Browsing by Author "Choi, Yook-Wah"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Cellular Characterization of SARS Coronavirus Nucleocapsid
    (Leibniz Research Centre for Working Environment and Human Factors, 2004) Goh, Phuay-Yee; Choi, Yook-Wah; Shen, Shuo; Tan, Yee-Joo; Fielding, Burtram C.; Tan, Timothy H.P.; O, Eng-Eong; Lim, Seng Gee; Hong, Wanjin
    The Severe and Acute Respiratory Syndrome coronavirus (SARS CoV) is a newly-emerged virus that caused an outbreak of atypical pneumonia in the winter of 2002-2003. Polyclonal antibodies raised against the nucleocapsid (N) of the SARS CoV showed the localization of N to the cytoplasm and the nucleolus in virus-infected and N-expressing Vero E6 cells. Like other coronavirus N proteins, the SARS N is probably a phosphoprotein. N protein expressed in mammalian cells is apparently able to “spread” to neighboring cells. For N to spread to neighboring cells, it must be exported out of the expressing cells. This is shown by the immunoprecipitation of N from the culture medium of a stable cell line expressing myc-N. Deletion studies showed that the 27 kD C-terminal domain of N (C1/2) is the minimal region of N that can spread to other cells. The nucleolar localization and spreading of N are artefacts of fixation, reminiscent of other protein-transduction domain (PTD)-containing proteins
  • Thumbnail Image
    Item
    Expression, purification and preliminary crystallographic analysis of recombinant human DEAD-box polypeptide 5
    (International Union of Crystallography, 2010) Choi, Yook-Wah; Dutta, Sujit; Fielding, Burtram C.; Tan, Yee-Joo
    The DEAD-box RNA helicase DDX5 is involved in many aspects of RNA processing and has been implicated in a number of cellular processes involving alteration of RNA secondary structure. The N-terminal region of DDX5, which contains the conserved domain 1 of the DEAD-box helicases, has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of this region is reported. X-ray diffraction data were processed to a resolution of 2.7 A ° . The crystals belonged to space group I222, with unit-cell parameters a = 66.18, b = 73.80, c = 104.00 A ° , = = = 90 .
  • No Thumbnail Available
    Item
    The variable N-terminal region of DDX5 contains structural elements and auto-inhibits its interaction with NS5B of hepatitis C virus
    (Portland Press, 2012) Dutta, Sujit; Gupta, Garvita; Choi, Yook-Wah; Kotaka, Masayo; Fielding, Burtram C.; Song, Jianxing; Tan, Yee-Joo
    RNA helicases of the DEAD (Asp-Glu-Ala-Asp)-box family of proteins are involved in many aspects of RNA metabolism from transcription to RNA decay, but most of them have also been shown to be multifunctional. The DEAD-box helicase DDX5 of host cells has been shown to interact with the RNA-dependent RNA polymerase (NS5B) of HCV (hepatitis C virus). In the present study, we report the presence of two independent NS5Bbinding sites in DDX5, one located at the N-terminus and another at the C-terminus. The N-terminal fragment of DDX5, which consists of the first 305 amino acids and shall be referred as DDX5-N, was expressed and crystallized. The crystal structure shows that domain 1 (residues 79–303) of DDX5 contains the typical features found in the structures of other DEADbox helicases. DDX5-N also contains the highly variable NTR (N-terminal region) of unknown function and the crystal structure reveals structural elements in part of the NTR, namely residues 52–78. This region forms an extensive loop and an α-helix. From co-immunoprecipitation experiments, the NTR of DDX5-N was observed to auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1–51 residues in NTR and the NS5B-binding site in DDX5-N. Furthermore, NMR investigations reveal that there is a direct interaction between DDX5 and NS5B in vitro.