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

TitleConformational characterization of human eukaryotic initiation factor 2 alpha: a single tryptophan protein
Publication TypeJournal Article
Year of Publication2009
AuthorsSreejith, RK, Yadav, VNand, Varshney, NK, Berwal, SK, Suresh, CG, Gaikwad, SM, Pal, JK
JournalBiochemical and Biophysical Research Communications
Volume390
Issue2
Pagination273-279
Date PublishedDEC
ISSN0006-291X
KeywordsCircular dichroism, heIF2 alpha, K562 cells, Molten globule, Steady-state and time-resolved fluorescence
Abstract

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.

DOI10.1016/j.bbrc.2009.09.106
Type of Journal (Indian or Foreign)Foreign
Impact Factor (IF)2.595
Divison category: 
Biochemical Sciences