CONCERTED ACTION OF THE HIGH-AFFINITY CALCIUM-BINDING SITES IN SKELETAL-MUSCLE TROPONIN-C

Mutants of each of the four divalent cation binding sites of chicken skeletal muscle troponin C (TnC) were constructed using site directed mutagenesis to convert Asp to Ala at the first coordinating position in each site. With a view to evaluating the importance of site-site interactions both within and between the N- and C-terminal domains, in this study the mutants are examined for their ability to associate with other components of the troponin-tropomyosin regulatory complex and to regulate thin filaments. The functional effects of each mutation in reconstitution assays are largely confined to the domain in which it occurs, where the unmutated site is unable to compensate for the defect, Thus the mutants of sites I and II bind to the regulatory complex but are impaired in ability to regulate tension and actomyosin ATPase activity, whereas the mutants of sites III and IV regulate activity but are unable to remain bound to thin filaments unless Ca2+ is present. When all four sites are intact, free Mg2+ causes a 50-60-fold increase in TnC's affinity for the other components of the regulatory complex, allowing it to attach firmly to thin filaments. Calcium can replace Mg2+ at a concentration ratio of 1:5000, and at this ratio the Ca2 . TnC complex is more tightly bound to the filaments than the Mg2 . TnC form, In the C-terminal mutants, higher concentrations of Ca2+ (above tension threshold) are required to effect this transformation than in the recombinant wild-type protein, suggesting that the mutants reveal an attachment mediated by Ca2+ in the N-domain sites.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Conformational analyses and docking studies of a series of 5-nitrofuran- and 5-nitrothiophen-semicarbazone derivatives in three possible binding sites of trypanothione and glutathione reductases

To explore three possible binding sites of trypanothione and glutathione reductase, namely, the active, the dimer interface and the coenzyme NADPH binding site, a series of eight compounds, nitrofurans and nitrothiophenes derivatives, were docked, using their crystallographic and modeled conformations. Docking results showed that, for both families and both enzymes, compounds are more likely to bind in the interface site, even though there is some probability of binding in the active site. These studies are in agreement with experimental data, which suggest that these class of compounds can act either as uncompetitive or mixed type inhibitors, and also with the finding that there is an alpha-helix which connects the active with the interface site, thus allowing charge transference between them. (c) 2005 Elsevier B.V. All rights reserved.

Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation

Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrp1p is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using-functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrp1p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrp1p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.

Mapping eIF5A binding sites for Dys1 and Lia1: In vivo evidence for regulation of eIF5A hypusination

The evolutionarily conserved factor eIF5A is the only protein known to undergo hypusination, a unique posttranslational modification triggered by deoxyhypusine synthase (Dys1). Although eIF5A is essential for cell viability, the function of this putative translation initiation factor is still obscure. To identify eIF5A-binding proteins that could clarify its function, we screened a two-hybrid library and identified two eIF-5A partners in S. cerevisiae: Dys1 and the protein encoded by the gene YJR070C, named Lia1 (Ligand of eIF5A). The interactions were confirmed by GST pulldown. Mapping binding sites for these proteins revealed that both eIF5A domains can bind to Dys1, whereas the C-terminal domain is sufficient to bind Lia1. We demonstrate for the first time in vivo that the N-terminal α-helix of Dys1 can modulate enzyme activity by inhibiting eIF5A interaction. We suggest that this inhibition be abrogated in the cell when hypusinated and functional eIF5A is required. © 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites

Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Genomic regions harboring insecticide resistance-associated Cyp genes are enriched by transposable element fragments carrying putative transcription factor binding sites in two sibling Drosophila species

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Deconstructing the DGAT1 enzyme: Binding sites and substrate interactions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Albumin-induced circular dichroism in Congo red: applications for studies of amyloid-like fibril aggregates and binding sites

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nickel chloride: potential accumulation from the airway. Toxicokinetics in rats.

The rate removal of nickel from the airway was measured in vivo. Removal in vivo was studied by intratracheal injection of nickel chloride solutions. Regardless of time after injection, the lungs and heart retained the greatest concentration of nickel and 40 days after 1.68 mumol administration they were the organs where nickel was still significantly measurable. The slow removal of nickel may indicate the presence of high affinity binding sites in the lung. Nickel can interact with others metals, such as copper and zinc, so that nickel exposure may have public health implications.

Water regulation of actinomycin-D binding to DNA: the interplay among drug affinity, DNA long-range conformation, and hydration

Actiaomycin-D (actD) binds to natural DNA at two different classes of binding sites, weak and strong. The affinity for these sites is highly dependent on DNA se(sequence and solution conditions, and the interaction appears to be purely entropic driven Although the entropic character of this reaction has been attributed to the release of water molecules upon drug to DNA complex formation, the mechanism by which hydration regulates actD binding and discrimination between different classes of binding sites on natural DNA is still unknown. In this work, we investigate the role of hydration on this reaction using the osmotic stress method. We skew that the decrease of solution water activity, due to the addition of sucrose, glycerol ethylene glycol, and betaine, favors drug binding to the strong binding sites on DNA by increasing both the apparent binding affinity Delta G, and the number of DNA base pairs apparently occupied by the bound drug n(bp/actD). These binding parameters vary linearly with the logarithm of the molar fraction of water in solution log(X-w), which indicates the contribution of water binding to the energetic of the reaction. It is demonstrated that the hydration change measured upon binding increases proportionally to the apparent size of the binding site n(bp/uctD). This indicates that n(bp/actD) measured from the Scatchard plod is a measure of the size of the DNA molecule changing conformation due to ligand binding. We also find that the contribution of DNA deformation, gauged by n(bp/act) to the total free energy of binding Delta G, is given by Delta G = Delta G(local) + n(bp/actD) x delta G(DNA), where Delta G(local), = -8020 +/- 51 cal/mol of actD bound and delta G(DNa) = -24.1 +/- 1.7cal/mol of base pair at 25 degrees C. We interpret Delta G(local), as the energetic contribution due to the direct interactions of actD with the actual tetranucleotide binding site, and it n(bp/actB) X delta G(DNA) as that due to change inconformation, induced by binding, of it n(bp/actD) DNA base pairs flanking the local site. This interpretation is supported by the agreement found between the value of delta G(DNA) and the torsional free energy change measured independently. We conclude suggesting an allosteric model for ligand binding to DNA, such that the increase in binding affinity is achieved by increasing the relaxation of the unfavorable free energy of binding storage at the local site through a larger number of DNA base pairs. The new aspect on this model is that the size of the complex is not fixed but determined by solutions conditions, such as water activity, which modulate the energetic barrier to change helix conformation. These results may suggest that long-range allosteric transitions of duplex DNA are involved in the inhibition of RNA synthesis by actD, and more generally, in the regulation of transcription. (C) 2000 John Wiley & Sons, Inc.

CHARACTERISTICS OF ZINC-BINDING TO HUMAN RED-BLOOD-CELL MEMBRANES

The objective of the present study was to standardize the analysis of zinc binding on human red blood cell (RBC) membranes in 20 normal adults. The displacement studies revealed that at the maximal stable zinc concentration tested (600 muM), 57% (mean) of the bound Zn-65 was displaced and to displace half maximal Zn-65, the stable zinc concentration was 300 muM. Scatchard plots revealed two classes of binding sites for zinc on RBC membranes: one with higher affinity, Kd = 1.20 x 10(-5) M (site I), and the other with lower affinity, Kd = 2.77 x 10(-4) M (site II). Binding sites occupancy was 97% means and 58.5% means for sites I and 11, respectively. The displacement was affected by temperature, membrane protein concentration, freezing, thawing, and dialysis. Other metal cations, including Co++, Fe++, and Mn++, had very little effect on Zn-65 displacement, in contrast copper displaced Zn-65 from its binding sites on RBC membranes. Zinc binding to RBC membranes was rapid and readily reversible in a dynamic equilibrium with its binding sites. It is anticipated that this method will be applicable to studies of a wide variety of diseases specifically related to zinc metabolism in humans as well as in animals. (C) 1994 Wiley-Liss, Inc.

Charge distribution and calcium affinity of sulfated alpha-L-galactans from ascidians. Comparison between linear and highly branched polymers

Calcium binding and charge distribution on highly branched and linear sulfated L-galactans from ascidians have been studied using a metallochromic indicator and conductimetric titrations. The distance between charged groups of the linear and highly branched galactans does not vary despite their marked differences in sulfate/total sugar molar ratios. These results indicate that the sulfated L-galactose units are concentrated in the central polysaccharide core and not intercalated among non-sulfated units. This inference is consistent with the chemical studies of these galactans. Surprisingly, calcium affinity increases with increasing amounts of non-sulfated sugar branches in the molecule. Thus, calcium binding in these polymers is not a simple function of availability of anion binding sites but a more complex calcium-polysaccharide interaction. (C) 1998 Elsevier B.V. Ltd. All rights reserved.