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PubMed2001

Mitochondrial adaptations to NaCl. Complex I is protected by anti-oxidants and small heat shock proteins, whereas complex II is protected by proline and betaine.

High soil sodium (Na) is a common stress in natural and agricultural systems. Roots are usually the first tissues exposed to Na stress and Na stress-related impairment of mitochondrial function is likely to be particularly important in roots. However, neither the effects of NaCl on mitochondrial function, nor its protection by several potential adaptive mechanisms, have been well studied. This study investigated the effects of NaCl stress on maize (Zea mays) mitochondrial electron transport and its relative protection by osmoprotectants (proline, betaine, and sucrose), antioxidants (ascorbate, glutathione, and alpha-tocopherol), antioxidant enzymes (catalase and Cu/Zn-superoxide dismutase), and mitochondrial small heat shock proteins (sHsps). We demonstrate that Complex I electron transport is protected by antioxidants and sHsps, but not osmoprotectants, whereas Complex II is protected only by low concentrations of proline and betaine. These results indicate that NaCl stress damaged Complex I via oxidative stress and suggests that sHsps may protect Complex I as antioxidants, but NaCl damaged Complex II directly. This is the first study to demonstrate that NaCl stress differentially affects Complex I and II in plants and that protection of Complex I and II during NaCl stress is achieved by different mechanisms.

Adaptation, Physiological↗

PubMed2000

Complexation of polyvalent cyclodextrin ions with oppositely charged guests: entropically favorable complexation due to dehydration.

Thermodynamic parameters for complexation of polyvalent cyclodextrin (CD) cation and anion with oppositely charged guests have been determined in D2O containing 0.02 M NaCl by means of 1H-NMR spectroscopy. Protonated heptakis(6-amino-6-deoxy)-beta-CD (per-NH3+-beta-CD) forms stable inclusion complexes with monovalent guest anions. The enthalpy (deltaH) and entropy changes (deltaS) for complexation of per-NH3+-beta-CD with p-methylbenzoate anion (p-CH3-Ph-CO2-) are 3.8 +/- 0.7 kJ mol(-1) and 88.6 +/- 2.2 J mol(-1) K(-1), respectively. The deltaH and deltaS values for the native beta-CD-p-CH3-Ph-CO2- system are -8.6 +/- 0.1 kJ mol(-1) and 15.3 +/- 0.7 J mol(-1) K(-1), respectively. The thermodynamic parameters clearly indicate that dehydration from both the host and guest ions accounts for the entropic gain in inclusion process of p-CH3-Ph-CO2- into the per-NH3+-beta-CD cavity. The fact that the neutral guests such as 2,6-dihydroxynaphthalene and p-methylbenzyl alcohol hardly form the complexes with per-NH3+-beta-CD exhibits that van der Waals and/or hydrophobic interactions do not cause the complexation of the polyvalent CD cation with the monovalent anion. The acetate anion is not included into the per-NH3+-beta-CD cavity, while the butanoate and hexanoate anions form the inclusion complexes. The complexation of the alkanoate anions is entropically dominated. Judging from these results, it may be concluded that Coulomb interactions cooperated with inclusion are required for realizing the large entropic gain due to extended dehydration. Entropically favorable complexation was also observed for the anionic CD-cationic guest system. The present study might present a general mechanism for ion pairing in water.

Anions↗

PubMed2004

Nucleophilic de-coordination and electrophilic regeneration of "hemilabile" pincer-type complexes: formation of anionic dialkyl, diaryl, and dihydride Pt(II) complexes bearing no stabilizing pi-acceptors.

Novel anionic dialkyl, diaryl, and dihydride platinum(II) complexes based on the new "long-arm" hemilabile PCN-type ligand C6H4[CH2P(tBu)2](CH2)2N(CH3)2 with the general formula Li+[Pt(PCN)(R)2]- (R=Me (4), Ph (6) and H (9)) were prepared by reaction of [Pt(PCN)(R)] complexes (obtained from the corresponding chlorides) with an equivalent of RLi, as a result of the opening of the chelate ring. Alkylating agents based on other metals produce less stable products. These anionic d8 complexes are thermally stable although they bear no stabilizing pi acceptors. They were characterized by 1H, 31P[1H], 13C, and 7Li NMR spectroscopy; complex 9 was also characterized by single crystal X-ray crystallography, showing that the Li+ ion is coordinated to the nitrogen atom of the open amine arm and to the hydride ligand (trans to the P atom) of a neighboring molecule (H--Li=2.15 A), resulting in a dimeric structure. Complexes 4 and 9 exhibit high nucleophilic reactivity, upon which the pincer complex is regenerated. Reaction of 4 with water, methyl iodide, and iodobenzene resulted in the neutral complex [Pt(PCN)(CH3)] (3) and methane, ethane, or toluene, respectively. Labeling studies indicate that the reaction proceeds by direct electrophilic attack on the metal center, rather than attack on the alkyl ligand. The anionic dihydride complex 9 reacted with water and methyl iodide to yield [Pt(PCN)(H)] (8) and H2 or methane, respectively.

Journal Article↗

PubMed2006

Oxidative DNA cleavage promoted by multinuclear copper complexes: activity dependence on the complex structure.

Polynuclear copper complexes with two or three Cu(BPA) (BPA, bis(2-pyridylmethyl)amine) motifs, [Cu2(mTPXA)Cl4]3 H2O (1), [Cu2(pTPXA)Cl4]3 H2O (2), [Cu3(HPTAB)Cl5]Cl3 H2O (3) (mTPXA = N,N,N',N'-tetra-(2-pyridylmethyl)-m-xylylene diamine; pTPXA = N,N, N',N'-tetra-(2-pyridylmethyl)-p-xylylenediamine; HPTAB = N,N,N',N',N'',N''-hexakis(2-pyridylmethyl)-1,3,5-tris-(aminomethyl)benzene) have been synthesized and characterized. The crystal structures of compounds 2 and 3 showed each Cu(BPA) motif had a 4+1 square-pyramidal coordination environment with one chloride occupying the apical position and three N atoms from the same BPA moiety together with another Cl atom forming the basal plane. Fluorescence and circular dichroism (CD) spectroscopy studies indicated that the DNA binding followed an order of 3>2>1 in the compounds. These complexes cleave plasmid pUC19 DNA by using an oxidative mechanism with mercaptopropionic acid (MPA) as the reductant under aerobic conditions. Dinuclear Cu2+ complexes 1 and 2 showed much higher cleavage efficiency than their mononuclear analogue [Cu(bpa)Cl2] at the same [Cu2+] concentration, suggesting a synergistic effect of the Cu2+ centers. Moreover, the meta-dicopper centers in complex 1 facilitated the formation of linear DNA. Interestingly, the additional copper center to the meta-dicopper motif in complex 3 decreased the cleavage efficacy of meta-dicopper motif in complex 1, although it is able to cleave DNA to the linear form at higher [Cu2+] concentrations. Therefore, the higher DNA binding ability of complex 3 did not lead to higher cleavage efficiency. These findings have been correlated to the DNA binding mode and the ability of the Cu2+ complexes to activate oxygen (O2). This work is a good example of the rational design of multinuclear Cu2+ artificial nuclease and the activity of which can be manipulated by the geometry and the number of metal centers.

Amines↗

PubMed1995

Identification of a complex between centrin and heat shock proteins in CSF-arrested Xenopus oocytes and dissociation of the complex following oocyte activation.

Coimmunoprecipitation experiments using a monoclonal anti-centrin antibody (20H5) and cytostatic factor (CSF)-arrested Xenopus oocyte extracts specifically precipitates oocyte centrin (20-kDa) and two associated proteins of 70- and 90-kDa. Microsequence analysis of a tryptic peptide fragment of the 70-kDa protein reveals 100% identity with a 13-amino-acid peptide sequence from Xenopus heat shock protein hsp-70. Western blot analysis of immunoprecipitates using anti-hsp monoclonal antibodies (N27 and AC-88) confirms the identity of the 70-kDa protein as hsp-70 and identifies the 90-kDa protein as hsp-90. The centrin/hsp complex is also immunoprecipitated when anti-hsp-70 or anti-hsp-90 monoclonal antibodies (BB70 and 4F3, respectively) are used as primary antibodies during immunoprecipitation. The centrin/hsp complex is sensitive to pH and Ca2+ concentration. The complex shows differential dissociation of hsp-70 and hsp-90 under a variety of conditions, suggesting that each hsp can bind to centrin independently of the other. When oocytes are first activated by electric shock or ionophore treatment, followed by immunoprecipitation using anti-centrin monoclonal antibody 20H5, centrin precipitates with significantly reduced levels of hsp-70 in the complex, and these complexes contain no apparent hsp-90. We conclude that, in CSF-arrested oocytes, the centrosomal protein, centrin, is associated as a complex with the heat shock proteins, hsp-70 and hsp-90, and that this complex dissociates upon activation of the oocyte. The functional consequences of the formation of complexes between centrin and these hsps are unknown. However, based on the roles that have been defined for heat shock proteins in other systems, several possibilities are suggested.

Amino Acid Sequence↗

PubMedDate not supplied

Intrinsically driven changes in gene interaction complexity. I. Growth of regulatory complexes and increase in number of genes.

A two-step process, previously considered in the literature, and here named coadaptational drive, is deemed to be largely responsible for both increases in the complexity of transcriptional control and increases in the total gene number, along lines of descent leading to more complex organisms. Coadaptational drive consists in a succession of modifications in the interaction among informational macromolecules, namely, structural decay spread by genetic drift and repair spread by selection. Increased genetic complexity, drawing on the opportunities offered by gene duplication, may be considered to be a secondary effect of such processes of decay and repair. The evolution toward higher regulatory complexity is thus considered to be obligatorily founded in part on random genetic drift. Increases in this complexity would represent primarily a trend intrinsic to the internal molecular environment, with the external environment having only to concur. Direct selection of mutations that increase complexity without the intervention of a phase of genetic drift is acknowledged likely to be a significant process as well, but it is claimed that a sequence of events of direct selection cannot be unlimited and will eventually stall, and that the roots of such a sequence ultimately are to be traced to an episode of coadaptational drive. Controller gene diseases, mostly mild, therefore seem to be essential for the evolution of increased biological complexity. The attempt is made to show or to confirm that (i) a conservative force (repair) provides a mechanism for the generation of novelty, (ii) a prominent part of selection, counterpart to Darwinian selection, originates from the internal environment and derives from the mechanics of genomic processes, and (iii) this selection is at times directional and leads to increases in complexity.

Animals↗

PubMed2000

Characterization of the human complex I NDUFB7 and 17.2-kDa cDNAs and mutational analysis of 19 genes of the HP fraction in complex I-deficient-patients.

Deficiency of NADH:ubiquinone oxidoreductase, the first enzyme complex of the mitochondrial respiratory chain, is one of the most frequent causes of human mitochondrial encephalomyopathies. A relatively small percentage of human complex I deficiency is associated with mitochondrial DNA mutations. cDNA characterization and mutational analysis of the structural complex I genes in 19 complex I-deficient patients, in whom common mtDNA mutations have been excluded, has so far revealed five patients with alterations in evolutionary conserved nuclear-encoded proteins. In order to complete our knowledge about the expected 36 structural nuclear complex I genes, we characterized the NDUFB7 and the 17.2-kDa cDNA sequences of the hydrophobic (HP) fraction of the complex. Subsequently, we screened all subunits of this fraction for the presence of mutations in those 14 patients of our initial patient cohort in whom the underlying genetic cause had not been elucidated. Strikingly, no pathogenic mutations were found in the HP subunits that would explain the complex I deficiency in our patients. Other strategies are needed to unravel proteins involved in the pathogenesis of the complicated cellular network of transcription until correct assemblage of complex I.

Amino Acid Sequence↗

PubMed1978

Studies on the ATPase complex from beef-heart mitochondria. I. Isolation and characterization of an oligomycin-sensitive and an olgiomycin-insensitive ATPase complex from beef-heart mitochondria.

1. A new method for the isolation of the oliogomycin-sensitive ATPase from beef-heart mitochondria is described. 2. A Triton-soluble ATPase complex was isolated as a by-product of the standard procedure, or as the main product when the submitochondrial particles were pretreated with 1% Triton. The ATPase activity of this complex is sensitive neither to oligomycin nor to dicyclohexylcarbodiimide. 3. The ATPase activity of the oligomycin-sensitive ATPase complex is nearly completely dependent on added phospholipids. The highest activation was found with asolectin. 4. The oligomycin-sensitive complex can be integrated into phospholipid vesicles resulting in an ATP- and Mg2+-dependent energization of the vesicles as monitored with the fluorescent dye 9-amino-6-chloro-2-methoxyacridine. 5. Aurovertin-binding studies based on fluorescence measurement reveal the presence of 1.5 mumol aurovertin-binding sites per g protein for the oligomycin-sensitive complex and about 2.2 mumol for the oligomycin-insensitive complex. 6. The preparation of the oligomycin-sensitive complex contains at least 6--7 polypeptides in addition to those derived from F1. One of these polypeptides, with an apparent molecular weight of 31 000, is virtually absent from the oligomycin-insensitive complex. 7. Some of these polypeptides have been identified and isolated.

Adenosine Triphosphatases↗

PubMed1990

Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 26. Mixed ligand complexes of cadmium, nitrilotriacetic acid, glutathione, and related ligands.

The complexation of glutathione and related ligands by the nitrilotriacetic acid complex of Cd2+ (Cd(NTA)-) has been investigated by 1H NMR as a model for the coordination chemistry of Cd2+ and GSH in biological systems. Related ligands included glycine, glutamic acid, cysteine, N-acetylcysteine, penicillamine, N-acetylpenicillamine, mercaptosuccinic acid, and the S-methyl derivative of glutathione. The nature of the complexes formed was deduced from 1H NMR spectra of Cd(NTA)- and the ligands. Mixed ligand complexes (Cd(NTA)L) and single ligand complexes (CdLx) are formed with the thiol ligands, whereas only mixed ligand complexes form with glycine, glutamic acid and S-methylglutathione. Formation constants of the mixed and the single ligand complexes were determined from NMR data. The results indicate that formation constants for binding of a thiolate donor group by Cd2+, either as the free ion or in a coordinately unsaturated complex, are in the range 10(5)-10(6).

Acetates↗

PubMed1991

Topographic distribution of the neurons of the central complex (centre médian-parafascicular complex) and of other thalamic neurons projecting to the striatum in macaques.

The distribution of the neurons of the central complex (or "centre médian-parafascicular complex") and of other thalamic regions projecting to the striatum was studied using a cartographic technique based on ventricular landmarks. The brain of a macaque was used as a reference for the cytoarchitectonic study of the complex. Three parts were isolated: the pars parafascicularis (or medial part), the pars media (or middle part) and the pars paralateralis (or lateral part). Wheat germ agglutinin conjugated to horseradish peroxidase was stereotaxically injected into either the sensorimotor or the associative territory of the striatum (i.e. the striatal space occupied by the axonal endings coming from either the sensorimotor or the associative cortex) of four macaques. Neurons projecting to the sensorimotor territory of the striatum were found to be located within the pars media (middle part) of the central complex while neurons projecting to the associative territory of the striatum were located within the pars parafascicularis. In all experimental cases, labelled neurons were scarce or absent in the pars paralateralis (or lateral part). Outside the central complex, neurons projecting to the sensorimotor territory of the striatum were scattered within the lateral part of the lateral mass, in the intralaminar nuclei and in the posterior part of the internal lamina. Neurons projecting to the associative territory of the striatum were observed mainly in the paraventricular region, dorsal to the rostral part of the lateral mass, and in the dorsolateral part of the nucleus oralis medialis. Our three-dimensional analysis of the clusters of the central complex cells projecting to the two striatal territories justifies the partitioning of the central complex into three parts. The pars media (or middle part), which projects to the sensorimotor territory of the striatum, receives selectively pallidal afferent axons. It belongs to the Nauta-Mehler loop, a closed loop linking the central complex to the basal ganglia. The pars parafascicularis, which projects to the associative territory of the striatum, seems more related to oculomotor neuronal systems. The pars paralateralis (or lateral part) appears to have very little, if any, relation with the striatum.

Afferent Pathways↗

PubMed2005

Binding of (MTR)2Zn2+ complex to chromatin: a comparison with (MTR)2Mg2+ complex.

Mithramycin (MTR), a member of aureolic group of anticancer antibiotic, binds reversibly to double stranded DNA via minor groove with (G.C) base specificity. It leads to inhibition of replication and transcription. Results from different laboratories have shown that at and above physiological pH, Mg2+ is an obligatory factor for the DNA binding and subsequent transcription inhibitory property of mithramycin. Zn2+ is another physiologically important bivalent cation. Its coordination property leads to its important role as a cofactor in different enzymes and nucleosomal DNA binding proteins. Characterization of the complex between mithramycin and Zn2+ using spectroscopic methods shows that the drug forms single type of complex with Zn2+ in the mole ratio of 2:1 in terms of antibiotic: Zn2+. DNA binding properties of the (MTR)2Zn2+ complex has been studied using calf thymus DNA, rat liver chromatin and nucleosome core. (MTR)2Zn2+ complex binds to calf thymus DNA with affinity higher than the corresponding dimer complex with Mg2+ ion. The presence of histone proteins in chromatin and nucleosome reduces the accessibility and hence binding potential of (MTR)2Zn2+ complex to nucleosomal DNA. We have also examined the effect of (MTR)2Zn2+ complex upon the stability of nucleosome core particle. The complex disassembles nucleosome structure leading to the release of nucleosomal DNA. Significance of the results to understand the molecular basis of the action of the drug in vivo is discussed.

Animals↗

PubMed2000

Mn2+, Co2+, Cu2+ and Zn2+ complexes with two macrocyclic ligands bearing L-lactate-like functions: potentiometric studies and evaluation of superoxide-scavenging properties of the Mn2+ complex.

Some aerobic organisms devoid of SOD use Mn2+ chelates to scavenge the O2- radical. Since the Mn2+-bis(lactato)diaquo complex is known as having a high SOD-like activity, we prepared manganese(II) complexes with triazamacrocyclic ligands bearing L-lactate-like functions in order to obtain model compounds able to disproportionate the superoxide radical. Thus, two macrocyclic ligands, N,N',N"-tris[2(S)-hydroxybutyric acid]-1,4,7-triazacyclononane, L1, and N,N',N"-tris[2(S)-hydroxybutyric acid]-1,5,9-triazacyclododecane, L2, were prepared and their capacity to retain the Mn2+ ion in aqueous solution was determined from potentiometric experiments. The chelating properties in aqueous solution of each ligand towards Co2+, Cu2+ and Zn2+ ions were also determined. L1 forms complexes with Mn2+, Co2+, Cu2+ and Zn2+ ions with stability constants of 8.33(5), 15.78(5), 17.65(3) and 14.32(1), respectively. L2 forms complexes with Cu2+ and Zn2+ ions with stability constants of 10.67(1) and 6.98(3), respectively. But the constants related to the Mn2+ and Co2+ complexes were too low to be determined by the method used. The stability constants values calculated for L2 complexes are significantly lower than those for the corresponding complexes of L1. Additional spectroscopic measurements were carried out on the Mn2+-L1 system. The electronic spectrum of this system showed a pH-dependence that may be consistent with the formation of hydroxo-species as the ESR spectra recorded at 120 K did not show oxidation of the Mn2+ ion in the pH range studied. The superoxide-scavenging activity of the manganese(II)-L1 complex was investigated using the cytochrome c assay. The Mn2+-L1 system showed an IC50 value of 1.7 microM which indicates that it appears as a potent SOD mimic.

Cations, Divalent↗

PubMed2002

Biological activity of palladium(II) and platinum(II) complexes of the acetone Schiff bases of S-methyl- and S-benzyldithiocarbazate and the X-ray crystal structure of the [Pd(asme)2] (asme=anionic form of the acetone Schiff base of S-methyldithiocarbazate) complex.

Palladium(II) and platinum(II) complexes of general empirical formula, [M(NS)(2)] (NS=uninegatively charged acetone Schiff bases of S-methyl- and S-benzyldithiocarbazate; M=Pt(II) and Pd(II)) have been prepared and characterized by a variety of physicochemical techniques. Based on conductance, IR and electronic spectral evidence, a square-planar structure is assigned to these complexes. The crystal and molecular structure of the [Pd(asme)(2)] complex (asme=anionic form of the acetone Schiff base of S-methyldithiocarbazate) has been determined by X-ray diffraction. The complex has a distorted cis-square planar structure with the ligands coordinated to the palladium(II) ions as uninegatively charged bidentate NS chelating agents via the azomethine nitrogen and the mercaptide sulfur atoms. The distortion from a regular square-planar geometry is attributed to the restricted bite angles of the ligands. Antimicrobial tests indicate that the Schiff bases exhibit strong activities against the pathogenic bacteria, Bacillus subtilis (mutant defective DNA repair), methicillin-resistant Staphylococcus aureus, B. subtilis (wild type) and Pseudomonas aeruginosa and the fungi, Candida albicans (CA), Candida lypotica (2075), Saccharomyces cerevisiae (20341) and Aspergillus ochraceous (398)-the activities exhibited by these compounds being greater than that of the standard antibacterial and antifungal drugs, streptomycin and nystatin, respectively. The palladium(II) and platinum(II) complexes are inactive against most of these organisms but, the microbe, Pseudomonas aeruginosa shows strong sensitivity to the platinum(II) complexes. Screening of the compounds for their cytotoxicities against T-lymphoblastic leukemia cancer cells has shown that the acetone Schiff base of S-methyldithiocarbazate (Hasme) exhibits a very weak activity, whereas the S-benzyl derivative (Hasbz) is inactive. However, the palladium(II) complexes exhibit strong cytotoxicities against this cancer; their activities being more than that of the standard anticancer drug, tamoxifen. The [Pt(asme)(2)] complex exhibits a very weak cytotoxicity, whereas [Pt(asbz)(2)] is inactive against leukemic cells.

Acetone↗

PubMed1999

Redox characteristics of Schiff base manganese and cobalt complexes related to water-oxidizing complex of photosynthesis.

In an effort to obtain synthetic analogues of water-oxidizing complex (WOC) of photosystem II (PS II) of plant photosynthesis, a Schiff base manganese and a cobalt complex, employing Niten, a SALEN type ligand, have been prepared. Cyclic and square wave voltammetric measurements have been performed to assess their redox characteristics. Both complexes undergo several reduction processes in cathodic negative potential region at more or less similar potentials. In view of these reductions being independent of the nature of the metal, they are thought to be ligand-localized. Although similar in negative region, a marked difference in the behavior of the complexes is observed in anodic region. While the cobalt complex is electrochemically inactive in the positive potentials up to +1.0 V vs. Ag/AgCl, the manganese complex displays two oxidation waves at +0.25 and +0.5 V vs. Ag/AgCl. The presence of oxidation wave in manganese complex at +0.5 V vs. Ag/AgCl or +0.7 V vs. NHE suggests that this complex can catalyze the oxidation of water and can, thus, simulate the WOC of PS II.

Cobalt↗

PubMed1997

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP.

BACKGROUND: Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP) with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution. RESULTS: The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G.A mismatches are flanked by sheared G.A and reversed Hoogsteen G.G mismatch pairs. CONCLUSIONS: The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G.A mismatch formation. The recognition G.A mismatch stacks with a reversed Hoogsteen G.G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10(14) molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.

Adenosine Monophosphate↗

PubMed2002

Structural and functional properties of the HIV-1 RNA-tRNA(Lys)3 primer complex annealed by the nucleocapsid protein: comparison with the heat-annealed complex.

The conversion of the single-stranded RNA genome into double-stranded DNA by virus-coded reverse transcriptase (RT) is an essential step of the retrovirus life cycle. In human immunodeficiency virus type 1 (HIV-1), RT uses the cellular tRNA(Lys)3 to initiate the (-) strand DNA synthesis. Placement of the primer tRNA(Lys)3 involves binding of its 3'-terminal 18 nt to a complementary region of genomic RNA termed PBS. However, the PBS sequence is not the unique determinant of primer usage and additional contacts are important. This placement is believed to be achieved in vivo by the nucleocapsid domain of Gag or by the mature protein NCp. Up to now, structural information essentially arose from heat-annealed primer-template complexes (Isel et al., J Mol Biol, 1995, 247:236-250; Isel et al., EMBO J, 1999, 18:1038-1048). Here, we investigated the formation of the primer-template complex mediated by NCp and compared structural and functional properties of heat- and NCp-annealed complexes. We showed that both heat- and NCp-mediated procedures allow comparable high yields of annealing. Then, we investigated structural features of both kinds of complexes by enzymatic probing, and we compared their relative efficiency in (-) strong stop DNA synthesis. We did not find any significant differences between these complexes, suggesting that information derived from the heat-annealed complex can be transposed to the NCp-mediated complex and most likely to complexes formed in vivo.

Base Pairing↗

PubMed1994

Sequence specificity of quinoxaline antibiotics. 1. Solution structure of a 1:1 complex between triostin A and [d(GACGTC)]2 and comparison with the solution structure of the [N-MeCys3,N-MeCys7]TANDEM-[d(GATATC)]2 complex.

Triostin A, a naturally occurring quinoxaline antibiotic that contains N-methyl groups on the valine and cysteine residues, binds sequence specifically to DNA at NCGN sites. [N-MeCys3,N-MeCys7]-TANDEM (CysMeTANDEM), a synthetic quinoxaline antibiotic, differs in its chemical structure from triostin A only at the valine residues, which contain no N-methyl substituents. CysMeTANDEM has a sequence specificity different from triostin A, binding specifically to DNA at NTAN sites. To understand the factors that determine the sequence specificity of these quinoxaline antibiotics, the solution structure of a 1:1 complex of triostin A with the DNA hexamer [d(GACGTC)]2 has been determined using NMR-derived distance and dihedral angle restraints. The solution structure of the triostin A-[d(GACGTC)]2 complex is compared directly to the solution structure of a 1:1 complex of CysMeTANDEM with [d(GATATC)]2 and is also compared to the crystal structure of 2:1 complex of triostin A with [d(CGTACG)]2. Triostin A binds to [d(GACGTC)]2 as a bis-intercalator around the CpG step, and the peptide ring of the drug binds in the minor groove of the DNA. The central C.G base pairs of the complex are underwound with an average helical twist angle of approximately -9.0 degrees and buckle inward by about 25 degrees. There are intermolecular hydrogen bonds between each of the Ala NH and the GN3 protons of the CpG binding site. Similar structural features are observed in the solution structure of the CysMeTANDEM-[d(GATATC)]2 complex. However, in the structure of the triostin A-[d(GACGTC)]2 complex, two intermolecular hydrogen bonds between each of the Ala CO oxygens of the drug and the 2-amino protons of guanine are observed. These hydrogen bonds do not form in the CysMeTANDEM-DNA complex. Instead, CysMeTANDEM contains two intramolecular hydrogen bonds between the Ala CO atoms and the Val amide protons, making the Ala CO atoms unavailable to form two intermolecular hydrogen bonds. The role of these intermolecular hydrogen bonds in the CpG specificity of triostin A is discussed.

Anti-Bacterial Agents↗

PubMed1985

Active site specific cadmium(II)-substituted horse liver alcohol dehydrogenase: crystal structures of the free enzyme, its binary complex with NADH, and the ternary complex with NADH and bound p-bromobenzyl alcohol.

Three crystal structures have been determined of active site specific substituted Cd(II) horse liver alcohol dehydrogenase and its complexes. Intensities were collected for the free, orthorhombic enzyme to 2.4-A resolution and for a triclinic binary complex with NADH to 2.7-A resolution. A ternary complex was crystallized from an equilibrium mixture of NAD+ and p-bromobenzyl alcohol. The microspectrophotometric analysis of these single crystals showed the protein-bound coenzyme to be largely NADH, which proves the complex to consist of CdII-LADH, NADH, and p-bromobenzyl alcohol. Intensity data for this abortive ternary complex were collected to 2.9-A resolution. The coordination geometry in the free Cd(II)-substituted enzyme is highly similar to that of the native enzyme. Cd(II) is bound to Cys-46, Cys-174, His-67, and a water molecule in a distorted tetrahedral geometry. Binding of coenzymes induces a conformational change similar to that in the native enzyme. The interactions between the coenzyme and the protein in the binary and ternary complexes are highly similar to those in the native ternary complexes. The substrate binds directly to the cadmium ion in a distorted tetrahedral geometry. No large, significant structural changes compared to the native ternary complex with coenzyme and p-bromobenzyl alcohol were found. The implications of these results for the use of active site specific Cd(II)-substituted horse liver alcohol dehydrogenase as a model system for the native enzyme are discussed.

Alcohol Dehydrogenase↗