Conformational characterization of human eukaryotic initiation factor 2alpha: a single tryptophan protein

The alpha-subunit of the human eukaryotic initiation factor 2 (heIF2alpha), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2alpha gets phosphorylated by eIF2alpha-specific kinases resulting in inhibition of protein synthesis....

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Veröffentlicht in:Biochemical and biophysical research communications 2009-12, Vol.390 (2), p.273-279
Hauptverfasser: Sreejith, R K, Yadav, Viveka Nand, Varshney, Nishant K, Berwal, Sunil K, Suresh, C G, Gaikwad, Sushama M, Pal, Jayanta K
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container_end_page 279
container_issue 2
container_start_page 273
container_title Biochemical and biophysical research communications
container_volume 390
creator Sreejith, R K
Yadav, Viveka Nand
Varshney, Nishant K
Berwal, Sunil K
Suresh, C G
Gaikwad, Sushama M
Pal, Jayanta K
description The alpha-subunit of the human eukaryotic initiation factor 2 (heIF2alpha), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2alpha gets phosphorylated by eIF2alpha-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2alpha, 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)=1.13ns and tau(2)=4.74ns) 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 4M 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.
doi_str_mv 10.1016/j.bbrc.2009.09.106
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subjects Circular Dichroism
Cloning, Molecular
Eukaryotic Initiation Factor-2 - chemistry
Eukaryotic Initiation Factor-2 - genetics
Fluorescence
Humans
Hydrophobic and Hydrophilic Interactions
Protein Conformation
Protein Denaturation
Tryptophan - chemistry
title Conformational characterization of human eukaryotic initiation factor 2alpha: a single tryptophan protein
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