Vinay Kumar Singh

Publications

• 5.36

  Impact points

  Crystal structure of a novel prokaryotic Ser/Thr kinase and its implication in the Cpx stress response pathway.

  Jimin Zheng, Chunhua He, Vinay Kumar Singh, Nancy L Martin, Zongchao Jia

  Molecular microbiology. 04/2007; 63(5):1360-71.

  The Cpx signalling system of Escherichia coli and Salmonella enterica senses extracytoplasmic stress and controls expression of factors that allow the bacterium to adapt to these stressors and thereby enhance survival. Many of the Cpx-responsive genes products are of unknown function. We determined ... [more] The Cpx signalling system of Escherichia coli and Salmonella enterica senses extracytoplasmic stress and controls expression of factors that allow the bacterium to adapt to these stressors and thereby enhance survival. Many of the Cpx-responsive genes products are of unknown function. We determined the crystal structure of one of these gene products, called YihE in E. coli, which exhibits a eukaryotic kinase fold. Functional assays established that both YihE and the S. enterica YihE homologue, RdoA, undergo autophosphorylation and phosphorylate protein substrates at Ser/Thr residues in vitro, demonstrating that YihE/RdoA is a novel Ser/Thr protein kinase in prokaryotic cells. Phenotypic analysis of yihE/rdoA null strains indicates that this kinase is most abundant in stationary phase, and is important for long-term cell survival and for expression of surface appendages in both a Cpx-independent and -dependent manner. YihE/RdoA is therefore a previously unknown kinase component of a new type of bacterial phosphorelay mechanism, adding kinase activity as another response to the Cpx sensing system that functions to maintain cellular homeostasis.
• 5.90

  Impact points

  Identification of an ITPase/XTPase in Escherichia coli by structural and biochemical analysis.

  Jimin Zheng, Vinay Kumar Singh, Zongchao Jia

  Structure (London, England : 1993). 11/2005; 13(10):1511-20.

  Inosine triphosphate (ITP) and xanthosine triphosphate (XTP) are formed upon deamination of ATP and GTP as a result of exposure to chemical mutagens and oxidative damage. Nucleic acid synthesis requires safeguard mechanisms to minimize undesired lethal incorporation of ITP and XTP. Here, we present ... [more] Inosine triphosphate (ITP) and xanthosine triphosphate (XTP) are formed upon deamination of ATP and GTP as a result of exposure to chemical mutagens and oxidative damage. Nucleic acid synthesis requires safeguard mechanisms to minimize undesired lethal incorporation of ITP and XTP. Here, we present the crystal structure of YjjX, a protein of hitherto unknown function. The three-dimensional fold of YjjX is similar to those of Mj0226 from Methanococcus janschii, which possesses nucleotidase activity, and of Maf from Bacillus subtilis, which can bind nucleotides. Biochemical analyses of YjjX revealed it to exhibit specific phosphatase activity for inosine and xanthosine triphosphates and have a possible interaction with elongation factor Tu. The enzymatic activity of YjjX as an inosine/xanthosine triphosphatase provides evidence for a plausible protection mechanism by clearing the noncanonical nucleotides from the cell during oxidative stress in E. coli.
• 1.56

  Impact points

  Refolding and one-step purification of recombinant human ARA70 over-expressed in Escherichia coli.

  Vinay Kumar Singh, Zongchao Jia

  Protein expression and purification. 03/2005; 39(2):283-7.

  Androgen receptor (AR)-associated coregulator 70 (ARA70) is a cytoplasmic protein that has been characterized to have the ability to induce AR transcriptional activity in response to androgens and anti-androgens in prostate cancer cells. AR has been shown to have an important role in the progression... [more] Androgen receptor (AR)-associated coregulator 70 (ARA70) is a cytoplasmic protein that has been characterized to have the ability to induce AR transcriptional activity in response to androgens and anti-androgens in prostate cancer cells. AR has been shown to have an important role in the progression of prostate cancer and in normal male reproductive system development. To elucidate the molecular mechanisms and biological relevance of ARA70 to prostrate cancer using a variety of biochemical analyses, the cDNA encoding full length ARA70 was cloned into pET21b vector. Here, we report the refolding and one-step purification of ARA70 from inclusion bodies over-expressed in Escherichia coli. The protein was purified to homogeneity, yielding approximately 60 mg ARA70 from 1L of terrific broth media. Refolding process of ARA70 was monitored using far-UV CD analysis.
• 0.55

  Impact points

  Crystallization and preliminary X-ray analysis of the GST-fused human Bri3 N-terminal domain.

  Qilu Ye, Vinay Kumar Singh, James Daniel Blonde, Zongchao Jia

  Acta crystallographica. Section F, Structural biology and crystallization communications. 02/2005; 61(Pt 1):62-4.

  Bri3 is a recently identified proline-rich transmembrane polypeptide up-regulated during TNF-mediated inflammation and immunity. The polyproline-rich N-terminal (residues 1-60) domain of Bri3 was affinity-purified to homogeneity as a glutathione-S-transferase (GST) fusion protein. Crystals were obta... [more] Bri3 is a recently identified proline-rich transmembrane polypeptide up-regulated during TNF-mediated inflammation and immunity. The polyproline-rich N-terminal (residues 1-60) domain of Bri3 was affinity-purified to homogeneity as a glutathione-S-transferase (GST) fusion protein. Crystals were obtained in approximately 3 d by the equilibrium vapour-diffusion method from a solution containing 1.5-2.2 M ammonium sulfate and 0.1 M bis-tris pH 6.0. The crystals belong to space group P4(3)2(1)2, with unit-cell parameters a = b = 91.66, c = 57.53 A. An X-ray data set was collected to 1.6 A resolution using synchrotron radiation, with an Rsym of 0.058 and a completeness of 95.3%. There is one molecule of the fusion protein in the asymmetric unit, which corresponds to approximately 35% solvent content.

• Tri-cistronic cloning, overexpression and purification of human Rad9, Rad1, Hus1 protein complex

  Vinay Kumar Singh, Salima Nurmohamed, Scott K. Davey, Zongchao Jia

  Protein Expression and Purification.

  The least understood components of the DNA damage checkpoint are the DNA damage sensors. Genetic studies of Schizosaccharomyces pombe identified six yeast genes, Rad3, Rad17, Rad9, Rad1, Hus1, and Rad26, which encode proteins thought to sense DNA damage and activate the checkpoint-signaling cascade.... [more] The least understood components of the DNA damage checkpoint are the DNA damage sensors. Genetic studies of Schizosaccharomyces pombe identified six yeast genes, Rad3, Rad17, Rad9, Rad1, Hus1, and Rad26, which encode proteins thought to sense DNA damage and activate the checkpoint-signaling cascade. It has been suggested that Rad9, Rad1 and Hus1 make a heterotrimeric complex forming a PCNA-like structure. In order to carry out structural and biophysical studies of the complex and its associated proteins, the cDNAs encoding full length human Rad9, Rad1 and Hus1 were cloned together into the pET28a vector using a one-step ligation procedure. Here we report successful tri-cistronic cloning, overexpression and purification of this three-protein complex using a single hexa-histidine tag. The trimeric protein complex of Rad9, Rad1 and Hus1 was purified to near homogeneity, yielding ∼10 mg of protein from one liter of Escherichia coli culture.