ChemBioChem

Cover image for Vol. 10 Issue 8

May 25, 2009

Volume 10, Issue 8

Pages 1265–1415

  1. Cover Picture

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    2. Cover Picture
    3. Graphical Abstract
    4. News
    5. Minireview
    6. Highlight
    7. Communications
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    1. Cover Picture: A Chemical Approach to Immunoprotein Engineering: Chemoselective Functionalization of Thioester Proteins in Their Native State (ChemBioChem 8/2009) (page 1265)

      Michael A. Cole, Sarah E. Tully, Alister W. Dodds, James N. Arnold, Grant E. Boldt, Robert B. Sim, John Offer and Paul Wentworth Jr.

      Version of Record online: 14 MAY 2009 | DOI: 10.1002/cbic.200990027

      Thumbnail image of graphical abstract

      The cover picture shows chemoselective labeling of the “buried” macrocyclic thiolactone-containing component of complement protein C3 with a variety of hydrazide probes. C3, C4, and α2-macroglobulin play crucial roles within the complement system and as blood homeostasis regulators. Given their role in the immune system and disease, these proteins and their resulting processed fragments are highly studied, and the generation of chemically defined conjugates of these proteins offers the potential for significant advances in vaccine design and applied immunological studies. The hydrazide functionalities shown in the lower portion of this image have been used to chemoselectively label the thioester site with molecular tags, thus providing a new bioconjugation approach for thioester proteins and insight into the chemical accessibility of the thioester site. In the background of this cover image is a haystack landscape by Claude Monet representing the ability of these probes to reach these thioester “needles” within their protein scaffold and selectively label them within the complex plasma “haystack”. For further details, see the article by P. Wentworth et al. on p. 1340 ff.

  2. Graphical Abstract

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    3. Graphical Abstract
    4. News
    5. Minireview
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  3. News

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    3. Graphical Abstract
    4. News
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  4. Minireview

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    1. Artificial Restriction DNA Cutters as New Tools for Gene Manipulation (pages 1279–1288)

      Hitoshi Katada and Makoto Komiyama

      Version of Record online: 24 APR 2009 | DOI: 10.1002/cbic.200900040

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      The final cut. Two types of artificial tools (artificial restriction DNA cutter and zinc finger nuclease) that cut double-stranded DNA through hydrolysis of target phosphodiester linkages, have been recently developed. The chemical structures, preparation, properties, and typical applications of these two man-made tools are reviewed.

  5. Highlight

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    4. News
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    1. Rolling-Circle Amplification: Unshared Advantages in miRNA Detection (pages 1289–1291)

      Saskia Neubacher and Christoph Arenz

      Version of Record online: 16 APR 2009 | DOI: 10.1002/cbic.200900116

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      Roll with it: The quantitative analysis of specific miRNAs from biological samples is very likely to revolutionize diagnostics of human disease. A novel method for miRNA analysis employing rolling-circle amplification (RCA) can homogeneously detect miRNA, even at concentrations as low as 10 fM. The use of T4 RNA ligase 2 (T4 RnL2) at elevated temperatures enables very good discrimination of miRNAs differing by a single nucleotide.

  6. Communications

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    1. Engineered Two-Helix Small Proteins for Molecular Recognition (pages 1293–1296)

      Jack M. Webster, Rong Zhang, Sanjiv S. Gambhir, Zhen Cheng and Faisal A. Syud

      Version of Record online: 6 MAY 2009 | DOI: 10.1002/cbic.200900062

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      Less is more: By starting with a high-affinity HER2-binding 3-helix affibody molecule, we successfully developed 2-helix small protein binders with 5 nM affinities by using a combination of several different strategies. Our efforts clearly suggest that 2-helix small proteins against important tumor targets can be obtained by rational protein design and engineering.

    2. Functional Analysis of MycE and MycF, Two O-Methyltransferases Involved in the Biosynthesis of Mycinamicin Macrolide Antibiotics (pages 1297–1301)

      Shengying Li, Yojiro Anzai, Kenji Kinoshita, Fumio Kato and David H. Sherman

      Version of Record online: 4 MAY 2009 | DOI: 10.1002/cbic.200900088

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      Mg motors: We characterized the in vitro function of MycE and MycF, two O-methyltransferases involved in the biosynthesis of mycinamicin antibiotics. Each enzyme was confirmed to be an S-adenosyl-L-methionine (SAM)-dependent deoxysugar methyltransferase. Their optimal activities require the presence of Mg2+. With the reconstituted in vitro assays, the order of mycinamicin VI[RIGHTWARDS ARROW]III[RIGHTWARDS ARROW]IV in the post-PKS (polyketide synthase) tailoring pathway of mycinamicin was unambiguously determined.

    3. Site-specific Protein Cross-Linking with Genetically Incorporated 3,4-Dihydroxy-L-Phenylalanine (pages 1302–1304)

      Aiko Umeda, Gabrielle Nina Thibodeaux, Jie Zhu, YungAh Lee and Zhiwen Jonathan Zhang

      Version of Record online: 6 MAY 2009 | DOI: 10.1002/cbic.200900127

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      Come together right now with L-DOPA: Chemical cross-linking is widely used to study protein–protein interactions. However, many cross-linking agents suffer from low reactivity or selectivity. An efficient and selective reaction of site-specific protein cross-linking was achieved using genetically incorporated 3,4-dihydroxy-L-phenylalanine.

    4. GilR, an Unusual Lactone-Forming Enzyme Involved in Gilvocarcin Biosynthesis (pages 1305–1308)

      Madan Kumar Kharel, Pallab Pahari, Hui Lian and Jürgen Rohr

      Version of Record online: 22 APR 2009 | DOI: 10.1002/cbic.200900130

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      Last at last: The terminal step of the gilvocarcin V (GV) biosynthetic pathway is an unusual lactone formation. Here we show that the enzyme, GilR, dehydrogenates the hemiacetal moiety of pregilvocarcin V to the lactone found in GV by using covalently bound FAD.

      Corrected by:

      Corrigendum: Corrigendum: GilR, an Unusual Lactone-Forming Enzyme Involved in Gilvocarcin Biosynthesis

      Vol. 11, Issue 9, 1161, Version of Record online: 7 JUN 2010

    5. Position-Dependent Electrostatic Protection against Protein Aggregation (pages 1309–1312)

      Alexander K. Buell, Gian Gaetano Tartaglia, Neil R. Birkett, Christopher A. Waudby, Michele Vendruscolo, Xavier Salvatella, Mark E. Welland, Christopher M. Dobson and Tuomas P. J. Knowles

      Version of Record online: 4 MAY 2009 | DOI: 10.1002/cbic.200900144

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      Proteins with a high propensity to aggregate can be largely prevented from doing so with surprisingly small changes to their primary structure. By using a combination of rational design and quantitative measurements of aggregation rates, we show that adding a single charge in specific “gatekeeper” regions is sufficient to change the timescale for amyloid fibril growth from minutes to weeks, thereby dramatically reducing the efficiency of this process.

    6. Histone H3 N-Terminal Peptide Binds Directly to Its Own mRNA: A Possible Mode of Feedback Inhibition to Control Translation (pages 1313–1316)

      Kyung Hyun Lee, Nam Ju Lee, Soonsil Hyun, Yong Keun Park, Eun Gyeong Yang, Jun-Kyu Lee, Sunjoo Jeong and Jaehoon Yu

      Version of Record online: 29 APR 2009 | DOI: 10.1002/cbic.200900154

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      Give me some feedback: In vitro selection of aptamers against the H3 peptide provided specific hairpin RNAs that possess high homology with histone H3 mRNA. The identified H3 hairpin RNA binds specifically to the H3 peptide with micromolar affinity and dose-dependently inhibits in vitro translation of the H3 protein. Consequently, the hairpin RNA and H3 peptide are one of the rare cis- and trans-elements on coding regions found among housekeeping proteins in higher eukaryotes.

    7. The Engineering of Bacteria Bearing Azido-Pseudaminic Acid-Modified Flagella (pages 1317–1320)

      Feng Liu, Annie J. Aubry, Ian C. Schoenhofen, Susan M. Logan and Martin E. Tanner

      Version of Record online: 6 MAY 2009 | DOI: 10.1002/cbic.200900018

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      Catch a tiger by the tail: We have demonstrated that by feeding nonmotile mutant C. jejuni bacteria with a neutral azide-labelled pseudaminic acid precursor we can restore their ability to generate functional flagella. The presence of azido-pseudaminic acid on the surface of the flagella provides a bio-orthogonal chemical handle that can be used to modify the flagellar proteins.

    8. [18F]SiFA-isothiocyanate: A New Highly Effective Radioactive Labeling Agent for Lysine-Containing Proteins (pages 1321–1324)

      Pedro Rosa-Neto, Björn Wängler, Ljuba Iovkova, Guido Boening, Andrew Reader, Klaus Jurkschat and Esther Schirrmacher

      Version of Record online: 6 MAY 2009 | DOI: 10.1002/cbic.200900132

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      A highly efficient18F-labeling synthon for universal protein labeling is reported. Diverse 18F-labeled proteins of 66–144 kDa were prepared with [18F]SiFA-isothiocyanate synthesized by an isotopic 19F for 18F exchange at the silicon atom. Overall preparative radiochemical yields were 20–40 % after 40–50 min. No bone uptake of 18F radioactivity was detected until 90 min post-injection of 18F-SiFA-RSA; this demonstrates the metabolic stability of the [18F]SiFA moiety.

    9. Phenolic Oxime Oligomers Inhibit Alzheimer's Amyloid Fibril Formation and Disaggregate Fibrils In Vitro (pages 1325–1329)

      Gunnar T. Dolphin, Olivier Renaudet, Myriam Ouberai, Pascal Dumy, Julian Garcia and Jean-Louis Reymond

      Version of Record online: 4 MAY 2009 | DOI: 10.1002/cbic.200900044

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      See you later amyloid β: A screen of a small library of oxime oligomers with an HTS fluorescence assay for amyloid fibril inhibition and subsequent investigation by atomic force microscopy revealed two new micromolar inhibitors of amyloid fibril formation. These new inhibitors have IC50 values in the 10 μM range.

    10. Lactone Size Dependent Reactivity in Candida Antarctica Lipase B: A Molecular Dynamics and Docking Study (pages 1330–1334)

      Martijn A. J. Veld, Linda Fransson, Anja R. A. Palmans, E. W. Meijer and Karl Hult

      Version of Record online: 7 MAY 2009 | DOI: 10.1002/cbic.200900128

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      Size matters: Lactones have extensively been studied as monomers in enzymatic polymerization reactions. Large lactones showed an unexpectedly high reactivity in these reactions. A combination of docking and molecular dynamics studies have been used to explain this high reactivity in terms of productive binding due to the transoid nature of the ester bond in these substrates.

    11. Two Base Pair Duplexes Suffice to Build a Novel Material (pages 1335–1339)

      Martin Meng, Carolin Ahlborn, Matthias Bauer, Oliver Plietzsch, Shahid A. Soomro, Arunoday Singh, Thierry Muller, Wolfgang Wenzel, Stefan Bräse and Clemens Richert

      Version of Record online: 6 MAY 2009 | DOI: 10.1002/cbic.200900162

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      Tetrahedral DNA hybrids with tetrakis(p-hydroxyphenyl)methane cores hybridize in a sequence-specific fashion at much higher temperatures than isolated linear duplexes. Dinucleotide DNA arms suffice to induce the formation of a solid at room temperature; this demonstrates the strength of multivalent binding. The graphic shows a view of a modeled assembly.

    12. A Chemical Approach to Immunoprotein Engineering: Chemoselective Functionalization of Thioester Proteins in Their Native State (pages 1340–1343)

      Michael A. Cole, Sarah E. Tully, Alister W. Dodds, James N. Arnold, Grant E. Boldt, Robert B. Sim, John Offer and Paul Wentworth Jr.

      Version of Record online: 28 APR 2009 | DOI: 10.1002/cbic.200900168

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      Less than 6 feet under: Serum proteins C3, C4, and α2M each contain a thioester domain buried within a hydrophobic pocket, which is thought to shield the labile thioester from hydrolysis. Herein, we make use of the inherent reactivity of the hydrazide for thioester moieties to chemoselectively label these crucial serum regulators in their native conformation; this demonstrates that access to the thioester site is much greater than previously supposed.

    13. The Natural Products Beauveriolide I and III: a New Class of β-Amyloid-Lowering Compounds (pages 1344–1347)

      Daniel P. Witter, Yanping Chen, Joseph K. Rogel, Grant E. Boldt and Paul Wentworth Jr.

      Version of Record online: 25 APR 2009 | DOI: 10.1002/cbic.200900139

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      Attacking Alzheimer's by ACAT: The aggregation of β-amyloid peptides, especially Aβ42, into senile plaques is a hallmark of Alzheimer's disease (AD). We show that the fungal natural products beauveriolides I and III can potently decrease Aβ secretion from cells expressing human amyloid precursor protein; this offers a potential new scaffold for the development of compounds with proven bioavailability for the treatment of AD.

  7. Full Papers

    1. Top of page
    2. Cover Picture
    3. Graphical Abstract
    4. News
    5. Minireview
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    1. Disulfide Bond Substitution by Directed Evolution in an Engineered Binding Protein (pages 1349–1359)

      Antoine Drevelle, Agathe Urvoas, Mériam Ben Hamida-Rebaï, Gérard Van Vooren, Magali Nicaise, Marie Valerio-Lepiniec, Michel Desmadril, Charles H. Robert and Philippe Minard

      Version of Record online: 4 MAY 2009 | DOI: 10.1002/cbic.200800745

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      Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug-carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed-evolution approach starting from NCS-3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide-free NCS variants suitable for a variety of purposes, including intracellular applications.

    2. Thermodynamic and Computational Studies on the Binding of p53-Derived Peptides and Peptidomimetic Inhibitors to HDM2 (pages 1360–1368)

      Anja Grässlin, Celine Amoreira, Kim K. Baldridge and John A. Robinson

      Version of Record online: 30 APR 2009 | DOI: 10.1002/cbic.200900008

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      Helix power: The binding interactions of linear and constrained β-hairpin-shaped peptides with HDM2 were compared by using experimental and theoretical methods. The entropic advantages enjoyed by the constrained peptides were found to be largely offset by reduced enthalpic contributions to binding of the cyclic mimetics. Formation of hydrogen bonds upon helix folding could contribute significantly to the enhanced enthalpy observed in binding of the linear peptides.

    3. Design of Triazole-Tethered Glycoclusters Exhibiting Three Different Spatial Arrangements and Comparative Study of their Affinities towards PA-IL and RCA 120 by Using a DNA-Based Glycoarray (pages 1369–1378)

      Lisa Moni, Gwladys Pourceau, Jing Zhang, Albert Meyer, Sébastien Vidal, Eliane Souteyrand, Alessandro Dondoni, François Morvan, Yann Chevolot, Jean-Jacques Vasseur and Alberto Marra

      Version of Record online: 29 APR 2009 | DOI: 10.1002/cbic.200900024

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      Sugar-coated chips: Glycoside clusters are valuable tools for carbohydrate–lectin recognition studies. However, the spatial arrangement of the sugar residues is a key issue in the design of high-affinity glycoclusters. Here the affinities of linear and antenna- and calixarene-based galactoside clusters towards two lectins derived from Pseudomonas aeruginosa and Ricinus communis were compared by means of glycoarrays.

    4. Parallel Pathways and Free-Energy Landscapes for Enzymatic Hydride Transfer Probed by Hydrostatic Pressure (pages 1379–1384)

      Christopher R. Pudney, Tom McGrory, Pierre Lafite, Jiayun Pang, Sam Hay, David Leys, Michael J. Sutcliffe and Nigel S. Scrutton

      Version of Record online: 29 APR 2009 | DOI: 10.1002/cbic.200900071

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      Mutation of an active-site residue in morphinone reductase leads to a conformationally rich landscape that enhances the rate of hydride transfer from NADH to FMN at standard pressure (1 bar). Increasing the pressure causes interconversion between different conformational substates in the mutant enzyme. While high pressure reduces the donor–acceptor distance in the wild-type enzyme, increased conformational freedom “dampens” its effect in the mutant.

    5. Probing the Role of Backbone Hydrogen Bonding in a Critical β Sheet of the Extracellular Domain of a Cys-Loop Receptor (pages 1385–1391)

      Kristin R. Gleitsman, Henry A. Lester and Dennis A. Dougherty

      Version of Record online: 29 APR 2009 | DOI: 10.1002/cbic.200900092

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      Probing the sheet: The network of hydrogen bonds formed in the outer β sheet of the nicotinic acetylcholine receptor (nAChR; see figure) is fairly robust and tolerates single amide-to-ester mutations throughout. However, eliminating two proximal hydrogen bonds completely destroys receptor function; this adds further support to gating models that ascribe important roles to these β strands of the nAChR extracellular domain.

    6. Cloning and Sequencing of the Biosynthetic Gene Cluster for Saquayamycin Z and Galtamycin B and the Elucidation of the Assembly of Their Saccharide Chains (pages 1392–1401)

      Annette Erb, Andriy Luzhetskyy, Uwe Hardter and Andreas Bechthold

      Version of Record online: 21 APR 2009 | DOI: 10.1002/cbic.200900054

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      Sweet ways: We have investigated the glycosyltransferase genes of the saquayamycin Z (shown) and galtamycin B biosynthetic gene cluster from Micromonospora sp. Tü6368. The results unambiguously show that both compounds are derived from the same cluster. Furthermore, the function of five glycosyltransferases was elucidated, and the results have shed light on the assembly of the sugar chains.

    7. Fluorescent Probes to Characterise FK506-Binding Proteins (pages 1402–1410)

      Christian Kozany, Andreas März, Christoph Kress and Felix Hausch

      Version of Record online: 5 MAY 2009 | DOI: 10.1002/cbic.200800806

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      Talented all-rounders: Fluorescence polarisation assays were developed for members of the FK506-binding protein family by using fluorescent rapamycin analogues (demonstrated in the figure). These tracers retain medium to high affinity to all tested proteins (FKBP12, -12.6, -13, -25, -51, -52). They can be used for active-site titrations, competition assays with unlabelled ligands and enable a robust, miniaturized assay adequate for high-throughput screening.

  8. Preview

    1. Top of page
    2. Cover Picture
    3. Graphical Abstract
    4. News
    5. Minireview
    6. Highlight
    7. Communications
    8. Full Papers
    9. Preview
    1. Preview: ChemBioChem 9/2009 (page 1415)

      Version of Record online: 14 MAY 2009 | DOI: 10.1002/cbic.200990030

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