02426nas a2200277   4500008004100000022001400041245011100055210006900166260009500235300001200330490000800342520139600350653002301746653001601769653001501785653001901800653004801819100002101867700002401888700002601912700002201938700001901960700002501979700002102004856012302025       2009                        eng d  a0006-291X00aConformational characterization of human eukaryotic initiation factor 2 alpha: a single tryptophan protein0 aConformational characterization of human eukaryotic initiation f  a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcDEC  a273-2790 v3903 a<p>The alpha-subunit of the human eukaryotic initiation factor 2 (heIF2 alpha), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2 alpha gets phosphorylated by eIF2 alpha-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2 alpha, a protein with a single tryptophan (trp) residue was examined for its conformational characteristics using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The steady-state fluorescence spectrum, fluorescence lifetimes (tau(1) = 1.13 ns and tau(2) = 4.74 ns) and solute quenching studies revealed the presence of trp conformers in hydrophobic and differential polar environment at any given time. Estimation of the alpha-helix and beta-sheet content showed: (i) more compact structure at pH 2.0, (ii) distorted alpha-helix and rearranged beta-sheet in presence of 4 M guanidine hydrochloride and (iii) retention of more than 50% ordered structure at 95 degrees C. Hydrophobic dye binding to the protein with loosened tertiary structure was observed at pH 2.0 indicating the existence of a molten globule-like structure. These observations indicate the inherent structural stability of the protein under various denaturing conditions. (C) 2009 Elsevier Inc. All rights reserved.</p>10aCircular dichroism10aheIF2 alpha10aK562 cells10aMolten globule10aSteady-state and time-resolved fluorescence1 aSreejith, R., K.1 aYadav, Viveka, Nand1 aVarshney, Nishant, K.1 aBerwal, Sunil, K.1 aSuresh, C., G.1 aGaikwad, Sushama, M.1 aPal, Jayanta, K.  uhttps://library.ncl.res.in/content/conformational-characterization-human-eukaryotic-initiation-factor-2-alpha-single-002072nas a2200205   4500008004100000022001500041245010100056210006900157260000800226300001400234490000800248520136500256100002201621700001901643700001901662700001901681700002501700700002101725856012001746       2018                        eng d  a 0141-813000aActivation of HRI is mediated by Hsp90 during stress through modulation of the HRI-Hsp90 complex0 aActivation of HRI is mediated by Hsp90 during stress through mod  cOCT  a1604-16130 v1183 aHeme Regulated Inhibitor (HRI) is known to get activated in various stresses such as heme deficiency, heat shock, heavy metal toxicity etc. Heat shock protein 90 (Hsp90), a ubiquitous cytoplasmic protein interacts with HRI in order to regulate protein synthesis. However, it still remains to establish this interaction of HRI and Hsp90 at cellular levels and how this modulation of HRI activity is mediated by Hsp90 during stress. In the present report, using co-immunoprecipitation analysis we show that HRI interacts with Hsp90 and this association is independent of other co-chaperones in in vitro conditions. Further, analysis using truncated domains of HRI revealed that the K1 subdomain is essential for HRI - Hsp90 complex formation. Our in silico protein - protein interaction studies also indicated interaction of Hsp90 with K1 subdomain of HRI. Mammalian two hybrid assay validated this HRI - Hsp90 interaction at cellular levels. When the in vitro kinase assay was carried out with the co-immunoprecipitated complex of HRI - Hsp90, an increase in the kinase activity was observed resulting elevated levels of eIF2 alpha phosphorylation upon heavy metal stress and heat shock. Thus, our results clearly indicate modulation of HRI kinase activity with simultaneous Hsp90 association under stress conditions. (C) 2018 Elsevier B.V. All rights reserved.1 aBerwal, Sunil, K.1 aBhatia, Varsha1 aBendre, Arneya1 aSuresh, C., G.1 aChatterjee, Sangeeta1 aPal, Jayanta, K.  uhttps://library.ncl.res.in/content/activation-hri-mediated-hsp90-during-stress-through-modulation-hri-hsp90-complex