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Journal of Structural and Functional Genomics

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https://www.readbyqxmd.com/read/27507291/successful-completion-of-a-semi-automated-enzyme-free-cloning-method
#1
Stefano Bonacci, Scilla Buccato, Domenico Maione, Roberto Petracca
Nowadays, in scientific fields such as Structural Biology or Vaccinology, there is an increasing need of fast, effective and reproducible gene cloning and expression processes. Consequently, the implementation of robotic platforms enabling the automation of protocols is becoming a pressing demand. The main goal of our study was to set up a robotic platform devoted to the high-throughput automation of the polymerase incomplete primer extension cloning method, and to evaluate its efficiency compared to that achieved manually, by selecting a set of bacterial genes that were processed either in the automated platform (330) or manually (94)...
September 2016: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/27400688/structural-mapping-of-kelch13-mutations-associated-with-artemisinin-resistance%C3%A2-in-malaria
#2
Gajinder Pal Singh, Preeti Goel, Amit Sharma
Mutations in Plasmodium falciparum gene kelch13 (pfkelch13) are strongly and causally associated with resistance to anti-malarial drug artemisinin, but their effects on PfKelch13 structure and function remain unclear. Utilizing the publicly available three-dimensional structure of PfKech13 (PDB ID: 4yy8), we find that most of the mutations in its propeller domain occur in two spatial clusters. Of these, one cluster is enriched in surface exposed residues which may drive PfKelch13-centered protein interactions, and the second cluster mostly contains residues which are buried and whose mutations may destabilize PfKelch13 structure...
September 2016: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/27400687/development-of-a-protein-ligand-binding-site-prediction-method-based-on-interaction-energy-and-sequence-conservation
#3
Hiroto Tsujikawa, Kenta Sato, Cao Wei, Gul Saad, Kazuya Sumikoshi, Shugo Nakamura, Tohru Terada, Kentaro Shimizu
We present a new method for predicting protein-ligand-binding sites based on protein three-dimensional structure and amino acid conservation. This method involves calculation of the van der Waals interaction energy between a protein and many probes placed on the protein surface and subsequent clustering of the probes with low interaction energies to identify the most energetically favorable locus. In addition, it uses amino acid conservation among homologous proteins. Ligand-binding sites were predicted by combining the interaction energy and the amino acid conservation score...
September 2016: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/27530928/nldb-a-database-for-3d-protein-ligand-interactions-in-enzymatic-reactions
#4
Yoichi Murakami, Satoshi Omori, Kengo Kinoshita
NLDB (Natural Ligand DataBase; URL: http://nldb.hgc.jp ) is a database of automatically collected and predicted 3D protein-ligand interactions for the enzymatic reactions of metabolic pathways registered in KEGG. Structural information about these reactions is important for studying the molecular functions of enzymes, however a large number of the 3D interactions are still unknown. Therefore, in order to complement such missing information, we predicted protein-ligand complex structures, and constructed a database of the 3D interactions in reactions...
August 16, 2016: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/27522608/homcos-an-updated-server-to-search-and-model-complex-3d-structures
#5
Takeshi Kawabata
The HOMCOS server ( http://homcos.pdbj.org ) was updated for both searching and modeling the 3D complexes for all molecules in the PDB. As compared to the previous HOMCOS server, the current server targets all of the molecules in the PDB including proteins, nucleic acids, small compounds and metal ions. Their binding relationships are stored in the database. Five services are available for users. For the services "Modeling a Homo Protein Multimer" and "Modeling a Hetero Protein Multimer", a user can input one or two proteins as the queries, while for the service "Protein-Compound Complex", a user can input one chemical compound and one protein...
August 13, 2016: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/27023709/expression-purification-and-crystallization-of-schizosaccharomyces-pombe-eif2b
#6
Kazuhiro Kashiwagi, Tomoaki Shigeta, Hiroaki Imataka, Takuhiro Ito, Shigeyuki Yokoyama
Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter...
March 2016: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/26984848/premeltons-in-dna
#7
Henry M Sobell
Premeltons are examples of emergent-structures (i.e., structural-solitons) that arise spontaneously in DNA due to the presence of nonlinear-excitations in its structure. They are of two kinds: B-B (or A-A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally-nontopological, undergo breather-motions that allow drugs and dyes to intercalate into DNA. B-A (or A-B) premeltons, on the other hand, are mobile, and being globally-topological, act as phase-boundaries transforming B- into A-DNA during the structural phase-transition...
March 2016: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/26935210/the-impact-of-structural-genomics-the-first-quindecennial
#8
REVIEW
Marek Grabowski, Ewa Niedzialkowska, Matthew D Zimmerman, Wladek Minor
The period 2000-2015 brought the advent of high-throughput approaches to protein structure determination. With the overall funding on the order of $2 billion (in 2010 dollars), the structural genomics (SG) consortia established worldwide have developed pipelines for target selection, protein production, sample preparation, crystallization, and structure determination by X-ray crystallography and NMR. These efforts resulted in the determination of over 13,500 protein structures, mostly from unique protein families, and increased the structural coverage of the expanding protein universe...
March 2016: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/26671275/gene-selection-and-cloning-approaches-for-co-expression-and-production-of-recombinant-protein-protein-complexes
#9
György Babnigg, Robert Jedrzejczak, Boguslaw Nocek, Adam Stein, William Eschenfeldt, Lucy Stols, Norman Marshall, Alicia Weger, Ruiying Wu, Mark Donnelly, Andrzej Joachimiak
Multiprotein complexes play essential roles in all cells and X-ray crystallography can provide unparalleled insight into their structure and function. Many of these complexes are believed to be sufficiently stable for structural biology studies, but the production of protein-protein complexes using recombinant technologies is still labor-intensive. We have explored several strategies for the identification and cloning of heterodimers and heterotrimers that are compatible with the high-throughput (HTP) structural biology pipeline developed for single proteins...
December 2015: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/26573864/automated-protein-motif-generation-in-the-structure-based-protein-function-prediction-tool-promol
#10
Mikhail Osipovitch, Mitchell Lambrecht, Cameron Baker, Shariq Madha, Jeffrey L Mills, Paul A Craig, Herbert J Bernstein
ProMOL, a plugin for the PyMOL molecular graphics system, is a structure-based protein function prediction tool. ProMOL includes a set of routines for building motif templates that are used for screening query structures for enzyme active sites. Previously, each motif template was generated manually and required supervision in the optimization of parameters for sensitivity and selectivity. We developed an algorithm and workflow for the automation of motif building and testing routines in ProMOL. The algorithm uses a set of empirically derived parameters for optimization and requires little user intervention...
December 2015: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/25854603/expression-platforms-for-producing-eukaryotic-proteins-a-comparison-of-e-coli-cell-based-and-wheat-germ-cell-free-synthesis-affinity-and-solubility-tags-and-cloning-strategies
#11
David J Aceti, Craig A Bingman, Russell L Wrobel, Ronnie O Frederick, Shin-Ichi Makino, Karl W Nichols, Sarata C Sahu, Lai F Bergeman, Paul G Blommel, Claudia C Cornilescu, Katarzyna A Gromek, Kory D Seder, Soyoon Hwang, John G Primm, Grzegorz Sabat, Frank C Vojtik, Brian F Volkman, Zsolt Zolnai, George N Phillips, John L Markley, Brian G Fox
Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E...
June 2015: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/25832174/crystal-structures-of-mycobacterial-meab-and-mmaa-like-gtpases
#12
Thomas E Edwards, Loren Baugh, Jameson Bullen, Ruth O Baydo, Pam Witte, Kaitlin Thompkins, Isabelle Q H Phan, Jan Abendroth, Matthew C Clifton, Banumathi Sankaran, Wesley C Van Voorhis, Peter J Myler, Bart L Staker, Christoph Grundner, Donald D Lorimer
The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis. However, the identity of the mycobacterial MeaB homolog is presently unclear...
June 2015: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/25801860/solution-structures-of-the-dna-binding-domains-of-immune-related-zinc-finger-protein-zfat
#13
Naoya Tochio, Takashi Umehara, Kazuhiko Nakabayashi, Misao Yoneyama, Kengo Tsuda, Mikako Shirouzu, Seizo Koshiba, Satoru Watanabe, Takanori Kigawa, Takehiko Sasazuki, Senji Shirasawa, Shigeyuki Yokoyama
ZFAT is a transcriptional regulator, containing eighteen C2H2-type zinc-fingers and one AT-hook, involved in autoimmune thyroid disease, apoptosis, and immune-related cell survival. We determined the solution structures of the thirteen individual ZFAT zinc-fingers (ZF) and the tandemly arrayed zinc-fingers in the regions from ZF2 to ZF5, by NMR spectroscopy. ZFAT has eight uncommon bulged-out helix-containing zinc-fingers, and six of their structures (ZF4, ZF5, ZF6, ZF10, ZF11, and ZF13) were determined. The distribution patterns of the putative DNA-binding surface residues are different among the ZFAT zinc-fingers, suggesting the distinct DNA sequence preferences of the N-terminal and C-terminal zinc-fingers...
June 2015: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/25758186/crystal-structure-of-the-mazg-related-nucleoside-triphosphate-pyrophosphohydrolase-from-thermotoga-maritima-msb8
#14
Balasundaram Padmanabhan, Prashant Deshmukh, Shigeyuki Yokoyama, Yoshitaka Bessho
The MazG family proteins, which are highly conserved in bacteria, are nucleoside triphosphate pyrophosphohydrolases that hydrolyze all canonical nucleoside triphosphates, and are also involved in removing noncanonical nucleoside triphosphates to prevent their incorporation into DNA or RNA. The primary structure of TM0360 from Thermotoga maritima MSB8 suggested that TM0360 is a MazG-related nucleoside triphosphate pyrophosphohydrolase. The crystal structure of the TM0360 protein was determined by the MAD technique at 2...
June 2015: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/25630330/annotation-of-proteins-of-unknown-function-initial-enzyme-results
#15
Talia McKay, Kaitlin Hart, Alison Horn, Haeja Kessler, Greg Dodge, Keti Bardhi, Kostandina Bardhi, Jeffrey L Mills, Herbert J Bernstein, Paul A Craig
Working with a combination of ProMOL (a plugin for PyMOL that searches a library of enzymatic motifs for local structural homologs), BLAST and Pfam (servers that identify global sequence homologs), and Dali (a server that identifies global structural homologs), we have begun the process of assigning functional annotations to the approximately 3,500 structures in the Protein Data Bank that are currently classified as having "unknown function". Using a limited template library of 388 motifs, over 500 promising in silico matches have been identified by ProMOL, among which 65 exceptionally good matches have been identified...
March 2015: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/25618148/a-selb-ef-tu-aif2%C3%AE-like-protein-from-methanosarcina-mazei-in-the-gtp-bound-form-binds-cysteinyl-trna-cys
#16
Tatsuo Yanagisawa, Ryohei Ishii, Yasushi Hikida, Ryuya Fukunaga, Toru Sengoku, Shun-ichi Sekine, Shigeyuki Yokoyama
The putative translation elongation factor Mbar_A0971 from the methanogenic archaeon Methanosarcina barkeri was proposed to be the pyrrolysine-specific paralogue of EF-Tu ("EF-Pyl"). In the present study, the crystal structures of its homologue from Methanosarcina mazei (MM1309) were determined in the GMPPNP-bound, GDP-bound, and apo forms, by the single-wavelength anomalous dispersion phasing method. The three MM1309 structures are quite similar (r.m.s.d. < 0.1 Å). The three domains, corresponding to domains 1, 2, and 3 of EF-Tu/SelB/aIF2γ, are packed against one another to form a closed architecture...
March 2015: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/25575462/expression-purification-crystallization-and-preliminary-x-ray-crystallographic-studies-of-the-human-adiponectin-receptors-adipor1-and-adipor2
#17
Hiroaki Tanabe, Kanna Motoyama, Mariko Ikeda, Motoaki Wakiyama, Takaho Terada, Noboru Ohsawa, Toshiaki Hosaka, Masakatsu Hato, Yoshifumi Fujii, Yoshihiro Nakamura, Satoshi Ogasawara, Tomoya Hino, Takeshi Murata, So Iwata, Miki Okada-Iwabu, Masato Iwabu, Kunio Hirata, Yoshiaki Kawano, Masaki Yamamoto, Tomomi Kimura-Someya, Mikako Shirouzu, Toshimasa Yamauchi, Takashi Kadowaki, Shigeyuki Yokoyama
The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices. These receptors regulate glucose and fatty acid metabolism, thereby ameliorating type 2 diabetes. The full-length human AdipoR1 and a series of N-terminally truncated mutants of human AdipoR1 and AdipoR2 were expressed in insect cells. In small-scale size exclusion chromatography, the truncated mutants AdipoR1Δ88 (residues 89-375) and AdipoR2Δ99 (residues 100-386) eluted mostly in the intact monodisperse state, while the others eluted primarily as aggregates...
March 2015: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/25421040/x-ray-structure-of-the-mature-ectodomain-of-phogrin
#18
Martín E Noguera, María E Primo, Jean Jakoncic, Edgardo Poskus, Michele Solimena, Mario R Ermácora
Phogrin/IA-2β and ICA512/IA-2 are two paralogs receptor-type protein-tyrosine phosphatases (RPTP) that localize in secretory granules of various neuroendocrine cells. In pancreatic islet β-cells, they participate in the regulation of insulin secretion, ensuring proper granulogenesis, and β-cell proliferation. The role of their cytoplasmic tail has been partially unveiled, while that of their luminal region remains unclear. To advance the understanding of its structure-function relationship, the X-ray structure of the mature ectodomain of phogrin (ME phogrin) at pH 7...
March 2015: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/25398586/development-of-a-hexahistidine-3%C3%A3-flag-tandem-affinity-purification-method-for-endogenous-protein-complexes-in-pichia-pastoris
#19
Toshiaki Higo, Noriyuki Suka, Haruhiko Ehara, Masatoshi Wakamori, Shin Sato, Hideaki Maeda, Shun-ichi Sekine, Takashi Umehara, Shigeyuki Yokoyama
We developed a method for efficient chromosome tagging in Pichia pastoris, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from P. pastoris. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis...
December 2014: Journal of Structural and Functional Genomics
https://www.readbyqxmd.com/read/25306867/structural-characterization-of-the-putative-abc-type-2-transporter-from-thermotoga-maritima-msb8
#20
Ekaterina V Filippova, Karolina L Tkaczuk, Maksymilian Chruszcz, Xiaohui Xu, Alexei Savchenko, Aled Edwards, Wladek Minor
This study describes the structure of the putative ABC-type 2 transporter TM0543 from Thermotoga maritima MSB8 determined at a resolution of 2.3 Å. In comparative sequence-clustering analysis, TM0543 displays similarity to NatAB-like proteins, which are components of the ABC-type Na(+) efflux pump permease. However, the overall structure fold of the predicted nucleotide-binding domain reveals that it is different from any known structure of ABC-type efflux transporters solved to date. The structure of the putative TM0543 domain also exhibits different dimer architecture and topology of its presumed ATP binding pocket, which may indicate that it does not bind nucleotide at all...
December 2014: Journal of Structural and Functional Genomics
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