02631nas a2200277   4500008004100000022001400041245015800055210006900213260007500282300001200357490000700369520154700376653002801923653002201951653002901973653001902002653004802021100002102069700001902090700002702109700002102136700001902157700002502176700002102201856013102222       2012                        eng d  a1053-050900aConformational transitions of the catalytic domain of heme-regulated eukaryotic initiation factor 2 alpha kinase, a key translational regulatory molecule0 aConformational transitions of the catalytic domain of hemeregula  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScJAN  a431-4410 v223 a<p>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.</p>10aCircular dichroism (CD)10aeIF2 alpha kinase10aHeme-regulated inhibitor10aMolten globule10aSteady-state and time-resolved fluorescence1 aSreejith, R., K.1 aSuresh, C., G.1 aBhosale, Siddharth, H.1 aBhavnani, Varsha1 aKumar, Avinash1 aGaikwad, Sushama, M.1 aPal, Jayanta, K.  uhttps://library.ncl.res.in/content/conformational-transitions-catalytic-domain-heme-regulated-eukaryotic-initiation-factor-2-002446nas a2200277   4500008004100000022001400041245012200055210006900177260009900246300001200345490000700357520142700364653001801791653002701809653002201836653001301858653002301871100002101894700002501915700001801940700002101958700001701979700001701996700002402013856013102037       2015                        eng d  a1359-511300aCloning, expression and in silico studies of a serine protease from a marine actinomycete (Nocardiopsis sp NCIM 5124)0 aCloning expression and in silico studies of a serine protease fr  aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLANDbELSEVIER SCI LTDcMAR  a378-3870 v503 a<p>A serine protease (N. protease), from Nocardiopsis sp., was cloned and expressed in Escherichia coli and investigated for its potential kinetic stability. Protein expression using two vectors, pET-22b (+) and pET-39b (+) was compared based on proper folding and soluble expression of the protein. pET-39b (+) was found to be a better vector for soluble expression of this protease containing disulfide bonds. In silico studies were also carried out for N. protease. Homology modeling suggested N. protease to be a member of PA clan of proteases. The phylogenetic analysis showed relatedness of N. protease to kinetically stable proteases. Molecular docking studies performed exhibited interaction of a peptide substrate with catalytic pocket of the enzyme. High temperature MD simulations were performed on N. protease to study its unfolding behavior and comparisons were made with alpha LP. A novel approach to study `cooperativity' of protein unfolding was undertaken, wherein `P' value analysis based on phi and psi values of the protein was performed. Data showed sharper P value transition for alpha LP when compared to N. protease thus indicating relatively less kinetic stability of N. protease. Present study holds significance as the non-streptomycete actinomycetes group is least explored and ensures industrially important enzymes with exceptional stabilities. (C) 2015 Elsevier Ltd. All rights reserved.</p>10aActinomycetes10aCloning and expression10aKinetic stability10aProtease10aThermal simulation1 aRohamare, Sonali1 aGaikwad, Sushama, M.1 aJones, Dafydd1 aBhavnani, Varsha1 aPal, Jayanta1 aSharma, Ranu1 aChatterjee, Prathit  uhttps://library.ncl.res.in/content/cloning-expression-and-silico-studies-serine-protease-marine-actinomycete-nocardiopsis-sp-0