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Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation

NATURE 1999年第6737期399卷 670-673页

作者： Joo, H Lin, ZL Arnold, FH CALTECH Div Chem & Chem Engn 210 41 Pasadena CA 91125 USA

Enzyme-based chemical transformations typically proceed with high selectivity under mild conditions, and are;becoming increasingly important in the pharmaceutical and chemical industries. Cytochrome P450 monooxygenases (P450s) constitute a large family(1) of enzymes of particular interest in this regard. Their biological functions, such as detoxification of xenobiotics and steroidogenesis(2-5), are based on the ability to catalyse the insertion of oxygen into:a wide variety of compounds(6). Such a catalytic transformation might find technological applications in areas ranging from gene therapy and environmental remediation to the selective synthesis of pharmaceuticals and chemicals(7-10). But relatively low turnover rates (particularly towards non-natural substrates), low stability and the need for electron-donating cofactors prohibit the practical use of P450s as isolated enzymes. Here we report the directed evolution(11) of the P450 from Pseudomonas putida to create mutants that hydroxylate naphthalene in the absence of cofactors through the 'peroxide shunt' pathway(12,13) with more than 20-fold higher activity than the native enzyme. We are able to screen efficiently for improved mutants by coexpressing them with horseradish peroxidase, which converts the products of the P450 reaction into fluorescent compounds amenable to digital imaging screening. This system should allow us to select and develop mono- and di-oxygenases into practically useful biocatalysts for the hydroxylation of a wide range of aromatic compounds.

关键词： Catalysis Cloning, Molecular Cytochrome P-450 Enzyme System/genetics Cytochrome P-450 Enzyme System/metabolism Directed Molecular Evolution Escherichia coli Fluorescence Fluorescence Horseradish Peroxidase/genetics Horseradish Peroxidase/metabolism Hydrogen Peroxide/metabolism Hydroxylation image processing, computer-assisted Mutagenesis Pseudomonas putida/enzymology Pseudomonas putida/genetics Recombination, Genetic

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Molecular architecture of the multiprotein splicing factor SF3b

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SCIENCE 2003年第5621期300卷 980-984页

作者： Golas, MM Sander, B Will, CL Lührmann, R Stark, H Max Planck Inst Biophys Chem D-37077 Gottingen Germany

The splicing factor SF3b is a multiprotein complex essential for the accurate excision of introns from pre-messenger RNA. As an integral component of the U2 small nuclear ribonucleoprotein (snRNP) and the U11/U12 di-snRNP, SF3b is involved in the recognition of the pre-messenger RNA's branch site within the major and minor spliceosomes. We have determined the three-dimensional structure of the human SF3b complex by single-particle electron cryomicroscopy at a resolution of less than 10 angstroms, allowing identification of protein domains with known structural folds. The best fit of a modeled RNA-recognition motif indicates that the protein p14 is located in the central cavity of the complex. The 22 tandem helical repeats of the protein SF3b155 are located in the outer shell of the complex enclosing p14.

关键词： Amino Acid Motifs Cryoelectron Microscopy Hela Cells Humans image processing, computer-assisted Macromolecular Substances Models, Molecular Multiprotein Complexes Phosphoproteins/chemistry Protein Conformation Protein Folding Protein Structure, Secondary Protein Structure, Tertiary Repetitive Sequences, Amino Acid Ribonucleoprotein, U2 Small Nuclear/chemistry RNA Precursors/chemistry RNA Precursors/metabolism RNA Splicing RNA-Binding Proteins Spliceosomes/chemistry Spliceosomes/metabolism

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Structure, function, and activator-induced conformations of the CRSP coactivator

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SCIENCE 2002年第5557期295卷 1058-1062页

作者： Taatjes, DJ Näär, AM Andel, F Nogales, E Tjian, R Univ Calif Berkeley Howard Hughes Med Inst Berkeley CA 94720 USA Univ Calif Berkeley Lawrence Berkeley Lab Dept Mol & Cell Biol Berkeley CA 94720 USA

The human cofactor complexes ARC (activator-recruited cofactor) and CRSP (cofactor required for Sp1 activation) mediate activator-dependent transcription in vitro. Although these complexes share several common subunits, their structural and functional relationships remain unknown. Here, we report that affinity-purified ARC consists of two distinct multisubunit complexes: a larger complex, denoted ARC-L, and a smaller coactivator, CRSP. Reconstituted in vitro transcription with biochemically separated ARC-L and CRSP reveals differential cofactor functions. The ARC-L complex is transcriptionally inactive, whereas the CRSP complex is highly active. Structural determination by electron microscopy (EM) and three-dimensional reconstruction indicate substantial differences in size and shape between ARC-L and CRSP. Moreover, EM analysis of independently derived CRSP complexes reveals distinct conformations induced by different activators. These results suggest that CRSP may potentiate transcription via specific activator-induced conformational changes.

关键词： CCAAT-Enhancer-Binding Proteins/chemistry CCAAT-Enhancer-Binding Proteins/metabolism Chromatin/metabolism DNA-Binding Proteins/chemistry DNA-Binding Proteins/metabolism Hela Cells Herpes Simplex Virus Protein Vmw65/metabolism Humans image processing, computer-assisted Imaging, Three-Dimensional Macromolecular Substances Microscopy, Electron Models, Genetic Precipitin Tests Precipitin Tests Protein Binding Protein Structure, Quaternary Protein Structure, Tertiary Protein Subunits Recombinant Fusion Proteins/chemistry Recombinant Fusion Proteins/metabolism Recombinant Proteins/metabolism Sterol Regulatory Element Binding Protein 1 Trans-Activators/chemistry Trans-Activators/isolation & purification Trans-Activators/metabolism Transcription Factors/metabolism Transcription, Genetic Transcriptional Activation

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Endocranial capacity in an early hominid cranium from Sterkfontein, South Africa

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SCIENCE 1998年第5370期280卷 1730-1731页

作者： Conroy, GC Weber, GW Seidler, H Tobias, PV Kane, A Brunsden, B Washington Univ Sch Med Dept Anat & Neurobiol St Louis MO 63110 USA Washington Univ Sch Med Dept Anthropol St Louis MO 63110 USA Univ Vienna Inst Human Biol A-1090 Vienna Austria Univ Witwatersrand Sch Med Dept Anat Sci Johannesburg South Africa Washington Univ Sch Med Div Plast Surg St Louis MO 63110 USA Washington Univ Sch Med Edward Mallinckrodt Inst Radiol St Louis MO 63110 USA

Two- and three-dimensional computer imaging shows that endocranial capacity in an similar to 2.8- to 2.6-million-year-old early hominid cranium (Stw 505) from Sterkfontein, South Africa, tentatively assigned to Australopithecus africanus, is similar to 515 cubic centimeters. Although this is the largest endocranial capacity recorded for this species, it is still markedly less than anecdotal reports of endocranial capacity exceeding 600 cubic centimeters. No australopithecine has an endocranial capacity approaching, let alone exceeding, 600 cubic centimeters. Some currently accepted estimates of early hominid endocranial capacity may be inflated, suggesting that the tempo and mode of early hominid brain evolution may need reevaluation.

关键词： Animals Brain/anatomy & histology computer Simulation Evolution Fossils History, Ancient Hominidae/anatomy & histology Humans image processing, computer-assisted Models, Anatomic Skull/anatomy & histology South Africa Tomography, X-Ray Computed

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Cryo-electron tomography reveals the cytoskeletal structure of Spiroplasma melliferum

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SCIENCE 2005年第5708期307卷 436-438页

作者： Kürner, J Frangakis, AS Baumeister, W European Mol Biol Lab D-69117 Heidelberg Germany Max Planck Inst Biochem Dept Biol Struct D-82152 Martinsried Germany

Evidence has accumulated recently that not only eukaryotes but also bacteria can have a cytoskeleton. We used cryo-electron tomography to study the three-dimensional structure of Spiroplasma melliferum cells in a close-to-native state at similar to4-nanometer resolution. We showed that these cells possess two types of filaments arranged in three parallel ribbons underneath the cell membrane. These two filamentous structures are built of the fibril protein and possibly the actin-like protein MreB. On the basis of our structural data, we could model the motility modes of these cells and explain how helical Mollicutes can propel themselves by means of coordinated length changes of their cytoskeletal ribbons.

关键词： Bacterial Proteins/analysis Blotting, Western Blotting, Western Cell Membrane/ultrastructure computer Simulation Cryoelectron Microscopy Cytoskeleton/chemistry Cytoskeleton/ultrastructure image processing, computer-assisted Imaging, Three-Dimensional Models, Biological Movement Spiroplasma/chemistry Spiroplasma/chemistry Spiroplasma/physiology Spiroplasma/physiology Spiroplasma/ultrastructure Spiroplasma/ultrastructure Tomography

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Infrared photorefractive polymers and their applications for imaging

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SCIENCE 1998年第5347期279卷 54-57页

作者： Kippelen, B Marder, SR Hendrickx, E Maldonado, JL Guillemet, G Volodin, BL Steele, DD Enami, Y Sandalphon Yao, YJ Wang, JF Röckel, H Erskine, L Peyghambarian, N Univ Arizona Ctr Opt Sci Tucson AZ 85721 USA CALTECH Beckman Inst Pasadena CA 91125 USA CALTECH Jet Prop Lab Pasadena CA 91109 USA

Photorefractive polymers with high diffraction efficiency in the visible and near-infrared regions of the electromagnetic spectrum have been developed, These polymers, which have a large dynamic range because of their high orientational birefringence, incorporate a dye designed to have a large dipole moment and a high linear polarizability anisotropy, Such polymers have enabled demonstrations of imaging through scattering media, using a holographic time-gating technique at a wavelength that is compatible with the transparency of biological tissues and with the emission of low-cost semiconductor laser diodes.

关键词： Birefringence Coloring Agents Diagnostic Imaging/methods image processing, computer-assisted Infrared Rays Infrared Rays Lasers Polymers Refractometry Scattering, Radiation Temperature

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High-probability uniquantal transmission at excitatory synapses in barrel cortex

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SCIENCE 2003年第5652期302卷 1981-1984页

作者： Silver, RA Lübke, J Sakmann, B Feldmeyer, D UCL Dept Physiol London WC1E 6BT England Forschungszentrum Julich Inst Med D-52425 Julich Germany Max Planck Inst Med Res Zellphysiol Abt D-69120 Heidelberg Germany

The number of vesicles released at excitatory synapses and the number of release sites per synaptic connection are key determinants of information processing in the cortex, yet they remain uncertain. Here we show that the number of functional release sites and the number of anatomically identified synaptic contacts are equal at connections between spiny stellate and pyramidal cells in rat barrel cortex. Moreover, our results indicate that the amount of transmitter released per synaptic contact is independent of release probability and the intrinsic release probability is high. These properties suggest that connections between layer 4 and layer 2/3 are tuned for reliable transmission of spatially distributed, timing-based signals.

关键词： Action Potentials Analysis of Variance Animals Axons/physiology Axons/ultrastructure Calcium/pharmacology Dendrites/physiology Dendrites/ultrastructure Dipeptides/pharmacology Excitatory Amino Acid Antagonists/pharmacology Excitatory Postsynaptic Potentials/drug effects Glutamic Acid/metabolism Glutamic Acid/metabolism image processing, computer-assisted Kynurenic Acid/pharmacology Lysine/analogs & derivatives Patch-Clamp Techniques Patch-Clamp Techniques Probability Pyramidal Cells/physiology Rats Rats, Wistar Receptors, Glutamate/metabolism Somatosensory Cortex/cytology Somatosensory Cortex/physiology Synapses/physiology Synaptic Transmission/physiology

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EVALUATION OF STORAGE PHOSPHOR IMAGING FOR QUANTITATIVE-ANALYSIS OF 2-D GELS USING THE QUEST-II SYSTEM

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BIOTECHNIQUES 1993年第6期15卷 1076-&页

作者： PATTERSON, SD LATTER, GI COLD SPRING HARBOR LAB BOX 1001 BUNGTOWN RDCOLD SPRING HARBORNY 11724

The advent of storage phosphor technology has been of considerable benefit to the imaging of gel-separated radiolabeled proteins due to the rapid and quantitative nature of the data acquisition process. Previously, times over one month were required to obtain fluorographs of the same gel to yield data o sufficient dynamic range for quantitative analysis of high-resolution two-dimensional (2-D) gels. As we are in the process of building a human 2-D gel protein database, and therefore have a high throughput of 2-D gels both to image and quantitate using the Quest II software, we undertook an evaluation of a storage phosphor imager, including an evaluation of signal fade. The results of this evaluation demonstrate the feasibility of using such a system, and we describe the procedures that allow us to use this technique for quantitative analysis of many complex 2-D gel patterns. These procedures include a useful batch printing program that allows printing of many images in a non-interactive mode. Examples will be presented of how autoradiography, using storage phosphor plates and the Quest II system, have enabled us to begin building a human 2-D gel protein database including posttranslational modification information, without the previous time constraints associated with such a project.

关键词： Autoradiography Autoradiography Cell Line Electrophoresis, Gel, Two-Dimensional Humans image processing, computer-assisted Luminescent Measurements Phosphoproteins/analysis Phosphorylation Software T-Lymphocytes/chemistry

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RELAXATION OF A SINGLE DNA MOLECULE OBSERVED BY OPTICAL MICROSCOPY

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SCIENCE 1994年第5160期264卷 822-826页

作者： PERKINS, TT QUAKE, SR SMITH, DE CHU, S STANFORD UNIV DEPT PHYSSTANFORDCA 94305

Single molecules of DNA, visualized in video fluorescence microscopy, were stretched to full extension in a flow, and their relaxation was measured when the flow stopped. The molecules, attached by one end to a 1-micrometer bead, were manipulated in an aqueous solution with optical tweezers. Inverse Laplace transformations of the relaxation data yielded spectra of decaying exponentials with distinct peaks, and the longest time component (tau) increased with length (L) as tau similar to L(1.66 +/- 0.10). A rescaling analysis showed that most of the relaxation curves had a universal shape and their characteristic times (lambda(t)) increased as lambda t similar to L(1.65 +/- 0.13). These results are in qualitative agreement with the theoretical prediction of dynamical scaling.

关键词： Algorithms Bacteriophage lambda DNA, Viral/chemistry Fluorescent Dyes image processing, computer-assisted Microscopy, Fluorescence Microspheres Models, Chemical Nucleic Acid Conformation

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High-resolution analysis of DNA replication in released chromatin fibers containing 5-bromodeoxyuridine

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BIOTECHNIQUES 1996年第6期21卷 1050-1054页

作者： Haaf, T Max-Planck-Institute of Molecular Genetics Berlin Germany. haaf@mping-berlin-dahlem.mpg.de

A strategy has been devised to physically map replication sites in released chromatin of mammalian cells. When added to the culture medium, 5-bromodeoxyuridine (BrdU) is incorporated into replicating DNA, partially replacing thymidine. BrdU pulses as short as one minute can be visualized on preparations of straightened chromatin fibers from protein-extracted nuclei by means of monoclonal anti-BrdU antibody. Short BrdU pulses (< 10 min) appear as strings of fluorescent signals that are 50-300 kb in length. This corresponds to the estimated size of individual replication units. Pulse chase experiments reveal that replicating DNA is tightly associated with the residual nuclear matrix, whereas newly replicated DNA is positioned on the released loop chromatin of nuclear halo preparations. Simultaneous fluorescence in situ hybridization (FISH) on BrdU-substituted released chromatin fibers suggests that replication can initiate at multiple sites anywhere within an alpha-satellite array of several Mb.

关键词： Bromodeoxyuridine/metabolism Cells, Cultured Chromatin/metabolism DNA/analysis DNA Replication/genetics Fibroblasts Fluorescent Antibody Technique, Indirect Fluorescent Antibody Technique, Indirect Humans image processing, computer-assisted

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