@article { ISI:000271552400019, title = {Conformational characterization of human eukaryotic initiation factor 2 alpha: a single tryptophan protein}, journal = {Biochemical and Biophysical Research Communications}, volume = {390}, number = {2}, year = {2009}, month = {DEC}, pages = {273-279}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, 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.

}, keywords = {Circular dichroism, heIF2 alpha, K562 cells, Molten globule, Steady-state and time-resolved fluorescence}, issn = {0006-291X}, doi = {10.1016/j.bbrc.2009.09.106}, author = {Sreejith, R. K. and Yadav, Viveka Nand and Varshney, Nishant K. and Berwal, Sunil K. and Suresh, C. G. and Gaikwad, Sushama M. and Pal, Jayanta K.} } @article { ISI:000291061500032, title = {Conformational transitions in ariesaema curvatum lectin: characterization of an acid induced active molten globule}, journal = {Journal of Fluorescence}, volume = {21}, number = {2}, year = {2011}, month = {MAR}, pages = {753-763}, publisher = {SPRINGER/PLENUM PUBLISHERS}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, abstract = {

Biophysical characterization of a lectin from Ariesaema curvatum (ACL) was carried out using steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. An intermediate with altered tryptophan microenvironment was detected in the phase diagram, which exibited pronounced secondary structure and hemagglutinating activity in presence of 0.25 M Gdn-HCl. An acid induced molten- globule like structure possessing activity and higher thermostability was detected. Transition to the molten globule state was reversible in nature. The lectin retained hemagglutinating activity even after incubation at 95 A degrees C. Both chemical and thermal unfolding of the lectin were found to consist of multistate processes. Fluorescence quenching of ACL was strong with acrylamide and KI. The single tryptophan was found to be surrounded by high density of the positively charged amino acid residues as shown by a ten fold higher K(sv) for KI compared to that for CsCl. The average lifetime of tryptophan fluorescence increased from 1.24 ns in the native state to 1.72 ns in the denatured state.

}, keywords = {Araceae, Fluorescence lifetime, Hemagglutinin, Molten globule, Secondary structure, Thermostability, Unfolding}, issn = {1053-0509}, doi = {10.1007/s10895-010-0766-2}, author = {Sharma, Urvashi and Gaikwad, Sushama M. and Suresh, C. G. and Dhuna, Vikram and Singh, Jatinder and Kamboj, Sukhdev Singh} } @article {Dutta Chowdhury, title = {Conformational transitions of the catalytic domain of heme-regulated eukaryotic initiation factor 2 alpha kinase, a key translational regulatory molecule}, journal = {Journal of Fluorescence}, volume = {22}, number = {1}, year = {2012}, month = {JAN}, pages = {431-441}, publisher = {SPRINGER/PLENUM PUBLISHERS}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, abstract = {

In mammalian cells, the heme-regulated inhibitor (HRI) plays a critical role in the regulation of protein synthesis at the initiation step through phosphorylation of a-subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have cloned and performed biophysical characterization of the kinase catalytic domain (KD) of rabbit HRI. The KD described here comprises kinase 1, the kinase insertion domain (KI) and kinase 2. We report here the existence of an active and stable monomer of HRI (KD). The HRI (KD) containing three tryptophan residues was examined for its conformational transitions occurring under various denaturing conditions using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The parameter A and phase diagram analysis revealed multi-state unfolding and existence of three stable intermediates during guanidine hydrochloride (Gdn-HCl) induced unfolding of HRI (KD). The protein treated with 6 M Gdn-HCl showed collisional and static mechanism of acrylamide quenching and the constants (K-sv=3.08 M-1 and K-s=5.62 M-1) were resolved using time resolved fluorescence titration. Based on pH, guanidine hydrochloride and temperature mediated transitions, HRI (KD) appears to exemplify a rigid molten globule-like intermediate with compact secondary structure, altered tertiary structure and exposed hydrophobic patches at pH 3.0. The results indicate the inherent structural stability of HRI (KD), a member of the class of stress response proteins.

}, keywords = {Circular dichroism (CD), eIF2 alpha kinase, Heme-regulated inhibitor, Molten globule, Steady-state and time-resolved fluorescence}, issn = {1053-0509}, doi = {10.1007/s10895-011-0976-2}, author = {Sreejith, R. K. and Suresh, C. G. and Bhosale, Siddharth H. and Bhavnani, Varsha and Kumar, Avinash and Gaikwad, Sushama M. and Pal, Jayanta K.} }