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Redesign of high-affinity nonspecific nucleases with altered sequence preference

Journal of the American Chemical Society 2009年第47期131卷 17345-17353页

作者： Wang, Yi-Ting Wright, Jon D. Doudeva, Lyudmila G. Jhang, Hua-Ci Lim, Carmay Yuan, Hanna S. Institute of Molecular Biology Academia Sinica Taipei Taiwan Institute of Biomedical Sciences Academia Sinica Taipei Taiwan Institute of Chemical Biology and Molecular Biophysics Taiwan International Graduate Program Academia Sinica Taipei Taiwan Institute of Bioinformatics and Structural Biology Department of Chemistry National Tsing Hua University HsinChu Taiwan Institute of Biochemistry and Molecular Biology National Taiwan University Taipei Taiwan

It is of crucial importance to elucidate the underlying principles that govern the binding affinity and selectivity between proteins and DNA. Here we use the nuclease domain of Colicin E7 (nColE7) as a model system to generate redesigned nucleases with improved DNA-binding affinities. ColE7 is a bacterial toxin, bearing a nonspecific endonuclease domain with a preference for hydrolyzing DNA phosphodiester bonds at the 3O-side after thymine and adenine;i.e., it prefers Thy and Ade at the -1 site. Using systematic computational screening, six nColE7 mutants were predicted to bind DNA with high affinity. Five of the redesigned single-point mutants were constructed and purified, and four mutants had a 3- to 5-fold higher DNA binding affinity than wild-type nColE7 as measured by fluorescence kinetic assays. Moreover, three of the designed mutants, D493N, D493Q, and D493R, digested DNA with an increased preference for guanine at +3 sites compared to the wild-type enzyme, as shown by DNA footprint assays. X-ray structure determination of the ColE7 mutant D493Q-DNA complex in conjunction with structural and free energy decomposition analyses provides a physical basis for the improved protein-DNA interactions: Replacing D493 at the protein-DNA interface with an amino acid residue that can maintain the native hydrogen bonds removes the unfavorable electrostatic repulsion between the negatively charged carboxylate and DNA phosphate groups. These results show that computational screening combined with biochemical, structural, and free energy analyses provide a useful means for generating redesigned nucleases with a higher DNA-binding affinity and altered sequence preferences in DNA cleavage. © 2009 American Chemical Society.

关键词： DNA

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Enhanced expression of β3-galactosyltransferase 5 activity is sufficient to induce in vivo synthesis of extended type 1 chains on lactosylceramides of selected human colonic carcinoma cell lines

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Glycobiology 2009年第4期19卷 418-427页

作者： Lin, Chi-Hung Fan, Yao-Yun Chen, Yen-Ying Wang, Shih-Hsin Chen, Chun-I Yu, Lung-Chih Khoo, Kay-Hooi Chemical Biology and Molecular Biophysics Program Taiwan International Graduate Program Academia Sinica Taipei 115 Taiwan Institute of Bioinformatics and Structural Biology National Tsing-Hua University Hsinchu 300 Taiwan Institute of Biological Chemistry Academia Sinica Nankang Taipei 115 Taiwan Taiwan and Institute of Biochemical Science National Taiwan University Taipei 106 Taiwan

In general, an elevated expression of β3-galactosyltrans-ferase (β3GalT) activity contributed by β3GalT5 correlates well with increased biosynthesis and expression of type 1 chain (Galβ1-3GlcNAcβ1-) derivatives such as Lewis A and sialyl Lewis A, which are mostly recognized as terminal epitopes and not further extended. Most known β3-N-acetylglucosaminyltransferases show a higher activity toward extending type 2 chain (Galβ1-4GlcNAcβ1-), and an over-expression of β3GalT5 could suppress the formation of the type 2 chain poly-N- acetyllactosaminoglycans. The potential of extending instead the predominant type 1 chain termini synthesized under such circumstances was, however, not investigated, partly due to technical difficulty in unambiguous identification of extended type 1 chains. Using an advanced mass spectrometry-based glycomic mapping and glycan sequencing approach, we show here that type 1 chains carried on the lacto-series glycosphingolipids of colonic carcinoma cells can be extended when the endogenous β3GalT activity relative to competing β4GalT activity, as defined against a common GlcNAcβ1-3Galβ1-4Glc acceptor, is sufficiently high, as found in Colo205 and SW1116, but not in DLD-1 cells. In support of this positive correlation, the lacto-series glycosphingolipids isolated from stably transfected DLD-1 clones over-expressing β3GalT5 were shown to comprise fucosylated dimeric type 1 chains, whereas a mock transfectant and the DLD-1 parent carried only fucosylated dimeric type 2 chains on their lactosylceramides. It suggests that while the natural expression of extended type 1 chain is likely to be determined by many contributing factors including the relative amounts of competing glycosyltransferases and the UDP-Gal level, the enhanced expression of β3GalT5 is sufficient to promote in vivo extension of type 1 chains by furnishing a significantly higher amount of type 1 chain precursors relative to competing type 2 chains. © The Author 2009. Published by Oxfo

关键词： β3-galactosyltransferase β3GalT5 Extended type 1 chain Glycomics Mass spectrometry

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structural insights into TDP-43, a new player in neurodegenerative diseases

Structural insights into TDP-43, a new player in neurodegene...

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亚洲晶体学联合大会

作者： Pan-Hsien Kuo Lyudmila G.Doudeva Che-Kun James Shen Hanna S.Yuan Institute of Bioinformatics and Structural Biology National Tsing Hua University Institute of Molecular Biology Academia Sinica Taiwan International Graduate Program Chemical Biology and Molecular BiophysicsAcademia Sinica Graduate Institute of Biochemistry and Molecular Biology National Taiwan University

TDP-43 is a pathogenic protein: its normal function in binding to UG-rich RNA is related to cystic fibrosis, and inclusion of its C-terminal fragments in brain cells is directly linked to frontotemporal lobar degenera... 详细信息

关键词： TDP RNA domain structural insights into TDP-43 a new player in neurodegenerative diseases

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Dual functions of an exosome component protein Rrp46/CRN-5 in RNA degradation and DNA fragmentation

Dual functions of an exosome component protein Rrp46/CRN-5 i...

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亚洲晶体学联合大会

作者： Yu-Yuan Hsiao Lyudmila G.Doudeva Hanna S.Yuan Institute of Molecular Biology Academia Sinica Institute of Bioinformatics and Structural Biology National Tsing Hua University Graduate Institute of Biochemistry and Molecular Biology National Taiwan University Taiwan Institute of Chemical Biology and Molecular Biophysics Taiwan International Graduate ProgramAcademia Sinica

Rrp46 is one of the RNase PH family proteins, first identified as a component protein of the eukaryotic exosome, a protein complex involved in RNA turnover, surveillance and processing. The Rrp46 homolog, CRN-5, was s... 详细信息

关键词： DNase Dual functions of an exosome component protein Rrp46/CRN-5 in RNA degradation and DNA fragmentation DNA exosome RNA

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ϕ-Value Analysis for Ultrafast Folding Proteins by NMR Relaxation Dispersion

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Journal of the American Chemical Society 2009年第2期132卷 450-451页

作者： Jae-Hyun Cho Nichole O’Connell Daniel P. Raleigh Arthur G. Palmer, III Department of Biochemistry and Molecular Biophysics Columbia University 630 West 168th Street New York New York 10032 and Department of Chemistry and Graduate Program in Biochemistry and Structural Biology State University of New York at Stony Brook Stony Brook New York 11794

Proteins that fold rapidly, on the (sub-) microsecond time scale, offer the prospect of direct comparison between experimental data and molecular dynamics simulations. However, experimental studies for such proteins frequently are hindered because folding rates are too fast to measure using conventional stopped-flow methods. To overcome this impediment, NMR spin relaxation dispersion experiments are used to quantify mutational effects on kinetics (ΔΔG°), stability (ΔΔG°), and ϕ-values (ΔΔG†/ΔΔG°) for proteins exhibiting chemical exchange line broadening that is fast on the NMR chemical shift time scale. The accuracy of ϕ-value analysis is enhanced because mutational effects on denatured or intermediate states can be detected through changes in line broadening. The transition and intermediate states of the villin headpiece domain, HP67, are characterized in varying solvent conditions to validate the method.

关键词： Kinetic parameters Genetics Colloids Quantum mechanics Nuclear magnetic resonance spectroscopy

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Glycans on influenza hemagglutinin affect receptor binding and immune response

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Proceedings of the National Academy of Sciences of the United States of America 2009年第43期106卷 18137-18142页

作者： Wang, Cheng-Chi Chen, Juine-Ruey Tseng, Yung-Chieh Hsu, Che-Hsiung Hung, Yu-Fu Chen, Shih-Wei Chen, Chin-Mei Khoo, Kay-Hooi Cheng, Ting-Jen Cheng, Yih-Shyun E. Jan, Jia-Tsrong Wu, Chung-Yi Ma, Che Wong, Chi-Huey Genomics Research Center Taipei 115 Taiwan Institute of Biochemical Sciences National Taiwan University Taipei 106 Taiwan Institute of Biochemistry and Molecular Biology National Yang-Ming University Taipei 112 Taiwan Chemical Biology and Molecular Biophysics Taiwan International Graduate Program Taipei 115 Taiwan Institute of Bioinformatics and Structural Biology National Tsing Hua University Hsinchu 300 Taiwan Institute of Biological Chemistry Academia Sinica Taipei 115 Taiwan

Recent cases of avian influenza H5N1 and the swine-origin 2009 H1N1 have caused a great concern that a global disaster like the 1918 influenza pandemic may occur again. Viral transmission begins with a critical interaction between hemagglutinin (HA) glycoprotein, which is on the viral coat of influenza, and sialic acid (SA) containing glycans, which are on the host cell surface. To elucidate the role of HA glycosylation in this important interaction, various defined HA glycoforms were prepared, and their binding affinity and specificity were studied by using a synthetic SA microarray. Truncation of the N-glycan structures on HA increased SA binding affinities while decreasing specificity toward disparate SA ligands. The contribution of each monosaccharide and sulfate group within SA ligand structures to HA binding energy was quantitatively dissected. It was found that the sulfate group adds nearly 100-fold (2.04 kcal/mol) in binding energy to fully glycosylated HA, and so does the biantennary glycan to the monoglycosylated HA glycoform. Antibodies raised against HA protein bearing only a single N-linked GlcNAc at each glycosylation site showed better binding affinity and neutralization activity against influenza subtypes than the fully glycosylated HAs elicited. Thus, removal of structurally nonessential glycans on viral surface glycoproteins may be a very effective and general approach for vaccine design against influenza and other human viruses.

关键词： Flu vaccine Glycan binding Glycosylation

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EXPERIMENTAL AND THEORETICAL STUDIES OF HYDROGEN BONDS IN FE-S PROTEINS

EXPERIMENTAL AND THEORETICAL STUDIES OF HYDROGEN BONDS IN FE...

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第十二届北京分析测试学术报告会暨展览会(BCEIA 2007)

作者： William M.WESTLER I-Jin LIN Erika B.GEBEL Timothy E.MACHONKIN Bin XIA John L.MARKLEY National Magnetic Resonance Facility at Madison University of Wisconsin-Madison Department of Biochemistry University of Wisconsin-Madison Graduate Program in Biophysics University of Wisconsin-Madison Program in Molecular and Computational Biophysics Johns Hopkins University Department of Chemical Biology College of Life Sciences Peking University

Iron-sulfur proteins are ubiquitous and essential to *** addition to their functional roles as electron transport proteins in photosynthesis and respiration,iron-sulfur proteins also are involved in nitrogen fixation,... 详细信息

关键词： NMR EXPERIMENTAL AND THEORETICAL STUDIES OF HYDROGEN BONDS IN FE-S PROTEINS

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Identifying interaction sites in "Recalcitrant" proteins: Predicted protein and RNA binding sites in REV proteins of HIV-1 and EIAV agree with experimental data

Identifying interaction sites in "Recalcitrant" proteins: Pr...

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11th Pacific Symposium on Biocomputing 2006, PSB 2006

作者： Terribllini, Michael Lee, Jae-Hyung Yan, Changhui Jernigan, Robert L. Carpenter, Susan Honavar, Vasant Dobbs, Drena Bioinformatics and Computational Biology Graduate Program LH. Baker Center for Bioinformatics and Biological Statistics Iowa State University Ames IA 50010 United States Department of Computer Science Iowa State University Ames IA 50010 United States Department of Biochemistry Biophysics and Molecular Biology Iowa State University Ames IA 50010 United States Department of Genetics Development and Cell Biology Iowa State University Ames IA 50010 United States Artificial Intelligence Research Laboratory Center for Computational Intelligence Learning and Discovery Iowa State University Ames IA 50010 United States Department of Veterinary Microbiology and Pathology Washington State University Pullman WA. 99164 United States

ISBN: (纸本)9812564632

Protein-protein and protein nucleic acid interactions are vitally important for a wide range of biological processes, including regulation or gene expression, protein synthesis, and replication and assembly of many viruses. We have developed machine learning approaches for predicting which amino acids of a protein participate in its interactions with other proteins and/or nucleic acids, using only the proiein sequence as input. In this paper, we describe an application of classifiers trained on datasets of well-characterized protein-protein and protein-RNA complexes for which experimental structures are available. We apply these classifiers to the problem of predicting protein and RNA binding sites in the sequence of a clinically important protein for which the structure is not known: the regulatory protein Rev, essential for the replication of HIV-I and other lentiviruses. We compare our predictions with published biochemical, genetic and partial structural information for HIV-1 and EIAV Rev and with our own published experimental mapping of RNA binding sites in EIAV Rev. The predicted and experimentally determined binding sites are in very good agreement. The ability to predict reliably the residues of a protein that directly contribute to specific binding events - without the requirement for structural information regarding either the protein or complexes in which it participates - can potentially generate new disease intervention strategies.

关键词： Binding sites

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Imaging Ice Embedded Single Particles With A 16 Megapixel CCD Camera

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Microscopy and Microanalysis 2003年第S2期9卷 962-963页

作者： Christopher R. Booth Donghua Chen Steven J. Ludtke Wah Chiu Program in Structural and Computational Biology and Molecular Biophysics National Center for Macromolecular Imaging Verna and Marrs McLean Department of Biochemistry and Molecular Biology Baylor College of Medicine Houston Texas 77030 USA

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Detection of virus particles in noisy spot-scan electron cryomicroscopy images

Detection of virus particles in noisy spot-scan electron cry...

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IEEE International Conference on Image Processing

作者： P. Thuman-Commike W. Chiu Program in Structural and Computational Biology and Molecular Biophysics Verna and Marrs McLean Department of Biochemistry The W. M. Keck Center for Computation Biology Baylor University College of Medicine Houston TX USA

We present a method to identify and extract virus particle images from noisy spot-scan electron cryomicroscopy images. We use a template matching algorithm to identify virus particles. Due to the high spatial variation and low contrast of these images we employ a series of preprocessing operations before performing the template matching. These preprocessing operations detect and remove the black areas present in spot-scan images using spatial histogram operations and binary mathematical morphology. Following the detailed description of our method we discuss the consistency and accuracy of particle selection.

关键词： Electrons Focusing computational biology Image processing molecular biophysics Biochemistry biology computing Educational institutions Biomedical imaging Histograms

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