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1.  Crystallization and preliminary X-ray analysis of a glucansucrase from the dental caries pathogen Streptococcus mutans  
In this study, the glucansucrase from the dental caries pathogen S. mutans was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant.
Glucansucrases encoded by Streptococcus mutans play essential roles in the synthesis of sticky dental plaques. Based on amino-acid sequence similarity, glucansucrases are classified as members of glycoside hydrolase family 70 (GH 70). Data on the crystal structure of GH 70 glucansucrases have yet to be reported. Here, the GH 70 glucansucrase GTF-SI from S. mutans was overexpressed in Escherichia coli strain BL21 (DE3), purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Orthorhombic GTF-SI crystals belonging to space group P21212 were obtained. A diffraction data set was collected to 2.1 Å resolution.
PMCID: PMC2935234  PMID: 20823533
glucansucrase; dental caries; Streptococcus mutans
2.  Export Pathway Selectivity of Escherichia coli Twin Arginine Translocation Signal Peptides*s 
The Journal of biological chemistry  2007;282(11):8309-8316.
The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a ≥ +1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.
PMCID: PMC2730154  PMID: 17218314
3.  Regulation of RraA, a Protein Inhibitor of RNase E-Mediated RNA Decay 
Journal of Bacteriology  2006;188(9):3257-3263.
The recently discovered RraA protein acts as an inhibitor of the essential endoribonuclease RNase E, and we demonstrated that ectopic expression of RraA affects the abundance of more than 700 transcripts in Escherichia coli (K. Lee, X. Zhan, J. Gao, J. Qiu, Y. Feng, R. Meganathan, S. N. Cohen, and G. Georgiou, Cell 114:623-634, 2003). We show that rraA is expressed from its own promoter, PrraA, located in the menA-rraA intergenic region. Primer extension and lacZ fusion analysis revealed that transcription from PrraA is elevated upon entry into stationary phase in a σs-dependent manner. In addition, the stability of the rraA transcript is dependent on RNase E activity, suggesting the involvement of a feedback circuit in the regulation of the RraA level in E. coli.
PMCID: PMC1447450  PMID: 16621818
4.  PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets 
Nucleic Acids Research  2005;33(17):e150.
We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale.
PMCID: PMC1251669  PMID: 16214800
5.  Exhaustive identification of interaction domains using a high-throughput method based on two-hybrid screening and PCR-convergence: molecular dissection of a kinetochore subunit Spc34p 
Nucleic Acids Research  2003;31(23):6953-6962.
The Dam1 complex, also known as DASH complex, is the outer kinetochore protein complex of yeast that plays a crucial role in attachment of kinetochore to microtubule. The Dam1 complex is formed by at least nine proteins including Dam1p, Duo1p, Dad1p, Spc19p and Spc34p. In this study, domains of Spc34p that physically interact with other subunits of the complex were mapped using a high-throughput methodology. The method is a combination of two-hybrid screening of a random truncation library of the Spc34 gene and a unique PCR-based amplification that converge the selected DNA fragments to a few short fragments. Duo1p, Dam1p, Dad1p and Spc19p binding domains of Spc34p were mapped on M1-E59, M1-D47, M1-D47 or T207-E295 and S154-Q294, respectively. Most of the boundaries were located at less conserved regions among fungal Spc34p homologs, which is consistent with the boundaries of the putative secondary structures. The accuracy of the mapped domain boundaries was verified using truncated Spc34p polypeptides. The results and methodology we demonstrated herein not only shed light on the molecular architecture of the protein complex but also pave the road to the high-throughput identification of specific interaction domains of proteins whose possible interaction partners have been identified in genome-scale analyses.
PMCID: PMC290257  PMID: 14627828
6.  Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers 
Nucleic Acids Research  2003;31(21):e126.
SCRATCHY is a methodology for the construction of libraries of chimeras between genes that display low sequence homology. We have developed a strategy for library creation termed enhanced crossover SCRATCHY, that significantly increases the number of clones containing multiple crossovers. Complementary chimeric gene libraries generated by incremental truncation (ITCHY) of two distinct parental sequences are created, and are then divided into arbitrarily defined sections. The respective sections are amplified by skewed sets of primers (i.e. a combination of gene A specific forward primer and gene B specific reverse primer, etc.) allowing DNA fragments containing non-homologous crossover points to be amplified. The amplified chimeric sections are then subjected to a DNA shuffling process generating an enhanced crossover SCRATCHY library. We have constructed such a library using the rat theta 2 glutathione transferase (rGSTT2) and the human theta 1 glutathione transferase (hGSTT1) genes (63% DNA sequence identity). DNA sequencing analysis of unselected library members revealed a greater diversity than that obtained by canonical family shuffling or with conventional SCRATCHY. Expression and high-throughput flow cytometric screening of the chimeric GST library identified several chimeric progeny that retained rat-like parental substrate specificity.
PMCID: PMC275483  PMID: 14576326
7.  Analysing the substrate multispecificity of a proton-coupled oligopeptide transporter using a dipeptide library 
Nature Communications  2013;4:2502.
Peptide uptake systems that involve members of the proton-coupled oligopeptide transporter (POT) family are conserved across all organisms. POT proteins have characteristic substrate multispecificity, with which one transporter can recognize as many as 8,400 types of di/tripeptides and certain peptide-like drugs. Here we characterize the substrate multispecificity of Ptr2p, a major peptide transporter of Saccharomyces cerevisiae, using a dipeptide library. The affinities (Ki) of di/tripeptides toward Ptr2p show a wide distribution range from 48 mM to 0.020 mM. This substrate multispecificity indicates that POT family members have an important role in the preferential uptake of vital amino acids. In addition, we successfully establish high performance ligand affinity prediction models (97% accuracy) using our comprehensive dipeptide screening data in conjunction with simple property indices for describing ligand molecules. Our results provide an important clue to the development of highly absorbable peptides and their derivatives including peptide-like drugs.
Proton-coupled oligopeptide transporters (POTs) can recognize and mediate the uptake of up to 8,400 di/tripeptides or peptide-like drugs. Ito et al. comprehensively map the substrate specificity of the yeast POT Ptr2p, and use this information to construct models for the prediction of ligand affinity.
PMCID: PMC3791473  PMID: 24060756

Results 1-7 (7)