Cover image for ChemBioChem

August 5, 2005

Volume 6, Issue 8

Pages 1297–1470

    1. Cover Picture: Fluorescence-Based Cloning of a Protein Tyrosine Kinase with a Yeast Tribrid System (ChemBioChem 8/2005) (page 1297)

      Daniel D. Clark and Blake R. Peterson

      Article first published online: 29 JUL 2005 | DOI: 10.1002/cbic.200590024

      The cover picture shows a new approach for the functional cloning of enzymes involved in the post-translational modification of proteins. This strategy employs a yeast tribrid system: a variant of the yeast two-hybrid system that couples activation of a reporter gene to expression of an enzyme. The image on the right is a combined differential interference contrast / confocal-laser scanning micrograph of S. cerevisiae transiently expressing the human Fyn protein tyrosine kinase. Cells expressing this enzyme trigger a reporter gene that encodes enhanced green fluorescent protein; this enables isolation of hits by fluorescence-activated cell sorting. This system is discussed in more detail on p. 1442 ff by B. R. Peterson and D. D. Clark.

    2. Graphical Abstract: ChemBioChem 8/2005 (pages 1299–1308)

      Article first published online: 29 JUL 2005 | DOI: 10.1002/cbic.200590025

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      A New Ligand for Immunoglobulin G Subdomains by Screening of a Synthetic Peptide Library (page 1307)

      Antonio Verdoliva, Daniela Marasco, Antonia De Capua, Angela Saporito, Piero Bellofiore, Vincenzo Manfredi, Roberto Fattorusso, Carlo Pedone and Menotti Ruvo

      Article first published online: 29 JUL 2005 | DOI: 10.1002/cbic.200590026

    4. Artificial Transcriptional Activation Domains (pages 1311–1315)

      Jenifer K. Lum and Anna K. Mapp

      Article first published online: 23 JUN 2005 | DOI: 10.1002/cbic.200500036

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      Artificial transcriptional activators show great promise as tools to probe activator function as well as provide a basis for future transcription-based therapeutics. This review highlights the current methods used to isolate artificial activation domains as well as novel peptidic and small-molecule activation domains recently identified. Considerations and challenges in artificial activator design will also be discussed.

    5. DNA Catalysis: Potential, Limitations, Open Questions (pages 1316–1322)

      Alessio Peracchi

      Article first published online: 8 JUL 2005 | DOI: 10.1002/cbic.200500098

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      In vitro selection methods are shedding light on the surprising functional versatility of DNA, and in particular on its catalytic aptitudes. The study of DNA enzymes (deoxyribozymes) helps address the principles and the limits of nucleic acid catalysis, while also leading to the creation of useful tools for biotechnology and nanotechnology.

    6. Multiparameter Imaging for the Analysis of Intracellular Signaling (pages 1323–1330)

      Carsten Schultz, Andreas Schleifenbaum, Joachim Goedhart and Theodorus W. J. Gadella Jr.

      Article first published online: 12 JUL 2005 | DOI: 10.1002/cbic.200500012

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      Paint it blue, green, and red. A holistic picture of a cell's function can be obtained only by visualizing interlocking cellular signaling pathways. For this purpose it is necessary to monitor as many parameters as possible in a single living cell (see picture). We discuss possibilities and limitations provided by multiparameter imaging of cellular events with the help of fluorescent probes.

    7. Pseudo 3D Single-Walled Carbon Nanotube Film for BSA-Free Protein Chips (pages 1331–1334)

      Hye Ryung Byon, Bong Jin Hong, Yong Song Gho, Joon Won Park and Hee Cheul Choi

      Article first published online: 5 JUL 2005 | DOI: 10.1002/cbic.200500081

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      Sweet home for proteins. Single-walled carbon nanotube (SWNT) film treated with CDI–Tween20 (CT) has been used as a substrate for a BSA-free protein chip. In the absence of BSA, which is conventionally applied as a nonspecific binding inhibitor, protein A, biotinylated BSA, and even small peptide such as 3×FLAG immobilized on the CT-functionalized SWNT film substrates recognize their respective counterparts, streptavidin, immunoglobulin G, and antiFLAG. with high specificity.

    8. Fluorescent Multiplex Analysis of Carrier Protein Post-Translational Modification (pages 1335–1337)

      Andrew C. Mercer, James J. La Clair and Michael D. Burkart

      Article first published online: 5 JUL 2005 | DOI: 10.1002/cbic.200500051

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      A multiplex assay to investigate the functional specificity between carrier proteins, phosphopantheinyl transferases, and coenzyme A (CoA) derivatives is shown. This system offers a multicolored tool for the biophysical study of biosynthetic pathways and orthogonally designed fusion proteins.

    9. Increased Thermal Stability of Site-Selectively Glycosylated Dihydrofolate Reductase (pages 1338–1340)

      Richard S. Swanwick, Alison M. Daines, Lai-Hock Tey, Sabine L. Flitsch and Rudolf K. Allemann

      Article first published online: 8 JUL 2005 | DOI: 10.1002/cbic.200500103

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      Heat protection. The native conformation of many proteins can be stabilised against thermal denaturation by glycosylation. Here we show that the thermal stability of the naturally nonglycosylated enzyme, dihydrofolate reductase, can be increased significantly by site-selective glycosylation (see figure). The data suggest that increases in thermal stability can be achieved even with the small carbohydrates used in this study.

    10. Bead-Based Cellular Analysis, Sorting and Multiplexing (pages 1341–1345)

      Rosario M. Sanchez-Martin, Mathilde Muzerelle, Nutcha Chitkul, Siew Eng How, Stifun Mittoo and Mark Bradley

      Article first published online: 23 JUN 2005 | DOI: 10.1002/cbic.200500059

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      Functionalized cross-linked polystyrene microspheres were synthesized, fluorescently labelled and delivered into cells to function as cellular tags and probes (see figure). This allowed the effective delivery of foreign materials into intact mammalian cells, without the need for delicate procedures such as micro-injection, and did not disrupt cell physiology.

    11. Design In Silico, Synthesis and Binding Evaluation of a Carbohydrate-Based Scaffold for Structurally Novel Inhibitors of Matrix Metalloproteinases (pages 1345–1349)

      Marco Fragai, Cristina Nativi, Barbara Richichi and Chiara Venturi

      Article first published online: 24 JUN 2005 | DOI: 10.1002/cbic.200400456

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      Bicyclic α-O-glycodipeptide mimetic scaffolds have been designed, synthesized and tested as new inhibitors of matrix metalloproteinases (MMPs). These compounds exhibit a wide synthetic versatility, suitable water solubility and peculiar bioavailability properties. The interaction with MMP-12, predicted by molecular docking (model shown), was assessed by NMR and fluorimetric assay.

    12. Luminescent Saccharide Biosensor by Using Lanthanide-Bound Lectin Labeled with Fluorescein (pages 1349–1352)

      Yoichiro Koshi, Eiji Nakata and Itaru Hamachi

      Article first published online: 29 JUL 2005 | DOI: 10.1002/cbic.200500072

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      A new luminescent biosensor for complicated glycoconjugates was engineered on the basis of a lanthanide-complexed sugar-binding protein (lectin) modified with a fluorophore. By using luminescence resonance energy transfer (LRET), ratiometric luminescent sensing can be carried out and successfully applied to a luminescent assay for an enzymatic trimming of a glycoprotein (see scheme).

    13. First Insight into the Symmetry and Flexibility of Membrane Efflux Pump P-Glycoprotein by Novel Bifunctional Modulators (pages 1353–1356)

      Jörg Wollmann, Martin Richter, Jósef Molnár and Andreas Hilgeroth

      Article first published online: 6 JUL 2005 | DOI: 10.1002/cbic.200400401

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      The other way round. The protein-target structure of P-glycoprotein is supposed to have an unexpectedly rigid symmetric binding region. Even the smallest deviations from symmetric inhibitor substitution lead to a decrease in inhibitory activity. However, the highest inhibitory activities shown towards HIV-1 protease are by asymmetric representatives, such as 1, thus demonstrating a high degree of flexibility of this protein-binding region.

    14. Converting Cytochrome b5 into Cytochrome c-Like Protein (pages 1356–1359)

      Ying-Wu Lin, Wen-Hu Wang, Qi Zhang, Hao-Jie Lu, Peng-Yuan Yang, Yi Xie, Zhong-Xian Huang and Hou-Ming Wu

      Article first published online: 24 JUN 2005 | DOI: 10.1002/cbic.200500030

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      Covalent attachment of a heme group to a protein matrix (see picture) allows a direct comparison of the properties of proteins in the same protein scaffold to be made. Using cytochrome b5 as the model will enable us to elucidate the precise role of the heme–protein covalent interaction that governs functional diversity and to delineate the “structure–property–reactivity–function” relationship of hemoproteins.

    15. Mechanism-Based Fluorescent Reporter for Protein Kinase A Detection (pages 1361–1367)

      Benedict Law, Ralph Weissleder and Ching-Hsuan Tung

      Article first published online: 12 JUL 2005 | DOI: 10.1002/cbic.200500027

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      The more the brighter. A peptide-based active-site-binding probe was designed to detect protein kinase A (PKA) in biological samples. Binding of the probe to PKA causes a fluorescence enhancement in a time- and dose-dependent manner. The probe detects the amount of PKA rather than the enzyme activity.

    16. Synthesis and Degradation of Nucleic Acid Components by Formamide and Cosmic Dust Analogues (pages 1368–1374)

      Raffaele Saladino, Claudia Crestini, Veronica Neri, John R. Brucato, Luigi Colangeli, Fabiana Ciciriello, Ernesto Di Mauro and Giovanna Costanzo

      Article first published online: 8 JUL 2005 | DOI: 10.1002/cbic.200500035

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      Cosmic-dust analogues catalyze the synthesis of nucleic bases from formamide. The synthesis of pyrimidines under prebiotic conditions has so far remained elusive. In this study, we have synthesized these in a rich mixture and good yield from formamide (see scheme). Cosmic-dust analogues were also found to selectively affect the stability of oligonucleotides.

    17. Partial Protection against Botulinum B Neurotoxin-Induced Blocking of Exocytosis by a Potent Inhibitor of Its Metallopeptidase Activity (pages 1375–1380)

      Christine Anne, Serge Turcaud, Armand G. S. Blommaert, François Darchen, Eric A. Johnson and Bernard P. Roques

      Article first published online: 29 JUN 2005 | DOI: 10.1002/cbic.200400398

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      A new way of possibly preventing and/or controlling botulism is provided by the first highly potent (Ki in the nM range) inhibitors of the metallopeptidase activity (light chain) responsible for the neurotoxicity of botulinum toxin B (BoNT/B). These compounds (see scheme) were shown to strongly reduce the cleavage of synaptobrevin with subsequent inhibition of the neurotransmitter release induced by native BoNT/B.

    18. An Inverse Substrate Orientation for the Regioselective Acylation of 3′,5′-Diaminonucleosides Catalyzed by Candida antarctica lipase B? (pages 1381–1390)

      Iván Lavandera, Susana Fernández, Julia Magdalena, Miguel Ferrero, Romas J. Kazlauskas and Vicente Gotor

      Article first published online: 24 JUN 2005 | DOI: 10.1002/cbic.200400422

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      Inverse substrate orientation in lipases. Hydrolase-catalyzed reactions with amines are often slower than related ones with alcohols, in spite of the higher nucleophilicity of amino groups. A combination of molecular modeling and kinetic experiments suggests that lipase-mediated processes of apparently close substrate analogues such as alcohols and amines might follow different pathways. Thus, the regioselective acylation of 3′,5′-diamino-3′,5′-dideoxythymidine has been rationalized by modeling inverse intermediates (see scheme).

    19. Transient Structural Ordering of the RNA-Binding Domain of Carnation Mottle Virus p7 Movement Protein Modulates Nucleic Acid Binding (pages 1391–1396)

      Marçal Vilar, Ana Saurí, Jose F. Marcos, Ismael Mingarro and Enrique Pérez-Payá

      Article first published online: 8 JUL 2005 | DOI: 10.1002/cbic.200400451

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      Stability pact. It is believed that nascent secondary-structure units could be stabilized early in the protein-folding pathway and could have functional and structure-forming capabilities. In this paper we show that the stabilization of a nascent α-helical conformation at the C terminus of the RNA-binding domain of a viral movement protein is important for the molecular mechanism associated with the interaction between the protein and the viral RNA (see picture).

    20. Guanine and Plasmid DNA binding of Mono- and Trinuclear fac-[Re(CO)3]+ Complexes with Amino Acid Ligands (pages 1397–1405)

      Fabio Zobi, Bernhard Spingler and Roger Alberto

      Article first published online: 16 JUN 2005 | DOI: 10.1002/cbic.200400453

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      Towards radiotherapeutic DNA-binding metal complexes. Does [Re(CO)3]+ interact with DNA in a cisplatin-like fashion? We have synthesized complexes of [Re(CO)3]+ with amino acids and studied their reaction with ΦX174 plasmid DNA (see example structure). The results show alteration of the electrophoretic mobility (see gel photograph) by stable adduct formation in a process that is likely to involve two bases. Potentially, chemo- and radiotoxicity can be combined in a single compound.

    21. LanGT2 Catalyzes the First Glycosylation Step during Landomycin A Biosynthesis (pages 1406–1410)

      Andriy Luzhetskyy, Takaaki Taguchi, Marta Fedoryshyn, Clemens Dürr, Sven-Eric Wohlert, Volodymyr Novikov and Andreas Bechthold

      Article first published online: 24 JUN 2005 | DOI: 10.1002/cbic.200500018

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      Switching from O- to C-glycosyl transfer: The specific functions of two glycosyltransferases (LanGT2 and LanGT1) have been elucidated. Substitution of LanGT2 with UrdGT2 from the urdamycin biosynthetic pathway leads to the new C-glycosylated tetrangulol (see scheme).

    22. Metabolic Engineering of Aminocoumarins: Inactivation of the Methyltransferase Gene cloP and Generation of New Clorobiocin Derivatives in a Heterologous Host (pages 1411–1418)

      Anja Freitag, Heike Rapp, Lutz Heide and Shu-Ming Li

      Article first published online: 24 JUN 2005 | DOI: 10.1002/cbic.200500019

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      New antibiotics through gene deletion. The methyltransferase gene cloP was deleted in the biosynthetic gene cluster of clorobiocin. The modified gene cluster was expressed in the heterologous host Streptomyces coelicolor M512 to give three new aminocoumarins that lack clorobiocin's methyl group at 4′′-OH in the noviose moiety (see scheme). The 5-methylpyrrole-2-carbonyl group was found to be attached to either 2′′-OH, 3′′-OH or 4′′-OH. This is the first time that an aminocoumarin antibiotic acylated at 4-OH of noviose has been detected.

    23. How Does DNA Compaction Favor Chiral Selectivity with Cationic Species? Higher Selectivity with Lower Cationic Charge (pages 1419–1422)

      Anatoly A. Zinchenko, Ning Chen, Shizuaki Murata and Kenichi Yoshikawa

      Article first published online: 8 JUL 2005 | DOI: 10.1002/cbic.200500032

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      Being too positive. In DNA compaction by chiral multications, chiral discrimination is manifested only if the charge on enantiomeric molecules is low. As shown in the picture, profound chiral discrimination between chiral dications vanishes completely in the case of tetravalent cations.

    24. An Enzyme Module System for the Synthesis of dTDP-activated Deoxysugars from dTMP and Sucrose (pages 1423–1430)

      Lothar Elling, Carsten Rupprath, Nicole Günther, Ulrike Römer, Stefan Verseck, Petra Weingarten, Gerald Dräger, Andreas Kirschning and Wolfgang Piepersberg

      Article first published online: 24 JUN 2005 | DOI: 10.1002/cbic.200500037

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      The right combination. A flexible enzyme module system gives preparative synthetic access to important dTDP-activated (deoxy)hexoses from dTMP and sucrose. Different biocatalytic combinations lead to dTDP-Glc, dTDP-4-keto-6-deoy-Glc and dTDP-L-Rha (see scheme). The modular character also allows the combination of enzymes from different dTDP-deoxyhexose pathways from microbial drug producers. This combinatorial biocatalytic approach may be extended to generate novel macrolides in vitro with in situ regeneration of dTDP-activated deoxysugars.

    25. Highly Productive Autocondensation and Transglycosylation Reactions with Sulfolobus solfataricus Glycosynthase (pages 1431–1437)

      Antonio Trincone, Assunta Giordano, Giuseppe Perugino, Mosè Rossi and Marco Moracci

      Article first published online: 12 JUL 2005 | DOI: 10.1002/cbic.200400430

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      Biocatalyst does the business: Preparative autocondensations of 2- and 4-nitrophenyl β-D-glucopyranosides and a series of transglycosylation reactions catalyzed by the genetically modified hyperthermophilic glycosyl hydrolase Ss-β-glyE387G (glycosynthase) from Sulfolobus solfataricus are reported, thereby demonstrating the synthetic potential of this biocatalyst (see scheme).

    26. Stereospecific Synthesis of Chiral 2,3-Dihydro-1,4-benzodithiine and Methyl-2,3-dihydro-1,4-benzodithiine Derivatives and their Toxic Effects on Trypanosoma brucei (pages 1438–1441)

      Syed Tasadaque Ali Shah, Patrick Merkel, Hubert Ragge, Michael Duszenko, Jörg Rademann and Wolfgang Voelter

      Article first published online: 29 JUL 2005 | DOI: 10.1002/cbic.200400375

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      Sights on parasites. Chiral 2,3-dihydro-1,4-benzodithiine and methyl-2,3-dihydro-1,4-benzodithiine derivatives (structure) were synthesized and found to efficiently kill bloodstream forms of Trypanosoma brucei (main picture). The parasites' energy metabolism and consumption of oxygen were found to be affected.

    27. Fluorescence-Based Cloning of a Protein Tyrosine Kinase with a Yeast Tribrid System (pages 1442–1448)

      Daniel D. Clark and Blake R. Peterson

      Article first published online: 8 JUL 2005 | DOI: 10.1002/cbic.200500047

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      Engineering yeast. Yeast two-hybrid systems have traditionally been used to identify specific protein interactions. To extend this approach to the discovery of enzymes that catalyze the phosphorylation of tyrosine residues, we screened a cDNA library against a yeast tribrid system by using fluorescence-activated cell sorting. By cloning Fyn kinase from a T-lymphocyte cDNA library, we demonstrate the utility of this approach for the isolation of enzymes involved in post-translational modification.

    28. Interaction of the Catalytic Domain of Inositol 1,4,5-Trisphosphate 3-Kinase A with Inositol Phosphate Analogues (pages 1449–1457)

      Alexandra Poinas, Katrien Backers, Andrew M. Riley, Stephen J. Mills, Colette Moreau, Barry V. L. Potter and Christophe Erneux

      Article first published online: 5 JUL 2005 | DOI: 10.1002/cbic.200400443

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      With or without OH: The 2-hydroxy group of inositol 1,4,5-triphosphate (Ins(1,4,5)P3) is not involved in binding to the Ins(1,4,5)P3 3-kinase A or in the mechanism of phosphoryl transfer. As shown here, D-2-deoxy-Ins(1,4,5)P3 can reproduce all the important features of Ins(1,4,5)P3 and is therefore a potent inhibitor and substrate of this enzyme.

    29. Guanine-Rich DNA Nanocircles for the Synthesis and Characterization of Long Cytosine-Rich Telomeric DNAs (pages 1458–1462)

      Jörg S. Hartig, Sara Fernandez-Lopez and Eric T. Kool

      Article first published online: 29 JUL 2005 | DOI: 10.1002/cbic.200500015

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      Around the bend. The synthesis of single-stranded circular DNAs composed of the human G-rich telomeric sequence was facilitated by developing a new protecting-group strategy. These telomeric circles were used in rolling-circle reactions (RCR, see figure) to synthesize very long repeats of the C-rich human telomeric strands, an activity that the enzyme telomerase does not have. Preliminary structural studies of these long artificial telomeres revealed four-stranded folds under certain conditions.

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      Preview: ChemBioChem 8/2005 (page 1470)

      Article first published online: 29 JUL 2005 | DOI: 10.1002/cbic.200590027