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. 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.
Molecular Cell Biology Laboratory, Department of Biotechnology, University of Pune, Pune, Maharashtra 411007, India.
R K
RK
Sreejith
Viveka Nand
VN
Yadav
Nishant K
NK
Varshney
Sunil K
SK
Berwal
C G
CG
Suresh
Sushama M
SM
Gaikwad
Jayanta K
JK
Pal
D002942:Circular Dichroism; D003001:Cloning, Molecular; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry* / Q000235:genetics; D005453:Fluorescence; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D011487:Protein Conformation; D011489:Protein Denaturation; D014364:Tryptophan / Q000737:chemistry*
D015852:Eukaryotic Initiation Factor-2; D014364:Tryptophan