Potential Cation Binding Sites Research Articles

Aqueous-Phase Synthesis of Copper Nanoparticles Using Organic Nanoparticles: Application of Assembly in Detection of Cr3+

A series of novel heterotripodal receptors were synthesized with potential cation binding sites. Their crystal structure revealed the presence of extensive intramolecular as well as intermolecular hydrogen bonding, which allowed them to process into organic nanoparticles (ONPs) in aqueous medium. The ONPs thus synthesized showed extraordinary ability for chromogenic recognition of Cu2+ ions, with the consequence that they could easily reduce Cu(II) into Cu(0) to produce copper nanocrystals (CuNCs) that grew on the surface of ONPs to generate hybrid nanomaterials, and were characterized by a number of studies. The hybrid NPs thus produced act as fluorescent probes for detection of Cr3+ in aqueous phase, whereas neither of its constituents (i.e., ONPs or CuNCs) showed this recognition behavior.

Solution structure of CyanoP from Synechocystis sp. PCC 6803: New insights on the structural basis for functional specialization amongst PsbP family proteins

The structure of the CyanoP subunit of photosystem II from the cyanobacterium Synechocystis sp. PCC 6803 has been determined in solution by Nuclear Magnetic Resonance spectroscopy. Combined with homology modeling of PsbP-like structures we have identified distinct structural differences between PsbP homologues which may account for the functional differences apparent between members of this protein family. A surface cleft containing a large number of conserved residues found only in CyanoP and PsbP-like homologues has been identified and our findings suggest that one of the potential cation binding sites found in CyanoP may be functionally significant. Evidence for the evolution and divergence of the PsbP super family is presented from a structural perspective including identification of residues which distinguish the PsbP family from unrelated proteins with a similar domain fold. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Observation of Water Molecules within the Bimolecular d(G3CT4G3C)2 G-Quadruplex

G-Rich oligonucleotides with cytosine residues in their sequences can form G-quadruplexes where G-quartets are flanked by G·C Watson-Crick base pairs. In an attempt to probe the role of cations in stabilization of a structural element with two G·C base pairs stacked on a G-quartet, we utilized solution state nuclear magnetic resonance to study the folding of the d(G(3)CT(4)G(3)C) oligonucleotide into a G-quadruplex upon addition of (15)NH(4)(+) ions. Its bimolecular structure exhibits antiparallel strands with edge-type loops. Two G-quartets in the core of the structure are flanked by a couple of Watson-Crick G·C base pairs in a sheared arrangement. The topology is equivalent to the solution state structure of the same oligonucleotide in the presence of Na(+) and K(+) ions [Kettani, A., et al. (1998) J. Mol. Biol.282, 619, and Bouaziz, S., et al. (1998) J. Mol. Biol.282, 637). A single ammonium ion binding site was identified between adjacent G-quartets, but three sites were expected. The remaining potential cation binding sites between G-quartets and G·C base pairs are occupied by water molecules. This is the first observation of long-lived water molecules within a G-quadruplex structure. The flanking G·C base pairs adopt a coplanar arrangement and apparently do not require cations to neutralize unfavorable electrostatic interactions among proximal carbonyl groups. A relatively fast movement of ammonium ions from the inner binding site to bulk with the rate constants of 21 s(-1) was attributed to the lack of hydrogen bonds between adjacent G·C base pairs and the flexibility of the T(4) loops.

The Effect of Site-Specific Modifications of DNA on Thermodynamic Stability, Ion Binding and Hydration

Cations, which associate with DNA in both the major and minor grooves, play a significant role in determining DNA conformation. In the major groove, cations are associated with the N7/O6 edge of guanines, while in the minor groove they are found at A-T pairs. Both G-C and A-T have potential cation binding sites that when modified should result in the reorganization of salts and water, which in turn would affect local conformation and stability. We report herein the biophysical characterization of DNA duplexes in which we altered the N-7 position in the major groove of purines (7-deaza-guanine, 7-aminomethyl-7-deazaguanine, 7-hydroxymethyl-7-deazaguanine and 7-deaza-adenine) and at N-3 position of adenine in the minor groove (3-deazaadenine and 3-methyl-3-deazaadenine). These modifications alter the electronic properties of the heterocyclic bases and specifically eliminate DNA cation binding sites in the different grooves, or in the case of 7-aminomethyl-7-deazaguanine places a cationic group in the major groove at the edge of a G-C pair. The low temperature NMR and x-ray crystal structures of some of these DNA appear identical to unmodified DNA; however, the thermodynamic analyses show that these modified bases have a significant impact on the dynamic structure of DNA. In most cases, a reduction in thermodynamic stability driven by enthalpy changes was observed. The only modification that is thermodynamically as, or more, stable than the corresponding unmodified DNA is the 7-aminomethyl-7-deaza-guanine. The thermodynamic effects of the different substitutions are associated with the folding enthalpies and hydration. Interpretation of how these base modifications affect DNA structure and stability will be discussed.Supported by RO1CA29088 from NIH and MCB-0315746 from NSF.

Cation localization and movement within DNA thrombin binding aptamer in solution

The thrombin binding aptamer, 15-mer oligonucleotide d[G(2)T(2)G(2)TGTG(2)T(2)G(2)], was folded into the well known antiparallel unimolecular G-quadruplex in the presence of (15)NH(4)(+) ions. Although the formed G-quadruplex is thermodynamically less stable than in the presence of K(+) ions, the loop conformations and folding topology are the same. On the other hand, titration of Na(+) ions into an aqueous solution of TBA resulted in the formation of one major and several minor species of G-quadruplexes. Solution-state NMR was used to localize (15)NH(4)(+) ions between the two G-quartets within the core of the structure, and to determine the equilibrium binding constant, which equals 190 M(-1). No other potential cation binding sites were resolved on the time-scale of NMR spectrometer. Exchange of (15)NH(4)(+) ions between the inner binding site and bulk solution is characterized by the exchange rate constant of 1.0 s(-1) at 15 degrees C. T4 and T13 form a noncanonical base pair, which greatly affects access of bulk ions into the cation binding site in the G-quadruplex core. G2 and G11 exhibit out of plane bending towards the two TT loops away from the bound (15)NH(4)(+) ions, which in turn exposes them to more efficient chemical exchange processes with bulk ions and water.

Identification of Functional Segments within the β2I-domain of Integrin αMβ2

The alpha(M)beta(2) integrin plays an important role in leukocyte biology through its interactions with a diverse set of ligands. Efficient ligand binding requires the involvement of both the alpha(M) and beta(2) subunits. Past ligand binding studies have focused mainly on the alpha(M) subunit, with the beta(2) subunit being largely unexplored. Therefore, in this study we conducted homolog-scanning mutagenesis on the I-domain (residues 125-385) within the beta(2) subunit. We identified four noncontiguous sequences (Arg(144)-Lys(148), Gln(199)-Ala(203), Leu(225)-Leu(230), and Gly(305)-His(309)) that are critical for fibrinogen and C3bi binding to alpha(M)beta(2). Molecular modeling revealed that these four sequences reside within a narrow region on the surface of the beta(2)I-domain, in close proximity to three potential cation-binding sites. Among these sequences, Gln(199)-Ala(203), Leu(225)-Leu(230), and Gly(305)-His(309) are important for the binding of both ligands, whereas Arg(144)-Lys(148) is more critical for fibrinogen than for C3bi binding. These sequences within the beta(2)I-domain are directly involved in ligand binding, since 1) switching these segments to their corresponding beta(1) sequences destroyed ligand binding; 2) loss of function was not due to a nonspecific gross conformational change, since the defective alpha(M)beta(2) mutants reacted well with a panel of conformation-dependent mAbs; 3) mutation of these functional sequences did not effect Ca(2+) binding; and 4) synthetic peptides corresponding to sequences Gln(199)-Ala(203) and Gly(305)-His(309) blocked ligand binding to alpha(M)beta(2), and the peptides interacted directly with fibrinogen and C3bi. Given the similarity among all integrin beta subunits, our results may help us to understand the underlying mechanism of integrin-ligand interactions in general.

Site-specific cation binding mediates TATA binding protein-DNA interaction from a hyperthermophilic archaeon.

Pyrococcus woesei (Pw) is a hyperthermophilic archaeal organism that exists under conditions of high salt and elevated temperature. In a previous study [O'Brien, R., DeDecker, B., Fleming, K., Sigler, P. B., and Ladbury, J. E., (1998) J. Mol. Biol. 279, 117-125], we showed that, despite the similarity of primary and secondary structure, the TATA box binding protein (TBP) from Pw binds thermodynamically in a fundamentally different way to its mesophilic counterparts. The affinity of the interaction increases as the salt concentration is increased. The formation of the protein-DNA complex involves the release of water and the uptake of ions, which were hypothesized to be cations. Here we test this hypothesis by selecting potential cation binding sites at negatively charged, acidic residues in the complex interface. These were substituted using site-directed mutagenesis of specific residues. Changes in the thermodynamic parameters on formation of the mutant protein-DNA complex were determined using isothermal titration calorimetry and compared to the wild type interaction. Removal of a glutamate residue from the binding site resulted in the uptake of one less cation on formation of the complex. This glutamate (E12) is directly involved in the binding of cations in the complex interface. Substitution of another acidic residue proximal to the DNA binding site (D101) had no effect on cation uptake, suggesting that the location of the amino acid on the protein surface is important in dictating the potential to coordinate cations. Removal of the cation binding site provided a more favorable entropy of binding; however, this effect is significantly reduced at higher salt concentrations. The removal of the cation binding site led to an increase in affinity with respect to the wild-type TBP at low salt concentrations.

Using meta computing tools to facilitate large-scale analyses of biological databases.

Given the high rate at which biological data are being collected and made public, it is essential that computational tools be developed that are capable of efficiently accessing and analyzing these data. High-performance distributed computing resources can play a key role in enabling large-scale analyses of biological databases. We use a distributed computing environment, Legion, to enable large-scale computations on the Protein Data Bank (PDB). In particular, we employ the Feature program to scan all protein structures in the PDB in search for unrecognized potential cation binding sites. We evaluate the efficiency of Legion's parallel execution capabilities and analyze the initial biological implications that result from having a site annotation scan of the entire PDB. We discuss four interesting proteins with unannotated, high-scoring candidate cation binding sites.

A structural motif for the voltage-gated potassium channel pore.

Mutation studies have identified a region of the S5-S6 loop of voltage-gated K+ channels (P region) responsible for teraethylammonium (TEA) block and permeation/selectivity properties. We previously modeled a similar region of the Na+ channel as four beta-hairpins with the C strands from each of the domains forming the external vestibule and with charged residues at the beta-turns forming the selectivity filter. However, the K+ channel P region amino acid composition is much more hydrophobic in this area. Here we propose a structural motif for the K+ channel pore based on the following postulates (Kv2.1 numbering). (i) The external TEA binding site is formed by four Tyr-380 residues; P loop residues participating in the internal TEA binding site are four Met-371 and Thr-372 residues. (ii) P regions form extended hairpins with beta-turns in sequence ITMT. (iii) only C ends of hairpins form the inner walls of the pore. (iv) They are extended nonregular strands with backbone carbonyl oxygens of segment VGYGD facing the pore with the conformation BRLRL. (v) Juxtaposition of P loops of the four subunits forms the pore. Fitting the external and internal TEA sites to TEA molecules predicts an hourglass-like pore with the narrowest point (GYG) as wide as 5.5 A, suggesting that selectivity may be achieved by interactions of carbonyls with partially hydrated K+. Other potential cation binding sites also exist in the pore.

Characterization of cation-binding sequences in the platelet integrin GPIIb-IIIa (alpha IIb beta 3) by terbium luminescence.

The binding of cations to purified GPIIb-IIIa (alpha IIb beta 3) and synthetic peptides corresponding to the potential cation-binding sites within this integrin has been assessed by terbium luminescence spectroscopy. Tb3+ supported fibrinogen binding to purified GPIIb-IIIa, at lower concentrations than Ca2+, consistent with its higher affinity for cation-binding motifs. Titration analyses indicated the presence of five Tb(3+)-binding sites of relatively high affinity in the receptor. These sites also could be filled by divalent cations. Six sequences within GPIIb-IIIa have the appropriate spacing of five of the usual six coordination sites for cations in functional Ca(2+)-binding EF-hand motifs. Peptides containing Tyr and/or Trp at selected positions as fluorescence energy donors were synthesized, and their Tb(3+)-binding capacity was assessed. The four potential Ca(2+)-binding sequences in the GPIIb subunit, GPIIb 242-255, 296-309, 364-377, and 425-438, were functional, despite lacking the usual Glu residue at the terminal coordination position. These peptides bound Tb3+ with the same affinity as typical Ca(2+)-binding loop peptides and also bound Ca2+ and other divalent cations without preference. Of the two candidate GPIIIa sequences, 118-131 and 208-221, the former bound Tb3+ and divalent cations with an affinity similar to that of the GPIIb peptides, whereas the latter peptide was not functional. This functional difference, as well as data obtained with substituted peptides, emphasizes the importance of the first coordination position for interaction of synthetic peptide loops with cations. Together, these data identify the five cation-binding sites within intact GPIIb-IIIa.