title: Conformational transitions of the catalytic domain of heme-regulated eukaryotic initiation factor 2α kinase, a key translational regulatory molecule.
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 α-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.
authors.affiliation: Department of Biotechnology, University of Pune, Pune, Maharashtra, 411007, India.
authors.forename: R K
authors.initials: RK
authors.lastname: Sreejith
authors.forename: C G
authors.initials: CG
authors.lastname: Suresh
authors.forename: Siddharth H
authors.initials: SH
authors.lastname: Bhosale
authors.forename: Varsha
authors.initials: V
authors.lastname: Bhavnani
authors.forename: Avinash
authors.initials: A
authors.lastname: Kumar
authors.forename: Sushama M
authors.initials: SM
authors.lastname: Gaikwad
authors.forename: Jayanta K
authors.initials: JK
authors.lastname: Pal
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D000818:Animals; D020134:Catalytic Domain; D003001:Cloning, Molecular; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D014176:Protein Biosynthesis; D058767:Protein Unfolding / Q000187:drug effects; D011817:Rabbits; D013057:Spectrum Analysis; D019892:eIF-2 Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D020106:Acrylamide; D019892:eIF-2 Kinase; D019791:Guanidine