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Author (up) Alonso, A.; Almendral, M.J.; Curto, Y.; Criado, J.J.; Rodriguez, E.; Manzano, J.L. file  url
openurl 
  Title Determination of the DNA-binding characteristics of ethidium bromide, proflavine, and cisplatin by flow injection analysis: usefulness in studies on antitumor drugs Type Journal Article
  Year 2006 Publication Analytical Biochemistry Abbreviated Journal Anal Biochem  
  Volume 355 Issue 2 Pages 157-164  
  Keywords Antineoplastic Agents--chemistry, metabolism; Binding Sites; Cisplatin--chemistry, metabolism; DNA--chemistry, metabolism; Ethidium--chemistry, metabolism; Flow Injection Analysis--methods; Fluorescent Dyes; Kinetics; Nucleic Acid Conformation; Proflavine--chemistry, metabolism; Spectrometry, Fluorescence  
  Abstract Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.  
  Call Number Serial 363  
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Author (up) Baylis, H.A.; Furuichi, T.; Yoshikawa, F.; Mikoshiba, K.; Sattelle, D.B. file  url
openurl 
  Title Inositol 1,4,5-trisphosphate receptors are strongly expressed in the nervous system, pharynx, intestine, gonad and excretory cell of Caenorhabditis elegans and are encoded by a single gene (itr-1) Type Journal Article
  Year 1999 Publication Journal of Molecular Biology Abbreviated Journal J Mol Biol  
  Volume 294 Issue 2 Pages 467-476  
  Keywords Amino Acid Sequence; Animals; Animals, Genetically Modified; Binding Sites; Caenorhabditis elegans/*genetics; Calcium Channels/*genetics/*metabolism; Cell Membrane/genetics/metabolism; Conserved Sequence; Gene Expression Profiling; Gonads/metabolism; Helminth Proteins/*genetics/*metabolism; Inositol 1,4,5-Trisphosphate Receptors; Intestines/metabolism; Molecular Sequence Data; Nervous System/metabolism; Pharynx/metabolism; RNA, Messenger; Receptors, Cytoplasmic and Nuclear/*genetics/*metabolism; Rectum/cytology/metabolism  
  Abstract Inositol 1,4,5-trisphosphate (InsP3) activates receptors (InsP3Rs) that mediate intracellular Ca(2+ )release, thereby modulating intracellular calcium signals and regulating important aspects of cellular physiology and gene expression. To further our understanding of InsP3Rs we have characterised InsP3Rs and the InsP3R gene, itr-1, from the model organism Caenorhabditis elegans. cDNAs encoding InsP3Rs were cloned enabling us to: (a) identify three putative transcription start sites that result in alternative mRNA 5' ends: (b) detect alternative splicing at three sites and: (c) determine the full genomic organisation of the itr-1 gene. The InsP3R protein (ITR-1) is approximately 42 % identical with known InsP3Rs and possesses conserved structural features. When the putative InsP3 binding domain was expressed in Escherichia coli, specific binding of InsP3 was detected. Using antibodies against ITR-1 we detected a protein of 220 kDa in C. elegans membranes. These antibodies and itr-1::GFP (green fluorescent protein) reporter constructs were used to determine the expression pattern of itr-1 in C. elegans. Strong expression was observed in the intestine, pharynx, nerve ring, excretory cell and gonad. These results demonstrate the high degree of structural and functional conservation of InsP3Rs from nematodes to mammals and the utility of C. elegans as a system for studies on InsP3R mediated signalling.  
  Call Number Serial 309  
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Author (up) Bertrand, D.; Gopalakrishnan, M. file  url
openurl 
  Title Allosteric modulation of nicotinic acetylcholine receptors Type Journal Article
  Year 2007 Publication Biochemical Pharmacology Abbreviated Journal Biochem Pharmacol  
  Volume 74 Issue 8 Pages 1155-1163  
  Keywords Allosteric Regulation; Animals; Binding Sites; Dose-Response Relationship, Drug; Humans; Receptors, Nicotinic/*chemistry/*drug effects; alpha7 Nicotinic Acetylcholine Receptor  
  Abstract Allosteric modulation refers to the concept that proteins could exist in multiple conformational states and that binding of allosteric ligands alters the energy barriers or “isomerization coefficients” between various states. In the context of ligand gated ion channels such as nicotinic acetylcholine receptors (nAChRs), it implies that endogenous ligand acetylcholine binds at the orthosteric site, and that molecules that bind elsewhere on the nAChR subunit(s) acts via allosteric interactions. For example, studies with the homomeric alpha7 nAChRs indicate that such ligand interactions can be well described by an allosteric model, and that positive allosteric effectors can affect energy transitions by (i) predominantly affecting the peak current response (Type I profile) or, (ii) both peak current responses and time course of agonist-evoked response (Type II profile). The recent discovery of chemically heterogeneous group of molecules capable of differentially modifying nAChR properties without interacting at the ligand binding site illustrates the adequacy of the allosteric model to predict functional consequences. In this review, we outline general principles of the allosteric concept and summarize the profiles of novel compounds that are emerging as allosteric modulators at the alpha7 and alpha4beta2 nAChR subtypes.  
  Call Number Serial 1877  
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Author (up) Bertrand, D.; Gopalakrishnan, M. file  url
openurl 
  Title Allosteric modulation of nicotinic acetylcholine receptors Type Journal Article
  Year 2007 Publication Biochemical Pharmacology Abbreviated Journal Biochem Pharmacol  
  Volume 74 Issue 8 Pages 1155-1163  
  Keywords Allosteric Regulation; Animals; Binding Sites; Dose-Response Relationship, Drug; Humans; Receptors, Nicotinic/*chemistry/*drug effects; alpha7 Nicotinic Acetylcholine Receptor  
  Abstract Allosteric modulation refers to the concept that proteins could exist in multiple conformational states and that binding of allosteric ligands alters the energy barriers or “isomerization coefficients” between various states. In the context of ligand gated ion channels such as nicotinic acetylcholine receptors (nAChRs), it implies that endogenous ligand acetylcholine binds at the orthosteric site, and that molecules that bind elsewhere on the nAChR subunit(s) acts via allosteric interactions. For example, studies with the homomeric alpha7 nAChRs indicate that such ligand interactions can be well described by an allosteric model, and that positive allosteric effectors can affect energy transitions by (i) predominantly affecting the peak current response (Type I profile) or, (ii) both peak current responses and time course of agonist-evoked response (Type II profile). The recent discovery of chemically heterogeneous group of molecules capable of differentially modifying nAChR properties without interacting at the ligand binding site illustrates the adequacy of the allosteric model to predict functional consequences. In this review, we outline general principles of the allosteric concept and summarize the profiles of novel compounds that are emerging as allosteric modulators at the alpha7 and alpha4beta2 nAChR subtypes.  
  Call Number Serial 1887  
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Author (up) Giles, G.I.; Sharma, R.P. file  url
openurl 
  Title Topoisomerase enzymes as therapeutic targets for cancer chemotherapy Type Journal Article
  Year 2005 Publication Medicinal Chemistry (Shariqah (United Arab Emirates)) Abbreviated Journal Med Chem  
  Volume 1 Issue 4 Pages 383-394  
  Keywords Antineoplastic Agents/pharmacology/*therapeutic use; Binding Sites; *Drug Delivery Systems; *Drug Therapy; Enzyme Inhibitors/pharmacology/therapeutic use; Humans; Models, Molecular; Neoplasms/*drug therapy; *Topoisomerase I Inhibitors; *Topoisomerase II Inhibitors  
  Abstract The topoisomerase enzymes are essential for DNA metabolism, where they act to adjust the number of supercoils in DNA, a key requirement in the cellular processes of transcription and replication. Their enzymatic mechanism creates transient nicks (type I) or breaks (type II) in the double stranded DNA polymer, allowing DNA to be converted between topological isomers. Humans possess both types of topoisomerase enzymes, however the two types utilize very different enzymatic mechanisms. Both type I and type II topoisomerases have been identified as clinically important targets for cancer chemotherapy and their inhibitors are central components in many therapeutic regimes. Over the course of the last 30 years inhibitors with extensive structural diversity have been developed through a combination of drug screening and rational design programs. Simultaneously much emphasis has been placed upon establishing the mechanisms of action of both classes of topoisomerase enzyme. Crucial structural insights have come from the crystal structure of topoisomerase I, while modelling comparisons are beginning to map out a possible framework for topoisomerase II action. This review discusses these recent advances in the fields of enzyme mechanism and inhibitor design. We also address the development of drug resistance and dose-limiting side effects as well as cover alternative methods in drug delivery.  
  Call Number Serial 200  
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Author (up) Hamada, T.; Sakube, Y.; Ahnn, J.; Kim, D.H.; Kagawa, H. file  url
openurl 
  Title Molecular dissection, tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans Type Journal Article
  Year 2002 Publication Journal of Molecular Biology Abbreviated Journal J Mol Biol  
  Volume 324 Issue 1 Pages 123-135  
  Keywords Amino Acid Sequence; Animals; Binding Sites; Caenorhabditis elegans/genetics/*metabolism; Caenorhabditis elegans Proteins/genetics/*metabolism; Calcium/*metabolism; Cross Reactions; Gene Expression Regulation, Developmental; Molecular Sequence Data; Muscles/*metabolism; Mutation; Recombinant Proteins/genetics/immunology/metabolism; Ryanodine Receptor Calcium Release Channel/genetics/immunology/*metabolism; Sequence Homology, Amino Acid  
  Abstract The ryanodine receptor of Caenorhabditis elegans (CeRyR) which contains 5,071 amino acid residues, is encoded by a single gene, ryr-1/unc-68. The unc-68(kh30) mutation, isolated in an animal showing abnormal response to the anesthetic ketamine, has the substitution Ser1444Asn in CeRyR, predicted to be a phosphorylation site. To elucidate the function of the region of CeRyR, and to determine the localization of CeRyR in this animal, ten region-peptides were produced in Escherichia coli by using expression plasmids and eight antisera were raised against these fusion peptides. One antibody against the region corresponding to the kh30 mutation site enabled detection of CeRyR from mutant animals both in Western analysis and in situ. Specificity of this antiserum was demonstrated using Western analysis, which showed the full size and the partial size bands in wild-type and in the Tc1-induced deletion mutant animals, respectively, but no corresponding bands in unc-68 null mutant animals. CeRyR was detected in I-bands of muscle sarcomeres by double immunostaining. CeRyR was found in the body wall, pharyngeal, vulval, anal and sex muscles of adult worms and also found to be present in embryonic muscle, but not in non-muscle cells. Two EF-hand motifs and the C terminus were demonstrated to be Ca(2+) binding regions. On the basis of these results, we propose a model for the functional domains of CeRyR, which agrees well with the model of mammalian skeletal RyR, which is based on proteolysis and cross-linking analysis. We discuss the usefulness and limitations of the molecular dissection approach, which uses peptides and peptide-specific antibodies to determine the local structure and function of individual domains within a large molecule.  
  Call Number Serial 447  
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Author (up) Hassaine, G.; Deluz, C.; Grasso, L.; Wyss, R.; Tol, M.B.; Hovius, R.; Graff, A.; Stahlberg, H.; Tomizaki, T.; Desmyter, A.; Moreau, C.; Li, X.-D.; Poitevin, F.; Vogel, H.; Nury, H. file  url
openurl 
  Title X-ray structure of the mouse serotonin 5-HT3 receptor Type Journal Article
  Year 2014 Publication Nature Abbreviated Journal Nature  
  Volume 512 Issue 7514 Pages 276-281  
  Keywords Amino Acid Sequence; Animals; Binding Sites; Crystallography, X-Ray; Mice; Models, Molecular; Molecular Sequence Data; Neurotransmitter Agents/metabolism; Protein Structure, Quaternary; Protein Structure, Tertiary; Protein Subunits/chemistry/metabolism; Receptors, Serotonin, 5-HT3/*chemistry/metabolism  
  Abstract Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structure of a mammalian Cys-loop receptor, the mouse serotonin 5-HT3 receptor, at 3.5 A resolution. The structure of the proteolysed receptor, made up of two fragments and comprising part of the intracellular domain, was determined in complex with stabilizing nanobodies. The extracellular domain reveals the detailed anatomy of the neurotransmitter binding site capped by a nanobody. The membrane domain delimits an aqueous pore with a 4.6 A constriction. In the intracellular domain, a bundle of five intracellular helices creates a closed vestibule where lateral portals are obstructed by loops. This 5-HT3 receptor structure, revealing part of the intracellular domain, expands the structural basis for understanding the operating mechanism of mammalian Cys-loop receptors.  
  Call Number Serial 1007  
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Author (up) Huang, Y.; Lu, Z.; Liu, N.; Chen, Y. file  url
openurl 
  Title Identification of important residues in diketoreductase from Acinetobacter baylyi by molecular modeling and site-directed mutagenesis Type Journal Article
  Year 2012 Publication Biochimie Abbreviated Journal Biochimie  
  Volume 94 Issue 2 Pages 471-478  
  Keywords 3-Hydroxyacyl CoA Dehydrogenases/chemistry/genetics/metabolism; Acinetobacter/*enzymology/genetics; Amino Acid Motifs; Amino Acid Substitution; Bacterial Proteins/*chemistry/genetics/metabolism; Binding Sites; Biocatalysis; Esters/*metabolism; Humans; Hydrogen-Ion Concentration; Hydroxymethylglutaryl-CoA Reductase Inhibitors/metabolism; Kinetics; Molecular Dynamics Simulation; Molecular Sequence Data; Mutagenesis, Site-Directed; NAD/metabolism; Oxidation-Reduction; Oxidoreductases/*chemistry/genetics/metabolism; Protein Structure, Tertiary; Recombinant Proteins/chemistry/genetics/metabolism; Sequence Homology, Amino Acid; Stereoisomerism  
  Abstract Diketoreductase (DKR) from Acinetobacter baylyi exhibits a unique property of double reduction of a beta, delta-diketo ester with excellent stereoselectivity, which can serve as an efficient biocatalyst for the preparation of an important chiral intermediate for cholesterol lowering statin drugs. Taken the advantage of high homology between DKR and human heart 3-hydroxyacyl-CoA dehydrogenase (HAD), a molecular model was created to compare the tertiary structures of DKR and HAD. In addition to the possible participation of His-143 in the enzyme catalysis by pH profile, three key amino acid residues, Ser-122, His-143 and Glu-155, were identified and mutated to explore the possibility of involving in the catalytic process. The catalytic activities for mutants S122A/C, H143A/K and E155Q were below detectable level, while their binding affinities to the diketo ester substrate and cofactor NADH did not change obviously. The experimental results were further supported by molecular docking, suggesting that Ser-122 and His-143 were essential for the proton transfer to the carbonyl functional groups of the substrate. Moreover, Glu-155 was crucial for maintaining the proper orientation and protonation of the imidazole ring of His-143 for efficient catalysis.  
  Call Number Serial 1415  
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Author (up) Narayana, N.; Matthews, D.A.; Howell, E.E.; Nguyen-huu, X. file  url
openurl 
  Title A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site Type Journal Article
  Year 1995 Publication Nature Structural Biology Abbreviated Journal Nat Struct Biol  
  Volume 2 Issue 11 Pages 1018-1025  
  Keywords Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Folic Acid Antagonists/chemistry/metabolism; Models, Molecular; Molecular Sequence Data; NADP/chemistry/metabolism; Plasmids/*genetics; Protein Conformation; Recombinant Proteins/chemistry; Tetrahydrofolate Dehydrogenase/*chemistry/genetics; Trimethoprim/chemistry/metabolism; Trimethoprim Resistance/*genetics  
  Abstract Bacteria expressing R67-plasmid encoded dihydrofolate reductase (R67 DHFR) exhibit high-level resistance to the antibiotic trimethoprim. Native R67 DHFR is a 34,000 M(r) homotetramer which exists in equilibrium with an inactive dimeric form. The structure of native R67 DHFR has now been solved at 1.7 A resolution and is unrelated to that of chromosomal DHFR. Homotetrameric R67 DHFR has an unusual pore, 25 A in length, passing through the middle of the molecule. Two folate molecules bind asymmetrically within the pore indicating that the enzyme's active site consists of symmetry related binding surfaces from all four identical units.  
  Call Number Serial 1208  
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Author (up) Short, C.A.; Cao, A.T.; Wingfield, M.A.; Doers, M.E.; Jobe, E.M.; Wang, N.; Levandoski, M.M. file  url
openurl 
  Title Subunit interfaces contribute differently to activation and allosteric modulation of neuronal nicotinic acetylcholine receptors Type Journal Article
  Year 2015 Publication Neuropharmacology Abbreviated Journal Neuropharmacology  
  Volume 91 Issue Pages 157-168  
  Keywords Animals; Binding Sites; Cysteine/chemistry; Neurons/*metabolism; Nicotinic Agonists/*metabolism; Oxidation-Reduction; Protein Subunits/*chemistry/*metabolism; Rats; Receptors, Nicotinic/*chemistry/*metabolism; Xenopus; Allosteric regulation; Cys-loop receptors; Cysteine-mediating cross-linking; Interface; Mutagenesis site specific; Nicotinic acetylcholine receptors; Oocyte  
  Abstract Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the beta(+)/alpha(-) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of alpha3beta2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair alpha3Y168-beta2D190, involving the C loop region of the beta2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair alpha3S125-beta2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs alpha3Q37-beta2A127 and alpha3E173-beta2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation.  
  Call Number Serial 1890  
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