factors give high specificity for a target. We argue that selection for high-affinity binding automatically leads to highly specific binding. This principle can be used to simplify screening approaches aimed at generating useful drugs.

The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

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

Nemoto, Takayuki; Ohara-Nemoto, Yuko; Denis, M.

1990-02-20

High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, the authors decided to investigate the possible relationship between these two phenomena. The binding of ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas ({sup 3}H)TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR showed the presence of twomore » types of binding sites. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low ({sup 3}H)TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. These results indicate that the transformed GR has a reduced ({sup 3}H)TA-binding affinity as compared to the nontransformed GR. The steroid-binding domain (amino acids 477-777) and the DNA- and steroid-binding domains (amino acids 415-777) of the human GR were expressed in Escherichia coli as protein A fused proteins. Taken together, these results suggest that the component(s) associating with the nontransformed GR, possibly the heat shock protein hsp 90, play(s) an important role in stabilizing the GR in a high-affinity state for steroids.« less

Modal gating of muscle nicotinic acetylcholine receptors

NASA Astrophysics Data System (ADS)

Vij, Ridhima

Many ion channels exhibit multiple patterns of kinetic activity in single-channel currents. This behavior is rare in WT mouse muscle nicotinic acetylcholine receptors (AChRs), where A2C↔A2O gating events are well-described by single exponentials. Also, single-channel open probability (PO) is essentially homogeneous at a given agonist concentration in the WT receptors. Here I report that perturbations of almost all the residues in loop C (alpha188-alpha199, at the agonist binding site) generate heterogeneity in PO ('modes'). Such unsettled activity was apparent with an alanine substitution at all positions in loop C (except alphaY190 and alphaY198) and with different side chain substitutions at alphaP197 for both adult- and fetal-type AChRs. I used single channel electrophysiology along with site-directed mutagenesis to study modal gating in AChRs consequent to mutations/deletions in loop C. The multiple patterns of kinetic activity arose from the difference in agonist affinity rather than in intrinsic AChR gating. Out of the four different agonists used to study the modal behavior, acetylcholine (ACh) showed a higher degree of kinetic heterogeneity compared to others. The time constant for switching between modes was long (~mins), suggesting that they arise from alternative, stable protein conformations. By studying AChRs having only 1 functional binding site, I attempted to find the source of the affinity difference, which was traced mainly to the alphadelta agonist site. Affinity at the neurotransmitter binding site is mainly determined by a core of five aromatic residues (alphaY93, alphaW149, alphaY190, alphaY198 and deltaW57). Phenylalanine substitutions at all aromatic residues except alphaY93 resulted in elimination of modes. Modes were also eliminated by alanine mutation at deltaW57 on the complementary side but not at other aromatics. Also, by substituting four gamma subunit residues into the delta subunit on the complementary beta sheet, I found that modes were reduced. Based on our results, we propose that WT loop C has an important role in determining resting affinity, in part by making stable interactions with the complementary surface of the alphadelta binding pocket. We suggest a possible structural basis for the fluctuations caused by loop C perturbations and propose that at the alphadelta agonist binding site, both loop C and the complementary subunit surface can adopt alternative conformations and interact with each other with respect to the aromatic core, to cause the variations in affinity.

Determining ERβ Binding Affinity to Singly Mutant ERE Using Dual Polarization Interferometry

NASA Astrophysics Data System (ADS)

Song, Hong Yan; Su, Xiaodi

In a classic mode of estrogen action, estrogen receptors (ERs) bind to estrogen responsive element (ERE) to activate gene transcription. A perfect ERE contains a 13-base pair sequence of a palindromic repeat separated by a three-base spacer, 5‧-GGTCAnnnTGACC-3‧. In addition to the consensus or wild-type ERE (wtERE), naturally occurring EREs often have one or two base pairs’ alternation. Based on the newly constructed Thermodynamic Modeling of ChIP-seq (TherMos) model, binding energy between ERβ and a series of 34-bp mutant EREs (mutERE) was simulated to predict the binding affinity between ERs and EREs with single base pair deviation at different sites of the 13-bp inverted sequence. Experimentally, dual polarization interferometry (DPI) method was developed to measure ERβ-mutEREs binding affinity. On a biotin-NeutrAvidin (NA)-biotin treated DPI chip, wtERE is immobilized. In a direct binding assay, ERβ-wtERE binding affinity is determined. In a competition assay, ERβ was preincubated with mutant EREs before being added for competitive binding to the immobilized wtERE. This competition strategy provided a successful platform to evaluate the binding affinity variation among large number of ERE with different base mutations. The experimental result correlates well with the mathematically predicted binding energy with a Spearman correlation coefficient of 0.97.

Pertussis toxin modifies the characteristics of both the inhibitory GTP binding proteins and the somatostatin receptor in anterior pituitary tumor cells

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

Mahy, N.; Woolkalis, M.; Thermos, K.

1988-08-01

The effects of pertussis toxin treatment on the characteristics of somatostatin receptors in the anterior pituitary tumor cell line AtT-20 were examined. Pertussis toxin selectively catalyzed the ADP ribosylation of the alpha subunits of the inhibitory GTP binding proteins in AtT-20 cells. Toxin treatment abolished somatostatin inhibition of forskolin-stimulated adenylyl cyclase activity and somatostatin stimulation of GTPase activity. To examine the effects of pertussis toxin treatment on the characteristics of the somatostatin receptor, the receptor was labeled by the somatostatin analog (125I)CGP 23996. (125I)CGP 23996 binding to AtT-20 cell membranes was saturable and within a limited concentration range was tomore » a single high affinity site. Pertussis toxin treatment reduced the apparent density of the high affinity (125I)CGP 23996 binding sites in AtT-20 cell membranes. Inhibition of (125I)CGP 23996 binding by a wide concentration range of CGP 23996 revealed the presence of two binding sites. GTP predominantly reduced the level of high affinity sites in control membranes. Pertussis toxin treatment also diminished the amount of high affinity sites. GTP did not affect (125I)CGP 23996 binding in the pertussis toxin-treated membranes. The high affinity somatostatin receptors were covalently labeled with (125I) CGP 23996 and the photoactivated crosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. No high affinity somatostatin receptors, covalently bound to (125I)CGP 23996, were detected in the pertussis toxin-treated membranes. These results are most consistent with pertussis toxin uncoupling the inhibitory G proteins from the somatostatin receptor thereby converting the receptor from a mixed population of high and low affinity sites to only low affinity receptors.« less

Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels

PubMed Central

Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal; Arrigoni, Cristina; Wong, Stephanie; Nurva, Shailika Reddy; Loussouarn, Gildas; Minor, Daniel L.

2013-01-01

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail ‘neck’, are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the ‘outer ion’ site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies shows that this site forms a previously unknown determinant of CaV high affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. PMID:24120938

Transition metal ion capture using functional mesoporous carbon made with 1,10-phenanthroline

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

Chouyyok, Wilaiwan; Yantasee, Wassana; Shin, Yongsoon

2009-11-01

Functional mesoporous carbon has been built using 1,10-phenanthroline as the fundamental building block, resulting in a nanoporous, high surface area sorbent capable of selectively binding transition metal ions. This material had a specific surface area of 870 m2/g, an average pore size of about 30Å, and contained as much as 8.2 weight percent N. Under acidic conditions, where the 1,10-phenanthroline ligand is protonated, this material was found to be an effective anion exchange material for transition metal anions like PdCl4-2 and H2VO4-1. 1,10-phenanthroline functionalized mesoporous carbon (“Phen-FMC”) was found to have a high affinity for Cu(II), even down to amore » pH of 1. At pHs above 5, Phen-FMC was found to bind a variety of transition metal cations (e.g. Co(II), Ni(II), Zn(II), etc.) from filtered ground water, river water and seawater. Phen-FMC displayed rapid sorption kinetics with Co(II) in filtered river water, reaching equilibrium in less than an hour, and easily lowering the [Co(II)] to sub-ppb levels. Phen-FMC was found to be more effective for transition metal ion capture than ion exchange resin or activated carbon.« less

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva† †Electronic supplementary information (ESI) available: Optimization of Mg2+ and ATMND concentrations for our CBSA-based ATMND-binding assay; ATMND-reported calibration curve for CBSA-5325 at various cocaine concentrations; ATMND binding affinity for the cocaine-assembled CBSA-5325; K D of 38-GC and different 38-GC mutants for cocaine as characterized by ITC; stem length effects on cocaine-induced CBSA assembly; spectra of CBSA-5335-based fluorescence detection of cocaine in 1× binding buffer; characterization of cocaine binding affinity of CBSA-5335 and PSA using ITC; fluorescence detection of cocaine in saliva with our fluorophore/quencher modified CBSA-5335; calibration curve of our CBSA-5335-based fluorophore/quencher assay in 1× binding buffer and 10% saliva at cocaine concentrations ranging from 0 to 10 μM; bias and precision of the CBSA-5335-based fluorophore/quencher assay; comparison of amplification-free split-aptamer assays for cocaine detection; sequence ID and DNA sequences used in this work. See DOI: 10.1039/c6sc01833e Click here for additional data file.

PubMed Central

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples. PMID:28451157

Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

PubMed

Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2012-04-25

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels.

PubMed

Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal; Arrigoni, Cristina; Wong, Stephanie; Nurva, Shailika Reddy; Loussouarn, Gildas; Minor, Daniel L

2014-01-23

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail "neck", are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the "outer ion" site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of CaV high-affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. © 2013. Published by Elsevier Ltd. All rights reserved.

Expanding RNA binding specificity and affinity of engineered PUF domains.

PubMed

Zhao, Yang-Yang; Mao, Miao-Wei; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-05-18

Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way.

Expanding RNA binding specificity and affinity of engineered PUF domains

PubMed Central

Zhao, Yang-Yang; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-01-01

Abstract Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way. PMID:29490074

Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C

PubMed Central

Brandoli, Giulia; Lempinen, Antti; Artes, Sanna; Turku, Ainoleena; Jäntti, Maria Helena; Talman, Virpi; Yli-Kauhaluoma, Jari; Tuominen, Raimo K.; Boije af Gennäs, Gustav

2018-01-01

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain–targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC. PMID:29641588

Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C.

PubMed

Provenzani, Riccardo; Tarvainen, Ilari; Brandoli, Giulia; Lempinen, Antti; Artes, Sanna; Turku, Ainoleena; Jäntti, Maria Helena; Talman, Virpi; Yli-Kauhaluoma, Jari; Tuominen, Raimo K; Boije Af Gennäs, Gustav

2018-01-01

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain-targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.

Affinities of penicillins and cephalosporins for the penicillin-binding proteins of Escherichia coli K-12 and their antibacterial activity.

PubMed Central

Curtis, N A; Orr, D; Ross, G W; Boulton, M G

1979-01-01

The affinities of a range of penicillins and cephalosporins for ther penicillin-binding proteins of Escherichia coli K-12 have been studied, and the results were compared with the antibacterial activity of the compounds against E. coli K-12 and an isogenic permeability mutant. Different penicillins and cephalosporins exhibited different affinities for the "essential" penicillin-binding proteins of E. coli K-12, in a manner which directly correlated with their observed effects upon bacterial morphology. Furthermore, the affinities of the compounds for their "primary" lethal penicillin-binding protein targets showed close agreement with their antibacterial activities against the permeability mutant. Images PMID:393164

Accurate Evaluation Method of Molecular Binding Affinity from Fluctuation Frequency

NASA Astrophysics Data System (ADS)

Hoshino, Tyuji; Iwamoto, Koji; Ode, Hirotaka; Ohdomari, Iwao

2008-05-01

Exact estimation of the molecular binding affinity is significantly important for drug discovery. The energy calculation is a direct method to compute the strength of the interaction between two molecules. This energetic approach is, however, not accurate enough to evaluate a slight difference in binding affinity when distinguishing a prospective substance from dozens of candidates for medicine. Hence more accurate estimation of drug efficacy in a computer is currently demanded. Previously we proposed a concept of estimating molecular binding affinity, focusing on the fluctuation at an interface between two molecules. The aim of this paper is to demonstrate the compatibility between the proposed computational technique and experimental measurements, through several examples for computer simulations of an association of human immunodeficiency virus type-1 (HIV-1) protease and its inhibitor (an example for a drug-enzyme binding), a complexation of an antigen and its antibody (an example for a protein-protein binding), and a combination of estrogen receptor and its ligand chemicals (an example for a ligand-receptor binding). The proposed affinity estimation has proven to be a promising technique in the advanced stage of the discovery and the design of drugs.

A model of high-affinity antibody binding to type III group B Streptococcus capsular polysaccharide.

PubMed

Wessels, M R; Muñoz, A; Kasper, D L

1987-12-01

We recently reported that the single repeating-unit pentasaccharide of type III group B Streptococcus (GBS) capsular polysaccharide is only weakly reactive with type III GBS antiserum. To further elucidate the relationship between antigen-chain length and antigenicity, tritiated oligosaccharides derived from type III capsular polysaccharide were used to generate detailed saturation binding curves with a fixed concentration of rabbit antiserum in a radioactive antigen-binding assay. A graded increase in affinity of antigen-antibody binding was seen as oligosaccharide size increased from 2.6 repeating units to 92 repeating units. These differences in affinity of antibody binding to oligosaccharides of different molecular size were confirmed by immunoprecipitation and competitive ELISA, two independent assays of antigen-antibody binding. Analysis of the saturation binding experiment indicated a difference of 300-fold in antibody-binding affinity for the largest versus the smallest tested oligosaccharides. Unexpectedly, the saturation binding values approached by the individual curves were inversely related to oligosaccharide chain length on a molar basis but equivalent on a weight basis. This observation is compatible with a model in which binding of an immunoglobulin molecule to an antigenic site on the polysaccharide facilitates subsequent binding of antibody to that antigen.

Comprehensive insight into the binding of sunitinib, a multi-targeted anticancer drug to human serum albumin

NASA Astrophysics Data System (ADS)

Kabir, Md. Zahirul; Tee, Wei-Ven; Mohamad, Saharuddin B.; Alias, Zazali; Tayyab, Saad

2017-06-01

Binding studies between a multi-targeted anticancer drug, sunitinib (SU) and human serum albumin (HSA) were made using fluorescence, UV-vis absorption, circular dichroism (CD) and molecular docking analysis. Both fluorescence quenching data and UV-vis absorption results suggested formation of SU-HSA complex. Moderate binding affinity between SU and HSA was evident from the value of the binding constant (3.04 × 104 M-1), obtained at 298 K. Involvement of hydrophobic interactions and hydrogen bonds as the leading intermolecular forces in the formation of SU-HSA complex was predicted from the thermodynamic data of the binding reaction. These results were in good agreement with the molecular docking analysis. Microenvironmental perturbations around Tyr and Trp residues as well as secondary and tertiary structural changes in HSA upon SU binding were evident from the three-dimensional fluorescence and circular dichroism results. SU binding to HSA also improved the thermal stability of the protein. Competitive displacement results and molecular docking analysis revealed the binding locus of SU to HSA in subdomain IIA (Sudlow's site I). The influence of a few common ions on the binding constant of SU-HSA complex was also noticed.

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity.

PubMed

Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

2015-01-01

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

PubMed Central

Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

2014-01-01

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

Dicyanovinylnaphthalenes for neuroimaging of amyloids and relationships of electronic structures and geometries to binding affinities

PubMed Central

Petrič, Andrej; Johnson, Scott A.; Pham, Hung V.; Li, Ying; Čeh, Simon; Golobič, Amalija; Agdeppa, Eric D.; Timbol, Gerald; Liu, Jie; Keum, Gyochang; Satyamurthy, Nagichettiar; Kepe, Vladimir; Houk, Kendall N.; Barrio, Jorge R.

2012-01-01

The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer’s disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t-butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM Ki) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM Ki). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel. PMID:23012452

Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

PubMed Central

Hall Sedlak, Ruth; Hnilova, Marketa; Grosh, Carolynn; Fong, Hanson; Baneyx, Francois; Schwartz, Dan; Sarikaya, Mehmet; Tamerler, Candan

2012-01-01

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo. PMID:22286990

A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites

PubMed Central

Barykina, Natalia V.; Subach, Oksana M.; Doronin, Danila A.; Sotskov, Vladimir P.; Roshchina, Marina A.; Kunitsyna, Tatiana A.; Malyshev, Aleksey Y.; Smirnov, Ivan V.; Azieva, Asya M.; Sokolov, Ilya S.; Piatkevich, Kiryl D.; Burtsev, Mikhail S.; Varizhuk, Anna M.; Pozmogova, Galina E.; Anokhin, Konstantin V.; Subach, Fedor V.; Enikolopov, Grigori N.

2016-01-01

Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca2+-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope. PMID:27677952

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel

PubMed Central

Yang, Fan; Vu, Simon; Yarov-Yarovoy, Vladimir; Zheng, Jie

2016-01-01

Vanilloids activation of TRPV1 represents an excellent model system of ligand-gated ion channels. Recent studies using cryo-electron microcopy (cryo-EM), computational analysis, and functional quantification revealed the location of capsaicin-binding site and critical residues mediating ligand-binding and channel activation. Based on these new findings, here we have successfully introduced high-affinity binding of capsaicin and resiniferatoxin to the vanilloid-insensitive TRPV2 channel, using a rationally designed minimal set of four point mutations (F467S–S498F–L505T–Q525E, termed TRPV2_Quad). We found that binding of resiniferatoxin activates TRPV2_Quad but the ligand-induced open state is relatively unstable, whereas binding of capsaicin to TRPV2_Quad antagonizes resiniferatoxin-induced activation likely through competition for the same binding sites. Using Rosetta-based molecular docking, we observed a common structural mechanism underlying vanilloids activation of TRPV1 and TRPV2_Quad, where the ligand serves as molecular “glue” that bridges the S4–S5 linker to the S1–S4 domain to open these channels. Our analysis revealed that capsaicin failed to activate TRPV2_Quad likely due to structural constraints preventing such bridge formation. These results not only validate our current working model for capsaicin activation of TRPV1 but also should help guide the design of drug candidate compounds for this important pain sensor. PMID:27298359

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

Kolstoe, Simon E.; Jenvey, Michelle C.; Purvis, Alan

Serum amyloid P component is a pentameric plasma glycoprotein that recognizes and binds to amyloid fibres in a calcium-dependent fashion and is likely to contribute to their deposition and persistence in vivo. Five molecules of the drug CPHPC avidly cross-link pairs of protein pentamers and the decameric complex is rapidly cleared in vivo. Crystal structures of the protein in complex with a bivalent drug and cadmium ions, which improve crystal quality, allow the definition of the preferred bound drug isomers. Under physiological conditions, the pentameric human plasma protein serum amyloid P component (SAP) binds hexanoyl bis(d-proline) (R-1-(6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl) pyrrolidine-2-carboxylic acid; CPHPC)more » through its d-proline head groups in a calcium-dependent interaction. Cooperative effects in binding lead to a substantial enhancement of affinity. Five molecules of the bivalent ligand cross-link and stabilize pairs of SAP molecules, forming a decameric complex that is rapidly cleared from the circulation by the liver. Here, it is reported that X-ray analysis of the SAP complex with CPHPC and cadmium ions provides higher resolution detail of the interaction than is observed with calcium ions. Conformational isomers of CPHPC observed in solution by HPLC and by X-ray analysis are compared with the protein-bound form. These are discussed in relation to the development of CPHPC to provide SAP depletion for the treatment of amyloidosis and other indications.« less

( sup 3 H)-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and ( sup 3 H) ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

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

Branchek, T.; Adham, N.; Macchi, M.

1990-11-01

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to (3H)ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding themore » serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both (3H)DOB and (3H)ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p) to this system caused a rightward shift and steepening of agonist competition curves for (3H) ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity (3H)DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that (3H)DOB and (3H)ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein.« less

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

PubMed

Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

2016-05-01

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

Adenylosuccinate synthetase: recent developments.

PubMed

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

1999-01-01

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

Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-binding Module*

PubMed Central

Taylor, Courtney B.; Talib, M. Faiz; McCabe, Clare; Bu, Lintao; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2012-01-01

Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3–6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function. PMID:22147693

Surfactant-free Colloidal Particles with Specific Binding Affinity

PubMed Central

2017-01-01

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles. PMID:28847149

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

B Akabayov; C Richardson

Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstratemore » that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.« less

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1.

PubMed

Real Hernandez, Luis M; Gonzalez de Mejia, Elvira

2017-04-01

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (-7.2 to -7.0kcal/mol) and the cowpea bean dipeptide Lys-Asp (-7.0kcal/mol) had higher binding affinities than ezetimibe (-6.6kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (-7.2kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption. Copyright © 2017 Elsevier Inc. All rights reserved.

Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

PubMed Central

Zhao, Wenjing; McCallum, Scott A.; Xiao, Zhongping; Zhang, Fuming; Linhardt, Robert J.

2011-01-01

Heparin and heparan sulphate (HS) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS-binding to vascular endothelial growth factor (VEGF) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and that a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that VEGF binding affinity likely depends on the specific structural features of these oligosaccharides including their degree of sulphation and sugar ring stereochemistry and conformation. Notably, the unique 3-O-sulpho group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs. PMID:21658003

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri*

PubMed Central

Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H+ or Na+ electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri. Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4′,6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR.

PubMed

Gater, Deborah L; Saurel, Olivier; Iordanov, Iordan; Liu, Wei; Cherezov, Vadim; Milon, Alain

2014-11-18

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP

PubMed Central

Wijeyesakere, Sanjeeva Joseph; Gagnon, Jessica K.; Arora, Karunesh; Brooks, Charles L.; Raghavan, Malini

2015-01-01

The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin–substrate interactions and as key determinants of PLC dynamics. PMID:26420867

Determinants of the Differential Antizyme-Binding Affinity of Ornithine Decarboxylase

PubMed Central

Liu, Yen-Chin; Hsu, Den-Hua; Huang, Chi-Liang; Liu, Yi-Liang; Liu, Guang-Yaw; Hung, Hui-Chih

2011-01-01

Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The K d value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 μM, while the K d value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 μM. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the K d was 1.4 and 1.2 μM, respectively. These affinities are 6-fold higher than the WT_ODC K d, which suggests that residues 119 and 137 play a role in AZ binding. PMID:22073206

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

PubMed Central

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

2010-01-01

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

New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

PubMed Central

Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

2013-01-01

3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

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

PubMed

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

2015-07-01

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

A 23Na Multiple-Quantum-Filtered NMR Study of the Effect of the Cytoskeleton Conformation on the Anisotropic Motion of Sodium Ions in Red Blood Cells

NASA Astrophysics Data System (ADS)

Knubovets, Tatyana; Shinar, Hadassah; Eliav, Uzi; Navon, Gil

1996-01-01

Recently, it has been shown that23Na double-quantum-filtered NMR spectroscopy can be used to detect anisotropic motion of bound sodium ions in biological systems. The technique is based on the formation of the second-rank tensor when the quadrupolar interaction is not averaged to zero. Using this method, anisotropic motion of bound sodium in human and dog red blood cells was detected, and the effect was shown to depend on the integrity of the membrane cytoskeleton. In the present study, multiple-quantum-filtered techniques were applied in combination with a quadrupolar echo to measure the transverse-relaxation times,T2fandT2s. Line fitting was performed to obtain the values of the residual quadrupolar interaction, which was measured for sodium in a variety of mammalian erythrocytes of different size, shape, rheological properties, and sodium concentrations. Human unsealed white ghosts were used to study sodium bound at the anisotropic sites on the inner side of the RBC membrane. Modulations of the conformation of the cytoskeleton by the variation of either the ionic strength or pH of the suspending medium caused drastic changes in both the residual quadrupolar interaction andT2fdue to changes in the fraction of bound sodium ions as well as changes in the structure of the binding sites. By combining the two spectroscopic parameters, structural change can be followed. The changes in the structure of the sodium anisotropic binding sites deduced by this method were found to correlate with known conformational changes of the membrane cytoskeleton. Variations of the medium pH affected both the fraction of bound sodium ions and the structure of the anisotropic binding sites. Sodium and potassium were shown to bind to the anisotropic binding sites with the same affinity.

The binding of analogs of porphyrins and chlorins with elongated side chains to albumin

PubMed Central

Ben Dror, Shimshon; Bronshtein, Irena; Weitman, Hana; Smith, Kevin M.; O’Neal, William G.; Jacobi, Peter A.; Ehrenberg, Benjamin

2012-01-01

In previous studies, we demonstrated that elongation of side chains of several sensitizers endowed them with higher affinity for artificial and natural membranes and caused their deeper localization in membranes. In the present study, we employed eight hematoporphyrin and protoporphyrin analogs and four groups containing three chlorin analogs each, all synthesized with variable numbers of methylenes in their alkyl carboxylic chains. We show that these tetrapyrroles’ affinity for bovine serum albumin (BSA) and their localization in the binding site are also modulated by chain lengths. The binding constants of the hematoporphyrins and protoporphyrins to BSA increased as the number of methylenes was increased. The binding of the chlorins depended on the substitution at the meso position opposite to the chains. The quenching of the sensitizers’ florescence by external iodide ions decreased as the side chains became longer, indicating to deeper insertion of the molecules into the BSA binding pocket. To corroborate this conclusion, we studied the efficiency of photodamage caused to tryptophan in BSA upon illumination of the bound sensitizers. The efficiency was found to depend on the side-chain lengths of the photosensitizer. We conclude that the protein site that hosts these sensitizers accommodates different analogs at positions that differ slightly from each other. These differences are manifested in the ease of access of iodide from the external aqueous phase, and in the proximity of the photosensitizers to the tryptophan. In the course of this study, we developed the kinetic equations that have to be employed when the sensitizer itself is being destroyed. PMID:19330323

Relative binding affinities of monolignols to horseradish peroxidase

DOE PAGES

Sangha, Amandeep K.; Petridis, Loukas; Cheng, Xiaolin; ...

2016-07-22

Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic –OH group andmore » a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic –OH group instead interacting with Pro139. Furthermore, since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate.« less

Identification of new ligands for the methionine biosynthesis transcriptional regulator (MetJ) by FAC-MS.

PubMed

Martí-Arbona, Ricardo; Teshima, Munehiro; Anderson, Penelope S; Nowak-Lovato, Kristy L; Hong-Geller, Elizabeth; Unkefer, Clifford J; Unkefer, Pat J

2012-01-01

We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector mixtures containing S-adenosyl-L-methionine (SAM) and S-adenosyl derivatives as potential ligands for MetJ binding. The differences in the elution time of different compounds allowed us to rank the binding affinity of each compound. Consistent with previous results, FAC-MS showed that SAM binds to MetJ with the highest affinity. In addition, adenine and 5'-deoxy-5'-(methylthio)adenosine bind to the effector binding site on MetJ. Our experiments with MetJ demonstrate that FAC-MS is capable of screening complex mixtures of molecules and identifying high-affinity binders to TRs. In addition, FAC-MS experiments can be used to discriminate between specific and nonspecific binding of the effectors as well as to estimate the dissociation constant (K(d)) for effector-TR binding. Copyright © 2012 S. Karger AG, Basel.

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi.

PubMed

Barykina, Natalia V; Subach, Oksana M; Piatkevich, Kiryl D; Jung, Erica E; Malyshev, Aleksey Y; Smirnov, Ivan V; Bogorodskiy, Andrey O; Borshchevskiy, Valentin I; Varizhuk, Anna M; Pozmogova, Galina E; Boyden, Edward S; Anokhin, Konstantin V; Enikolopov, Grigori N; Subach, Fedor V

2017-01-01

Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.

Solubilization and purification of melatonin receptors from lizard brain

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

Rivkees, S.A.; Conron, R.W. Jr.; Reppert, S.M.

Melatonin receptors in lizard brain were identified and characterized using {sup 125}I-labeled melatonin (({sup 125}I)MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resultedmore » in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.« less

Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry.

PubMed

Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

2016-01-15

Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu(2+) with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15K in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu(2+) ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu(2+) ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu(2+) ions are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry

NASA Astrophysics Data System (ADS)

Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

2016-01-01

Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu2 + with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15 K in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu2 + ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu2 + ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu2 + ions are discussed.

Exploring the Origin of Differential Binding Affinities of Human Tubulin Isotypes αβII, αβIII and αβIV for DAMA-Colchicine Using Homology Modelling, Molecular Docking and Molecular Dynamics Simulations

PubMed Central

Panda, Dulal; Kunwar, Ambarish

2016-01-01

Tubulin isotypes are found to play an important role in regulating microtubule dynamics. The isotype composition is also thought to contribute in the development of drug resistance as tubulin isotypes show differential binding affinities for various anti-cancer agents. Tubulin isotypes αβII, αβIII and αβIV show differential binding affinity for colchicine. However, the origin of differential binding affinity is not well understood at the molecular level. Here, we investigate the origin of differential binding affinity of a colchicine analogue N-deacetyl-N-(2-mercaptoacetyl)-colchicine (DAMA-colchicine) for human αβII, αβIII and αβIV isotypes, employing sequence analysis, homology modeling, molecular docking, molecular dynamics simulation and MM-GBSA binding free energy calculations. The sequence analysis study shows that the residue compositions are different in the colchicine binding pocket of αβII and αβIII, whereas no such difference is present in αβIV tubulin isotypes. Further, the molecular docking and molecular dynamics simulations results show that residue differences present at the colchicine binding pocket weaken the bonding interactions and the correct binding of DAMA-colchicine at the interface of αβII and αβIII tubulin isotypes. Post molecular dynamics simulation analysis suggests that these residue variations affect the structure and dynamics of αβII and αβIII tubulin isotypes, which in turn affect the binding of DAMA-colchicine. Further, the binding free-energy calculation shows that αβIV tubulin isotype has the highest binding free-energy and αβIII has the lowest binding free-energy for DAMA-colchicine. The order of binding free-energy for DAMA-colchicine is αβIV ≃ αβII >> αβIII. Thus, our computational approaches provide an insight into the effect of residue variations on differential binding of αβII, αβIII and αβIV tubulin isotypes with DAMA-colchicine and may help to design new analogues with higher binding affinities for tubulin isotypes. PMID:27227832

Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

PubMed

Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

2006-07-01

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

Pb-binding to various dissolved organic matter in urban aquatic systems: Key role of the most hydrophilic fraction

NASA Astrophysics Data System (ADS)

Pernet-Coudrier, Benoît; Companys, Encarnació; Galceran, Josep; Morey, Margalida; Mouchel, Jean-Marie; Puy, Jaume; Ruiz, Núria; Varrault, Gilles

2011-07-01

Dissolved organic matter (DOM) from the treated effluent of a wastewater treatment plant and from the river Seine under high human pressure has been separated into three fractions: hydrophobic (containing humic and fulvic substances), transphilic and hydrophilic using a two column array of XAD-8 and XAD-4 resins. The acid base properties and the binding characteristics with respect to Pb ions (using the new electroanalytical technique AGNES, Absence of Gradients and Nernstian Equilibrium Stripping) have been studied and fitted to NICA (Non-Ideal Competitive Isotherm). We evaluated the binding potential of each DOM fraction in order to better predict the speciation of Pb and, later, its bioavailability in the river. The total binding capacity of the different fractions to Pb, as well as the total titratable charge, reaches its maximum value at the most hydrophilic fraction from the treated effluent. Specific properties of the distribution of the complexing sites within each DOM fraction have been exposed by plotting the conditional affinity spectrum (CAS). The addition of these distributions, weighted according to the respective abundance of each organic fraction, allows for a full description of the Pb binding properties of the whole DOM of a sampling site. Despite its weak aromaticity, the hydrophilic fraction from the wastewater treatment plant effluent exhibits a high lead binding affinity, so that at typical environmental pH and free Pb levels (0.1 μg L -1), Pb is mainly bound to the most hydrophilic fraction of the treated effluent (49% of bound Pb at pH 7). This feature may greatly enhance the transport of Pb and highlights that Pb speciation should also consider other fractions apart from humic and/or fulvic acids when studying surface waters under high human pressure.

Hupresin Retains Binding Capacity for Butyrylcholinesterase and Acetylcholinesterase after Sanitation with Sodium Hydroxide.

PubMed

Onder, Seda; David, Emilie; Tacal, Ozden; Schopfer, Lawrence M; Lockridge, Oksana

2017-01-01

Hupresin is a new affinity resin that binds butyrylcholinesterase (BChE) in human plasma and acetylcholinesterase (AChE) solubilized from red blood cells (RBC). Hupresin is available from the CHEMFORASE company. BChE in human plasma binds to Hupresin and is released with 0.1 M trimethylammonium bromide (TMA) with full activity and 10-15% purity. BChE immunopurified from plasma by binding to immobilized monoclonal beads has fewer contaminating proteins than the one-step Hupresin-purified BChE. However, when affinity chromatography on Hupresin follows ion exchange chromatography at pH 4.5, BChE is 99% pure. The membrane bound AChE, solubilized from human RBC with 0.6% Triton X-100, binds to Hupresin and remains bound during washing with sodium chloride. Human AChE is not released in significant quantities with non-denaturing solvents, but is recovered in 1% trifluoroacetic acid. The denatured, partially purified AChE is useful for detecting exposure to nerve agents by mass spectrometry. Our goal was to determine whether Hupresin retains binding capacity for BChE and AChE after Hupresin is washed with 0.1 M NaOH. A 2 mL column of Hupresin equilibrated in 20 mM TrisCl pH 7.5 was used in seven consecutive trials to measure binding and recovery of BChE from 100 mL human plasma. Between each trial the Hupresin was washed with 10 column volumes of 0.1 M sodium hydroxide. A similar trial was conducted with red blood cell AChE in 0.6% Triton X-100. It was found that the binding capacity for BChE and AChE was unaffected by washing Hupresin with 0.1 M sodium hydroxide. Hupresin could be washed with sodium hydroxide at least seven times without losing binding capacity.

Calcium binding to an elastic portion of connectin/titin filaments.

PubMed

Tatsumi, R; Maeda, K; Hattori, A; Takahashi, K

2001-01-01

Alpha-connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the beta-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on beta-connectin. Purified beta-connectin was digested by trypsin into 1700- and 400-kDa fragments. which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 x 10(7) M(-1) and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 microM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of beta-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of beta-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 microM leupeptin to split connectin filaments into beta-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200+/-33 kDa (mean+/-SD), while alpha- and beta-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 microm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction-relaxation cycle of skeletal muscle.

Hollow fiber based affinity selection combined with high performance liquid chromatography-mass spectroscopy for rapid screening lipase inhibitors from lotus leaf.

PubMed

Tao, Yi; Zhang, Yufeng; Wang, Yi; Cheng, Yiyu

2013-06-27

A novel kind of immobilized enzyme affinity selection strategy based on hollow fibers has been developed for screening inhibitors from extracts of medicinal plants. Lipases from porcine pancreas were adsorbed onto the surface of polypropylene hollow fibers to form a stable matrix for ligand fishing, which was called hollow fibers based affinity selection (HF-AS). A variety of factors related to binding capability, including enzyme concentration, incubation time, temperature, buffer pH and ion strength, were optimized using a known lipase inhibitor hesperidin. The proposed approach was applied in screening potential lipase bound ligands from extracts of lotus leaf, followed by rapid characterization of active compounds using high performance liquid chromatography-mass spectrometry. Three flavonoids including quercetin-3-O-β-D-arabinopyranosyl-(1→2)-β-D-galactopyranoside, quercetin-3-O-β-D-glucuronide and kaempferol-3-O-β-d-glucuronide were identified as lipase inhibitors by the proposed HF-AS approach. Our findings suggested that the hollow fiber-based affinity selection could be a rapid and convenient approach for drug discovery from natural products resources. Copyright © 2013 Elsevier B.V. All rights reserved.

Interaction of fluorescent sensor with superparamagnetic iron oxide nanoparticles.

PubMed

Karunakaran, Chockalingam; Jayabharathi, Jayaraman; Sathishkumar, Ramalingam; Jayamoorthy, Karunamoorthy

2013-06-01

To sense superparamagnetic iron oxides (Fe2O3 and Fe3O4) nanocrystals a sensitive bioactive phenanthroimidazole based fluorescent molecule, 2-(4-fluorophenyl)-1-phenyl-1H-phenanthro [9,10-d] imidazole has been designed and synthesized. Electronic spectral studies show that phenanthroimidazole is bound to the surface of iron oxide semiconductors. Fluorescent enhancement has been explained on the basis of photo-induced electron transfer (PET) mechanism and apparent binding constants have been deduced. Binding of phenanthroimidazole with iron oxide nanoparticles lowers the HOMO and LUMO energy levels of phenanthroimidazole molecule. Chemical affinity between the nitrogen atom of the phenanthroimidazole and Fe(2+) and Fe(3+) ions on the surface of the nano-oxide may result in strong binding of the phenanthroimidazole derivative with the nanoparticles. The electron injection from the photoexcited phenanthroimidazole to the iron oxides conduction band explains the enhanced fluorescence. Copyright © 2013 Elsevier B.V. All rights reserved.

Two classes of binding sites for [3H]substance P in rat cerebral cortex.

PubMed

Geraghty, D P; Burcher, E

1993-01-22

The binding characteristics of [3H]substance P ([3H]SP) were investigated in membranes prepared from rat cerebral cortex. Binding of [3H]SP reached equilibrium after 50 min at 25 degrees C and was saturable at 8 nM. Saturation data could be resolved into high affinity (equilibrium dissociation constant, Kd, 0.22 nM) and low affinity sites (Kd, 2.65 nM). The low affinity sites were more numerous than the high affinity sites, with a ratio of 4:1. The non-hydrolyzable GTP analogue GppNHp had no effect on binding, indicating that the high and low affinity sites are not guanine nucleotide-regulated states of the same (NK-1) receptor. The low affinity sites are unlikely to represent NK-3 receptors since coincubation with the selective NK-3 receptor agonist senktide did not alter the biphasic nature of [3H]SP binding. The rank order of potency for inhibition of [3H]SP (2 nM) binding was SP > or = [Sar9, Met(O2)11]-SP > or = physalaemin > SP(3-11) > NP gamma = [Ala3]-SP > or = SP(4-11) > or = NPK > or = SP(5-11) > or = NKB approximately NKA > SP(1-9), compatible with binding to an NK-1 site. N-terminal fragments and non-amidated analogues were ineffective competitors for [3H]SP binding. However, competition data for several peptides including substance P (SP) and the NK-1 selective agonist [Sar9, Met(O2)11]-SP could be resolved into two components.(ABSTRACT TRUNCATED AT 250 WORDS)

Imprinting of metal receptors into multilayer polyelectrolyte films: fabrication and applications in marine antifouling† †Electronic supplementary information (ESI) available: FTIR, NMR spectra of synthesized polymers, XPS spectra and AFM images of non-cross linked and cross linked LBLA and LBLB films, UV-Visible absorption spectra of copper complexation with PAH-His, QCM data of LBLA and LBLB films and stability of the films are provided in the electronic supplementary information. See DOI: 10.1039/c4sc02367f Click here for additional data file.

PubMed Central

Puniredd, Sreenivasa Reddy; Go, Dewi Pitrasari; Zhu, Xiaoying; Guo, Shifeng; Ming Teo, Serena Lay; Chen Lee, Serina Siew

2015-01-01

Polymeric films constructed using the layer-by-layer (LbL) fabrication process were employed as a platform for metal ion immobilization and applied as a marine antifouling coating. The novel Cu2+ ion imprinting process described is based on the use of metal ion templates and LbL multilayer covalent cross-linking. Custom synthesized, peptide mimicking polycations composed of histidine grafted poly(allylamine) (PAH) to bind metal ions, and methyl ester containing polyanions for convenient cross-linking were used in the fabrication process. Two methods of LbL film formation have been investigated using alternate polyelectrolyte deposition namely non-imprinted LbLA, and imprinted LbLB. Both LbL films were cross linked at mild temperature to yield covalent bridging of the layers for improved stability in a sea water environment. A comparative study of the non-imprinted LbLA films and imprinted LbLB films for Cu2+ ion binding capacity, leaching rate and stability of the films was performed. The results reveal that the imprinted films possess enhanced affinity to retain metal ions due to the preorganization of imidazole bearing histidine receptors. As a result the binding capacity of the films for Cu2+ could be improved by seven fold. Antifouling properties of the resulting materials in a marine environment have been demonstrated against the settlement of barnacle larvae, indicating that controlled release of Cu ions was achieved. PMID:28966763

Channel architecture in maltoporin: dominance studies with lamB mutations influencing maltodextrin binding provide evidence for independent selectivity filters in each subunit.

PubMed Central

Ferenci, T; Lee, K S

1989-01-01

Maltoporin trimers constitute maltodextrin-selective channels in the outer membrane of Escherichia coli. To study the organization of the maltodextrin-binding site within trimers, dominance studies were undertaken with maltoporin variants of altered binding affinity. It has been established that amino acid substitutions at three dispersed regions of the maltoporin sequence (at residues 8, 82, and 360) resulted specifically in maltodextrin-binding defects and loss of maltodextrin channel selectivity; a substitution at residue 118 increased both binding affinity and maltodextrin transport. Strains heterodiploid for lamB were constructed in which these substitutions were encoded by chromosomal and plasmid-borne genes, and the relative level of maltoporin expression from these genes was estimated. Binding assays with bacteria forming maltoporin heterotrimers were performed in order to test for complementation between binding-negative alleles, negative dominance of negative over wild-type alleles, and possible dominance of negatives over the high-affinity allele. Double mutants with mutations affecting residues 8 and 118, 82 and 118, and 118 and 360 were constructed in vitro, and the dominance properties of the mutations in cis were also tested. There was no complementation between negatives and no negative dominance in heterotrimers. The high-affinity mutation was dominant over negatives in trans but not in cis. The affinity of binding sites in heterotrimer populations was characteristic of the high-affinity allele present and uninfluenced by the negative allele. These results are consistent with the presence of three discrete binding sites in a maltoporin trimer and suggest that the selectivity filter for maltodextrins is not at the interface between the three subunits. PMID:2521623

Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts.

PubMed

Kane, M A; Portanova, L B; Kelley, K; Holley, M; Ross, S E; Boose, D; Escobedo-Morse, A; Alvarado, B

1994-01-01

Swiss 3T3 cells contained substantial amounts of soluble and specific [125I]GRP binders. Like the membrane-associated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fractionation yielded different profiles, the M(rs) were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [125I]GRP. These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells.

Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose

PubMed Central

DiScipio, Richard G.; Liddington, Robert C.; Schraufstatter, Ingrid U.

2016-01-01

A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

(/sup 3/H)-(Thr4,Gly7)OT: a highly selective ligand for central and peripheral OT receptors

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

Elands, J.; Barberis, C.; Jard, S.

1988-01-01

Oxytocin receptors in rat hippocampal synaptic plasma membranes were compared with mammary gland and uterine oxytocin receptors. For this purpose, a highly specific oxytocic agonist (Thr4,Gly7)oxytocin was tritiated. We demonstrated that this ligand labels oxytocin receptors selectively. Scatchard analyses revealed a high affinity for all the oxytocin receptors investigated, with equilibrium dissociation constants between 1.0 and 2.0 nM. Binding appeared to take place at a single population of receptor sites. Competition experiments confirmed the high affinity of arginine vasopressin for hippocampal oxytocin receptors but also revealed that mammary gland and uterine oxytocin receptors do not discriminate more efficiently between oxytocinmore » and arginine vasopressin. This lack in specificity is not affected by applying different concentrations of Mg ions.« less

Dual Mechanism of Ion Permeation through VDAC Revealed with Inorganic Phosphate Ions and Phosphate Metabolites

PubMed Central

Krammer, Eva-Maria; Vu, Giang Thi; Homblé, Fabrice; Prévost, Martine

2015-01-01

In the exchange of metabolites and ions between the mitochondrion and the cytosol, the voltage-dependent anion channel (VDAC) is a key element, as it forms the major transport pathway for these compounds through the mitochondrial outer membrane. Numerous experimental studies have promoted the idea that VDAC acts as a regulator of essential mitochondrial functions. In this study, using a combination of molecular dynamics simulations, free-energy calculations, and electrophysiological measurements, we investigated the transport of ions through VDAC, with a focus on phosphate ions and metabolites. We showed that selectivity of VDAC towards small anions including monovalent phosphates arises from short-lived interactions with positively charged residues scattered throughout the pore. In dramatic contrast, permeation of divalent phosphate ions and phosphate metabolites (AMP and ATP) involves binding sites along a specific translocation pathway. This permeation mechanism offers an explanation for the decrease in VDAC conductance measured in the presence of ATP or AMP at physiological salt concentration. The binding sites occur at similar locations for the divalent phosphate ions, AMP and ATP, and contain identical basic residues. ATP features a marked affinity for a central region of the pore lined by two lysines and one arginine of the N-terminal helix. This cluster of residues together with a few other basic amino acids forms a “charged brush” which facilitates the passage of the anionic metabolites through the pore. All of this reveals that VDAC controls the transport of the inorganic phosphates and phosphate metabolites studied here through two different mechanisms. PMID:25860993

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

PubMed

Marsh, Lorraine

2015-01-01

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

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

PubMed

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

2016-10-01

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

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics

PubMed Central

King, Amy C.; Kavosi, Mania; Wang, Mengmeng; O'Hara, Denise M.; Tchistiakova, Lioudmila; Katragadda, Madan

2018-01-01

ABSTRACT A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1–5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1–3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics. PMID:28991504

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

PubMed

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

2014-07-11

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

Specific interaction between negative atmospheric ions and organic compounds in atmospheric pressure corona discharge ionization mass spectrometry.

PubMed

Sekimoto, Kanako; Sakai, Mami; Takayama, Mitsuo

2012-06-01

The interaction between negative atmospheric ions and various types of organic compounds were investigated using atmospheric pressure corona discharge ionization (APCDI) mass spectrometry. Atmospheric negative ions such as O(2)(-), HCO(3)(-), COO(-)(COOH), NO(2)(-), NO(3)(-), and NO(3)(-)(HNO(3)) having different proton affinities served as the reactant ions for analyte ionization in APCDI in negative-ion mode. The individual atmospheric ions specifically ionized aliphatic and aromatic compounds with various functional groups as atmospheric ion adducts and deprotonated analytes. The formation of the atmospheric ion adducts under certain discharge conditions is most likely attributable to the affinity between the analyte and atmospheric ion and the concentration of the atmospheric ion produced under these conditions. The deprotonated analytes, in contrast, were generated from the adducts of the atmospheric ions with higher proton affinity attributable to efficient proton abstraction from the analyte by the atmospheric ion.

Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor.

PubMed

Matulis, Daumantas; Kranz, James K; Salemme, F Raymond; Todd, Matthew J

2005-04-05

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

2017-08-01

The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis

PubMed Central

Adachi, Kengo; Oiwa, Kazuhiro; Yoshida, Masasuke; Nishizaka, Takayuki; Kinosita, Kazuhiko

2012-01-01

F1-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >104, followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >104. All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities. PMID:22929779

Sequence2Vec: a novel embedding approach for modeling transcription factor binding affinity landscape.

PubMed

Dai, Hanjun; Umarov, Ramzan; Kuwahara, Hiroyuki; Li, Yu; Song, Le; Gao, Xin

2017-11-15

An accurate characterization of transcription factor (TF)-DNA affinity landscape is crucial to a quantitative understanding of the molecular mechanisms underpinning endogenous gene regulation. While recent advances in biotechnology have brought the opportunity for building binding affinity prediction methods, the accurate characterization of TF-DNA binding affinity landscape still remains a challenging problem. Here we propose a novel sequence embedding approach for modeling the transcription factor binding affinity landscape. Our method represents DNA binding sequences as a hidden Markov model which captures both position specific information and long-range dependency in the sequence. A cornerstone of our method is a novel message passing-like embedding algorithm, called Sequence2Vec, which maps these hidden Markov models into a common nonlinear feature space and uses these embedded features to build a predictive model. Our method is a novel combination of the strength of probabilistic graphical models, feature space embedding and deep learning. We conducted comprehensive experiments on over 90 large-scale TF-DNA datasets which were measured by different high-throughput experimental technologies. Sequence2Vec outperforms alternative machine learning methods as well as the state-of-the-art binding affinity prediction methods. Our program is freely available at https://github.com/ramzan1990/sequence2vec. xin.gao@kaust.edu.sa or lsong@cc.gatech.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Concepts in receptor optimization: targeting the RGD peptide.

PubMed

Chen, Wei; Chang, Chia-en; Gilson, Michael K

2006-04-12

Synthetic receptors have a wide range of potential applications, but it has been difficult to design low molecular weight receptors that bind ligands with high, "proteinlike" affinities. This study uses novel computational methods to understand why it is hard to design a high-affinity receptor and to explore the limits of affinity, with the bioactive peptide RGD as a model ligand. The M2 modeling method is found to yield excellent agreement with experiment for a known RGD receptor and then is used to analyze a series of receptors generated in silico with a de novo design algorithm. Forces driving binding are found to be systematically opposed by proportionate repulsions due to desolvation and entropy. In particular, strong correlations are found between Coulombic attractions and the electrostatic desolvation penalty and between the mean energy change on binding and the cost in configurational entropy. These correlations help explain why it is hard to achieve high affinity. The change in surface area upon binding is found to correlate poorly with affinity within this series. Measures of receptor efficiency are formulated that summarize how effectively a receptor uses surface area, total energy, and Coulombic energy to achieve affinity. Analysis of the computed efficiencies suggests that a low molecular weight receptor can achieve proteinlike affinity. It is also found that macrocyclization of a receptor can, unexpectedly, increase the entropy cost of binding because the macrocyclic structure further restricts ligand motion.

Heavy metal ion inhibition studies of human, sheep and fish α-carbonic anhydrases.

PubMed

Demirdağ, Ramazan; Yerlikaya, Emrah; Şentürk, Murat; Küfrevioğlu, Ö İrfan; Supuran, Claudiu T

2013-04-01

Carbonic anhydrases (CAs, EC 4.2.1.1) were purified from sheep kidney (sCA IV), from the liver of the teleost fish Dicentrarchus labrax (dCA) and from human erythrocytes (hCA I and hCA II). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The kinetic parameters of these enzymes were determined for their esterase activity with 4-nitrophenyl acetate as substrate. The following metal ions, Pb(2+), Co(2+), Hg(2+), Cd(2+), Zn(2+), Se(2+), Cu(2+), Al(3+) and Mn(3+) showed inhibitory effects on these enzymes. The tested metal ions inhibited these CAs competitively in the low milimolar/submillimolar range. The susceptibility to various cations inhibitors differs significantly between these vertebrate α-CAs and is probably due to their binding to His64 or the histidine cluster.

Mapping the receptor site for alpha-scorpion toxins on a Na+ channel voltage sensor.

PubMed

Wang, Jinti; Yarov-Yarovoy, Vladimir; Kahn, Roy; Gordon, Dalia; Gurevitz, Michael; Scheuer, Todd; Catterall, William A

2011-09-13

The α-scorpions toxins bind to the resting state of Na(+) channels and inhibit fast inactivation by interaction with a receptor site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ~73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na(+) channels and reveals its mode of interaction with a gating modifier toxin.

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin.

PubMed

Nielsen, A D; Borch, K; Westh, P

2000-06-15

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.

Prostaglandin E and F2 alpha receptors in human myometrium during the menstrual cycle and in pregnancy and labor

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

Giannopoulos, G.; Jackson, K.; Kredentser, J.

The binding of prostaglandins E1 and F2 alpha has been studied in the human myometrium and cervix during the menstrual cycle and in the myometrium of pregnant patients at term before and during labor. Tritium-labeled prostaglandin E1 and F2 alpha binding was saturable and reversible. Scatchard analysis of tritium-labeled prostaglandin E1 binding was linear, which suggests a single class of high-affinity binding sites with an estimated apparent equilibrium dissociation constant of 2.5 to 5.4 nmol/L and inhibitor affinities of 0.9, 273, 273, and 217 nmol/L for prostaglandins E2, A1, B1, and F2 alpha, respectively. Scatchard analysis of tritium-labeled prostaglandin F2more » alpha, binding was also linear, but the affinity of these binding sites was much lower, with an average dissociation constant of 50 nmol/L and inhibitor affinities of 1.6, 2.2, and 11.2 nmol/L for prostaglandins E1, E2, and A1, respectively. In nonpregnant patients, the concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were similar in the myometrium during the proliferative and secretory phases of the menstrual cycle, but the concentration of these sites was much lower in the cervix. The concentration of the tritium-labeled prostaglandin E1 binding sites was significantly lower in the myometrium of pregnant patients at term than in the myometrium of nonpregnant patients. The concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were not significantly different in the upper and lower myometrium of pregnant patients at term or in the myometrium of such patients before and during labor. The concentrations of the tritium-labeled prostaglandin F2 alpha binding sites during the menstrual cycle and in pregnancy at term were similar to those of tritium-labeled prostaglandin E1 binding sites.« less

The binding properties of cycloxaprid on insect native nAChRs partially explain the low cross-resistance with imidacloprid in Nilaparvata lugens.

PubMed

Zhang, Yixi; Xu, Xiaoyong; Bao, Haibo; Shao, Xusheng; Li, Zhong; Liu, Zewen

2018-06-06

Neonicotinoids, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) to control Nilaparvata lugens, a major rice insect pest. High imidacloprid resistance has been reported in N. lugens in laboratory and in fields. Cycloxaprid, an oxabridged cis-nitromethylene neonicotinoid, showed high insecticidal activity against N. lugens and low cross-resistance in the imidacloprid resistant strains and field populations. Binding studies have demonstrated that imidacloprid had two binding sites with different affinities (Kd = 3.18 ± 0.43 pM and 1.78 ± 0.19 nM) in N. lugens nAChRs. Cycloxaprid was poor at displacing [ 3 H]imidacloprid at its high-affinity binding site (Ki = 159.38±20.43 nM), but quite efficient at the low-affinity binding site (Ki = 1.27±0.35 nM). These data showed that cycloxaprid had overlapping binding sites with imidacloprid only at its low-affinity binding site. Therefore, the low displacement ability of cycloxaprid against imidacloprid binding at its high affinity site could partially explain the low cross-resistance of cycloxaprid in the imidacloprid resistant populations. The high insecticidal activity, low cross-resistance and different binding properties on insect nAChRs of cycloxaprid demonstrating it a potential insecticide to control N. lugens and related insect pests, especially the ones with high resistance to neonicotinoids. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Large scale affinity calculations of cyclodextrin host-guest complexes: Understanding the role of reorganization in the molecular recognition process

PubMed Central

Wickstrom, Lauren; He, Peng; Gallicchio, Emilio; Levy, Ronald M.

2013-01-01

Host-guest inclusion complexes are useful models for understanding the structural and energetic aspects of molecular recognition. Due to their small size relative to much larger protein-ligand complexes, converged results can be obtained rapidly for these systems thus offering the opportunity to more reliably study fundamental aspects of the thermodynamics of binding. In this work, we have performed a large scale binding affinity survey of 57 β-cyclodextrin (CD) host guest systems using the binding energy distribution analysis method (BEDAM) with implicit solvation (OPLS-AA/AGBNP2). Converged estimates of the standard binding free energies are obtained for these systems by employing techniques such as parallel Hamitionian replica exchange molecular dynamics, conformational reservoirs and multistate free energy estimators. Good agreement with experimental measurements is obtained in terms of both numerical accuracy and affinity rankings. Overall, average effective binding energies reproduce affinity rank ordering better than the calculated binding affinities, even though calculated binding free energies, which account for effects such as conformational strain and entropy loss upon binding, provide lower root mean square errors when compared to measurements. Interestingly, we find that binding free energies are superior rank order predictors for a large subset containing the most flexible guests. The results indicate that, while challenging, accurate modeling of reorganization effects can lead to ligand design models of superior predictive power for rank ordering relative to models based only on ligand-receptor interaction energies. PMID:25147485

Interplay between binding affinity and kinetics in protein-protein interactions.

PubMed

Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

2016-07-01

To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Mechanistic Insights into the Allosteric Modulation of Opioid Receptors by Sodium Ions

PubMed Central

2015-01-01

The idea of sodium ions altering G-protein-coupled receptor (GPCR) ligand binding and signaling was first suggested for opioid receptors (ORs) in the 1970s and subsequently extended to other GPCRs. Recently published ultra-high-resolution crystal structures of GPCRs, including that of the δ-OR subtype, have started to shed light on the mechanism underlying sodium control in GPCR signaling by revealing details of the sodium binding site. Whether sodium accesses different receptor subtypes from the extra- or intracellular sides, following similar or different pathways, is still an open question. Earlier experiments in brain homogenates suggested a differential sodium regulation of ligand binding to the three major OR subtypes, in spite of their high degree of sequence similarity. Intrigued by this possibility, we explored the dynamic nature of sodium binding to δ-OR, μ-OR, and κ-OR by means of microsecond-scale, all-atom molecular dynamics (MD) simulations. Rapid sodium permeation was observed exclusively from the extracellular milieu, and following similar binding pathways in all three ligand-free OR systems, notwithstanding extra densities of sodium observed near nonconserved residues of κ-OR and δ-OR, but not in μ-OR. We speculate that these differences may be responsible for the differential increase in antagonist binding affinity of μ-OR by sodium resulting from specific ligand binding experiments in transfected cells. On the other hand, sodium reduced the level of binding of subtype-specific agonists to all OR subtypes. Additional biased and unbiased MD simulations were conducted using the δ-OR ultra-high-resolution crystal structure as a model system to provide a mechanistic explanation for this experimental observation. PMID:25073009

Mechanistic insights into the allosteric modulation of opioid receptors by sodium ions.

PubMed

Shang, Yi; LeRouzic, Valerie; Schneider, Sebastian; Bisignano, Paola; Pasternak, Gavril W; Filizola, Marta

2014-08-12

The idea of sodium ions altering G-protein-coupled receptor (GPCR) ligand binding and signaling was first suggested for opioid receptors (ORs) in the 1970s and subsequently extended to other GPCRs. Recently published ultra-high-resolution crystal structures of GPCRs, including that of the δ-OR subtype, have started to shed light on the mechanism underlying sodium control in GPCR signaling by revealing details of the sodium binding site. Whether sodium accesses different receptor subtypes from the extra- or intracellular sides, following similar or different pathways, is still an open question. Earlier experiments in brain homogenates suggested a differential sodium regulation of ligand binding to the three major OR subtypes, in spite of their high degree of sequence similarity. Intrigued by this possibility, we explored the dynamic nature of sodium binding to δ-OR, μ-OR, and κ-OR by means of microsecond-scale, all-atom molecular dynamics (MD) simulations. Rapid sodium permeation was observed exclusively from the extracellular milieu, and following similar binding pathways in all three ligand-free OR systems, notwithstanding extra densities of sodium observed near nonconserved residues of κ-OR and δ-OR, but not in μ-OR. We speculate that these differences may be responsible for the differential increase in antagonist binding affinity of μ-OR by sodium resulting from specific ligand binding experiments in transfected cells. On the other hand, sodium reduced the level of binding of subtype-specific agonists to all OR subtypes. Additional biased and unbiased MD simulations were conducted using the δ-OR ultra-high-resolution crystal structure as a model system to provide a mechanistic explanation for this experimental observation.

The Free Energy Landscape of GABA Binding to a Pentameric Ligand-Gated Ion Channel and Its Disruption by Mutations.

PubMed

Comitani, Federico; Limongelli, Vittorio; Molteni, Carla

2016-07-12

Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop superfamily are important neuroreceptors that mediate fast synaptic transmission. They are activated by the binding of a neurotransmitter, but the details of this process are still not fully understood. As a prototypical pLGIC, here we choose the insect resistance to dieldrin (RDL) receptor involved in resistance to insecticides and investigate the binding of the neurotransmitter GABA to its extracellular domain at the atomistic level. We achieve this by means of μ-sec funnel-metadynamics simulations, which efficiently enhance the sampling of bound and unbound states by using a funnel-shaped restraining potential to limit the exploration in the solvent. We reveal the sequence of events in the binding process from the capture of GABA from the solvent to its pinning between the charged residues Arg111 and Glu204 in the binding pocket. We characterize the associated free energy landscapes in the wild-type RDL receptor and in two mutant forms, where the key residues Arg111 and Glu204 are mutated to Ala. Experimentally these mutations produce nonfunctional channels, which is reflected in the reduced ligand binding affinities due to the loss of essential interactions. We also analyze the dynamical behavior of the crucial loop C, whose opening allows the access of GABA to the binding site and closure locks the ligand into the protein. The RDL receptor shares structural and functional features with other pLGICs; hence, our work outlines a valuable protocol to study the binding of ligands to pLGICs beyond conventional docking and molecular dynamics techniques.

Correlation of Local Effects of DNA Sequence and Position of Beta-Alanine Inserts with Polyamide-DNA Complex Binding Affinities and Kinetics

PubMed Central

Wang, Shuo; Nanjunda, Rupesh; Aston, Karl; Bashkin, James K.; Wilson, W. David

2012-01-01

In order to better understand the effects of β-alanine (β) substitution and the number of heterocycles on DNA binding affinity and selectivity, the interactions of an eight-ring hairpin polyamide (PA) and two β derivatives as well as a six-heterocycle analog have been investigated with their cognate DNA sequence, 5′-TGGCTT-3′. Binding selectivity and the effects of β have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV-melting, biosensor-surface plasmon resonance, isothermal titration calorimetry, circular dichroism and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs, and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔCp, and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that β substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in PA binding to the mutant DNAs further confirm the position-dependent effects on PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T•A versus A•T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of β substitution on binding tell a clear and very important story about sequence dependent binding of PAs to DNA. PMID:23167504

Modern Directions for Potentiometric Sensors

PubMed Central

Bakker, Eric; Chumbimuni-Torres, Karin

2009-01-01

This paper gives an overview of the newest developments of polymeric membrane ion-selective electrodes. A short essence of the underlying theory is given, emphasizing how the electromotive force may be used to assess binding constants of the ionophore, and how the selectivity and detection limit are related to the underlying membrane processes. The recent developments in lowering the detection limits of ISEs are described, including recent approaches of developing all solid state ISEs, and breakthroughs in detecting ultra-small quantities of ions at low concentrations. These developments have paved the way to use potentiometric sensors as in ultra-sensitive affinity bioanalysis in conjunction with nanoparticle labels. Recent results establish that potentiometry compares favorably to electrochemical stripping analysis. Other new developments with ion-selective electrodes are also described, including the concept of backside calibration potentiometry, controlled current coulometry, pulsed chronopotentiometry, and localized flash titration with ion-selective membranes to design sensors for the direct detection of total acidity without net sample perturbation. These developments have further opened the field for exciting new possibilities and applications. PMID:19890473

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.

PubMed

Tome, Jacob M; Ozer, Abdullah; Pagano, John M; Gheba, Dan; Schroth, Gary P; Lis, John T

2014-06-01

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

Alternative Affinity Ligands for Immunoglobulins.

PubMed

Kruljec, Nika; Bratkovič, Tomaž

2017-08-16

The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

[125I]2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a high-affinity radioligand selective for I1 imidazoline receptors.

PubMed

Greney, Hugues; Urosevic, Dragan; Schann, Stephan; Dupuy, Laurence; Bruban, Véronique; Ehrhardt, Jean-Daniel; Bousquet, Pascal; Dontenwill, Monique

2002-07-01

The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5'-O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway associated to I1R. Because [125I]LNP 911 was unable to bind to the I2 binding site and alpha2AR, our data indicate that [125I]LNP 911 is the first highly selective radioiodinated probe for I1R with a nanomolar affinity. This new tool should facilitate the molecular characterization of the I1 imidazoline receptor.

Binding of Phenazinium Dye Safranin T to Polyriboadenylic Acid: Spectroscopic and Thermodynamic Study

PubMed Central

Roy, Snigdha; Das, Suman

2014-01-01

Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na+] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure. PMID:24498422

Binding of phenazinium dye safranin T to polyriboadenylic acid: spectroscopic and thermodynamic study.

PubMed

Pradhan, Ankur Bikash; Haque, Lucy; Roy, Snigdha; Das, Suman

2014-01-01

Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na⁺] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure.

Host-Guest Complexes with Protein-Ligand-Like Affinities: Computational Analysis and Design

PubMed Central

Moghaddam, Sarvin; Inoue, Yoshihisa

2009-01-01

It has recently been discovered that guests combining a nonpolar core with cationic substituents bind cucurbit[7]uril (CB[7]) in water with ultra-high affinities. The present study uses the Mining Minima algorithm to study the physics of these extraordinary associations and to computationally test a new series of CB[7] ligands designed to bind with similarly high affinity. The calculations reproduce key experimental observations regarding the affinities of ferrocene-based guests with CB[7] and β-cyclodextrin and provide a coherent view of the roles of electrostatics and configurational entropy as determinants of affinity in these systems. The newly designed series of compounds is based on a bicyclo[2.2.2]octane core, which is similar in size and polarity to the ferrocene core of the existing series. Mining Minima predicts that these new compounds will, like the ferrocenes, bind CB[7] with extremely high affinities. PMID:19133781

Impaired binding affinity of electronegative low-density lipoprotein (LDL) to the LDL receptor is related to nonesterified fatty acids and lysophosphatidylcholine content.

PubMed

Benítez, Sonia; Villegas, Virtudes; Bancells, Cristina; Jorba, Oscar; González-Sastre, Francesc; Ordóñez-Llanos, Jordi; Sánchez-Quesada, José Luis

2004-12-21

The binding characteristics of electropositive [LDL(+)] and electronegative LDL [LDL(-)] subfractions to the LDL receptor (LDLr) were studied. Saturation kinetic studies in cultured human fibroblasts demonstrated that LDL(-) from normolipemic (NL) and familial hypercholesterolemic (FH) subjects had lower binding affinity than their respective LDL(+) fractions (P < 0.05), as indicated by higher dissociation constant (K(D)) values. FH-LDL(+) also showed lower binding affinity (P < 0.05) than NL-LDL(+) (K(D), sorted from lower to higher affinity: NL-LDL(-), 33.0 +/- 24.4 nM; FH-LDL(-), 24.4 +/- 7.1 nM; FH-LDL(+), 16.6 +/- 7.0 nM; NL-LDL(+), 10.9 +/- 5.7 nM). These results were confirmed by binding displacement studies. The impaired affinity binding of LDL(-) could be attributed to altered secondary and tertiary structure of apolipoprotein B, but circular dichroism (CD) and tryptophan fluorescence (TrpF) studies revealed no structural differences between LDL(+) and LDL(-). To ascertain the role of increased nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC) content in LDL(-), LDL(+) was enriched in NEFA or hydrolyzed with secretory phospholipase A(2). Modification of LDL gradually decreased the affinity to LDLr in parallel to the increasing content of NEFA and/or LPC. Modified LDLs with a NEFA content similar to that of LDL(-) displayed similar affinity. ApoB structure studies of modified LDLs by CD and TrpF showed no difference compared to LDL(+) or LDL(-). Our results indicate that NEFA loading or phospholipase A(2) lipolysis of LDL leads to changes that affect the affinity of LDL to LDLr with no major effect on apoB structure. Impaired affinity to the LDLr shown by LDL(-) is related to NEFA and/or LPC content rather than to structural differences in apolipoprotein B.

Engineering cofactor and ligand binding in an artificial neuroglobin

NASA Astrophysics Data System (ADS)

Zhang, Lei

HP-7 is one artificial mutated oxygen transport protein, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which has a neutral hydrophobicity, slows gaseous ligand binding 22-fold, increases the affinity of the distal histidine ligand by a factor of thirteen, and decreases the binding affinity of carbon monoxide, a nonreactive oxygen analogue, three-fold. Paradoxically, it also decreases heme binding affinity by a factor of three in the reduced state and six in the oxidized state. Application of a two-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. We have also examined the effects these mutations have on function. The K d of the nonnreactive oxygen analogue carbon monoxide (CO) is only decreased three-fold, despite the large increase in distal histidine affinity engendered by the 22-fold decrease in the histidine ligand off-rate. This is a result of the four-fold increase in affinity for CO binding to the pentacoordinate state. Oxygen binds to HP7 with a Kd of 117 µM, while the mutant rapidly oxidizes when exposed to oxygen. EPR analysis of both ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation causes a large increase in water penetration into the protein core. The inability of the mutant protein may thus either be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors.

Amyloid tracers detect multiple binding sites in Alzheimer's disease brain tissue.

PubMed

Ni, Ruiqing; Gillberg, Per-Göran; Bergfors, Assar; Marutle, Amelia; Nordberg, Agneta

2013-07-01

Imaging fibrillar amyloid-β deposition in the human brain in vivo by positron emission tomography has improved our understanding of the time course of amyloid-β pathology in Alzheimer's disease. The most widely used amyloid-β imaging tracer so far is (11)C-Pittsburgh compound B, a thioflavin derivative but other (11)C- and (18)F-labelled amyloid-β tracers have been studied in patients with Alzheimer's disease and cognitively normal control subjects. However, it has not yet been established whether different amyloid tracers bind to identical sites on amyloid-β fibrils, offering the same ability to detect the regional amyloid-β burden in the brains. In this study, we characterized (3)H-Pittsburgh compound B binding in autopsied brain regions from 23 patients with Alzheimer's disease and 20 control subjects (aged 50 to 88 years). The binding properties of the amyloid tracers FDDNP, AV-45, AV-1 and BF-227 were also compared with those of (3)H-Pittsburgh compound B in the frontal cortices of patients with Alzheimer's disease. Saturation binding studies revealed the presence of high- and low-affinity (3)H-Pittsburgh compound B binding sites in the frontal cortex (K(d1): 3.5 ± 1.6 nM; K(d2): 133 ± 30 nM) and hippocampus (K(d1):5.6 ± 2.2 nM; K(d2): 181 ± 132 nM) of Alzheimer's disease brains. The relative proportion of high-affinity to low-affinity sites was 6:1 in the frontal cortex and 3:1 in the hippocampus. One control showed both high- and low-affinity (3)H-Pittsburgh compound B binding sites (K(d1): 1.6 nM; K(d2): 330 nM) in the cortex while the others only had a low-affinity site (K(d2): 191 ± 70 nM). (3)H-Pittsburgh compound B binding in Alzheimer's disease brains was higher in the frontal and parietal cortices than in the caudate nucleus and hippocampus, and negligible in the cerebellum. Competitive binding studies with (3)H-Pittsburgh compound B in the frontal cortices of Alzheimer's disease brains revealed high- and low-affinity binding sites for BTA-1 (Ki: 0.2 nM, 70 nM), florbetapir (1.8 nM, 53 nM) and florbetaben (1.0 nM, 65 nM). BF-227 displaced 83% of (3)H-Pittsburgh compound B binding, mainly at a low-affinity site (311 nM), whereas FDDNP only partly displaced (40%). We propose a multiple binding site model for the amyloid tracers (binding sites 1, 2 and 3), where AV-45 (florbetapir), AV-1 (florbetaben), and Pittsburgh compound B, all show nanomolar affinity for the high-affinity site (binding site 1), as visualized by positron emission tomography. BF-227 shows mainly binding to site 3 and FDDNP shows only some binding to site 2. Different amyloid tracers may provide new insight into the pathophysiological mechanisms in the progression of Alzheimer's disease.

Identification, characterization and application of a G-quadruplex structured DNA aptamer against cancer biomarker protein anterior gradient homolog 2.

PubMed

Wu, Jie; Wang, Chi; Li, Xilan; Song, Yanling; Wang, Wei; Li, Cong; Hu, Jia; Zhu, Zhi; Li, Jiuxing; Zhang, Weiyun; Lu, Zhongxian; Yang, Chaoyong James

2012-01-01

Anterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism. Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with K(d) values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2. In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.

Copper Coordination in the Full-Length, Recombinant Prion Protein†

PubMed Central

Burns, Colin S.; Aronoff-Spencer, Eliah; Legname, Giuseppe; Prusiner, Stanley B.; Antholine, William E.; Gerfen, Gary J.; Peisach, Jack; Millhauser, Glenn L.

2010-01-01

The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment constitutes the fundamental binding unit in the octarepeat domain [Burns et al. (2002) Biochemistry 41, 3991–4001; Aronoff-Spencer et al. (2000) Biochemistry 39, 13760–13771]. Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92–96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin–echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion. PMID:12779334

Carbohydrate binding sites in a pancreatic alpha-amylase-substrate complex, derived from X-ray structure analysis at 2.1 A resolution.

PubMed Central

Qian, M.; Haser, R.; Payan, F.

1995-01-01

The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) that was soaked with the substrate maltopentaose showed electron density corresponding to two independent carbohydrate recognition sites on the surface of the molecule. Both binding sites are distinct from the active site described in detail in our previous high-resolution study of a complex between PPA and a carbohydrate inhibitor (Qian M, Buisson G, Duée E, Haser H, Payan F, 1994, Biochemistry 33:6284-6294). One of the binding sites previously identified in a 5-A-resolution electron density map, lies at a distance of 20 A from the active site cleft and can accommodate two glucose units. The second affinity site for sugar units is located close to the calcium binding site. The crystal structure of the maltopentaose complex was refined at 2.1 A resolution, to an R-factor of 17.5%, with an RMS deviation in bond distances of 0.007 A. The model includes all 496 residues of the enzyme, 1 calcium ion, 1 chloride ion, 425 water molecules, and 3 bound sugar rings. The binding sites are characterized and described in detail. The present complex structure provides the evidence of an increased stability of the structure upon interaction with the substrate and allows identification of an N-terminal pyrrolidonecarboxylic acid in PPA. PMID:7613472

No major role for binding by salivary proteins as a defense against dietary tannins in Mediterranean goats.

PubMed

Hanovice-Ziony, Michal; Gollop, Nathan; Landau, Serge Yan; Ungar, Eugene David; Muklada, Hussein; Glasser, Tzach Aharon; Perevolotsky, Avi; Walker, John Withers

2010-07-01

We investigated whether Mediterranean goats use salivary tannin-binding proteins to cope with tannin-rich forages by determining the affinity of salivary or parotid gland proteins for tannic acid or quebracho tannin. Mixed saliva, sampled from the oral cavity, or parotid gland contents were compared to the intermediate affinity protein bovine serum albumin with a competitive binding assay. Goats that consume tannin-rich browse (Damascus) and goats that tend to avoid tannins (Mamber) were sequentially fed high (Pistacia lentiscus L.), low (vetch hay), or zero (wheat hay) tannin forages. Affinity of salivary proteins for tannins did not differ between goat breeds and did not respond to presence or absence of tannins in the diet. Proteins in mixed saliva had slightly higher affinity for tannins than those in parotid saliva, but neither source contained proteins with higher affinity for tannins than bovine serum albumin. Similarly, 3 months of browsing in a tannin-rich environment had little effect on the affinity of salivary proteins for tannin in adult goats of either breed. We sampled mixed saliva from young kids before they consumed forage and after 3 months of foraging in a tannin-rich environment. Before foraging, the saliva of Mamber kids had higher affinity for tannic acid (but not quebracho tannin) than the saliva of Damascus kids, but there was no difference after 3 months of exposure to tannin-rich browse, and the affinity of the proteins was always similar to the affinity of bovine serum albumin. Our results suggest there is not a major role for salivary tannin-binding proteins in goats. Different tendencies of goat breeds to consume tannin-rich browse does not appear be related to differences in salivary tannin-binding proteins.