02648nas a2200241 4500008004100000022001400041245013000055210006900185260009500254300001200349490000800361520173000369653002202099653002302121653001702144653002802161653001902189653002002208653001402228100001902242700002502261856012002286 2010 eng d a0006-291X00aMultistate unfolding of alpha-mannosidase from Canavalia ensiformis (Jack Bean): evidence for the thermostable molten globule0 aMultistate unfolding of alphamannosidase from Canavalia ensiform a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcDEC a391-3970 v4033 a

The relevance of partially ordered states of proteins (such as the molten-globule state) in cellular processes is beginning to be understood. We examined the conformational transitions in a multimeric and high molecular weight class II alpha-mannosidase from Canavalia ensiformis (Jack Bean) (Jb alpha-man) utilizing intrinsic fluorescence, solute quenching, hydrophobic dye binding, size exclusion chromatography and circular dichroism (CD) spectroscopy for the protein in presence of Guanidine hydrochloride (GdnHCl). The decomposition analysis of the protein spectra obtained during unfolding showed progressive appearance of class S, I, II and III trp. The parameter A and spectral center of mass showed multi state unfolding of the protein and phase diagram analysis revealed formation of an intermediate of Jb alpha-man in the vicinity of 1 M GdnHCl. The intermediate exhibited compact secondary and distorted tertiary structure with exposed hydrophobic amino acids on the surface, indicating the molten-globule nature. The dissociation, partial unfolding and aggregation of Jb alpha-man occurred simultaneously during chemical denaturation. The molten-globule possessed slightly higher hydrodynamic radius, perturbance in the structure up to 60 degrees C and stability of the structure up to 80 degrees C unlike the native Jack Bean alpha-mannosidase. The modes of chemical and thermal denaturation of the native protein were different. The solute quenching parameters confirmed the altered confirmation of the intermediate. Taken together, our results constitute one of the early reports of formation of GdnHCl induced molten globule in a class II alpha-mannosidase. (C) 2010 Elsevier Inc. All rights reserved.

10aalpha-Mannosidase10aCircular dichroism10afluorescence10aGuanidine hydrochloride10aMolten globule10aThermostability10aUnfolding1 aKumar, Avinash1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/multistate-unfolding-alpha-mannosidase-canavalia-ensiformis-jack-bean-evidence-001667nas a2200229 4500008004100000022001400041245014200055210006900197260007300266300001400339490000700353520078800360653002101148653002201169653002501191653001501216653001501231653001801246100001901264700002501283856012901308 2011 eng d a0141-813000aJack bean alpha-mannosidase (Jb alpha-man): tolerance to alkali, chelating and reducing agents and energetics of catalysis and inhibition0 aJack bean alphamannosidase Jb alphaman tolerance to alkali chela aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcDEC a1066-10710 v493 a

Investigations of the catalytic and structural transitions of jack bean alpha-mannosidase (Jb alpha-man) are described in the present paper. The enzyme was maximally stable at pH 5.0; however, when incubated in the pH range of 11.0-12.0, showed 1.3 times higher activity and also stability for longer time. The free amino group at or near the active site was probably involved in the stability and activation mechanism. The active site is constituted by the association of two unidentical subunits connected by disulfide linkages. The metalloenzyme has Zn(2+) ions tightly bound and chelation reduces the thermal stability of the protein. Energetics of catalysis and thermodynamics of inhibition of the enzyme were also carried out. (C) 2011 Elsevier B.V. All rights reserved.

10aAlkali tolerance10aalpha-Mannosidase10abeta-Mercaptoethanol10aEnergetics10aInhibition10aMetalloenzyme1 aKumar, Avinash1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/jack-bean-alpha-mannosidase-jb-alpha-man-tolerance-alkali-chelating-and-reducing-agents-002631nas 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

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.

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-2-002613nas a2200361 4500008004100000245010900041210006900150260000800219490000600227520145100233100001901684700002801703700001701731700002001748700002001768700001701788700001901805700001901824700002001843700001801863700002501881700002101906700002101927700001801948700001801966700002901984700002302013700002102036700002502057700001602082700002402098856012902122 2017 eng d00aMapping architectural and transcriptional alterations in non-lesional and lesional epidermis in vitiligo0 aMapping architectural and transcriptional alterations in nonlesi cAUG0 v73 aIn vitiligo, chronic loss of melanocytes and consequent absence of melanin from the epidermis presents a challenge for long-term tissue maintenance. The stable vitiligo patches are known to attain an irreversible depigmented state. However, the molecular and cellular processes resulting in this remodeled tissue homeostasis is unclear. To investigate the complex interplay of inductive signals and cell intrinsic factors that support the new acquired state, we compared the matched lesional and non-lesional epidermis obtained from stable non-segmental vitiligo subjects. Hierarchical clustering of genome-wide expression of transcripts surprisingly segregated lesional and non-lesional samples in two distinct clades, despite the apparent heterogeneity in the lesions of different vitiligo subjects. Pathway enrichment showed the expected downregulation of melanogenic pathway and a significant downregulation of cornification and keratinocyte differentiation processes. These perturbations could indeed be recapitulated in the lesional epidermal tissue, including blunting of rete-ridges, thickening of stratum corneum and increase in the size of corneocytes. In addition, we identify marked increase in the putrescine levels due to the elevated expression of spermine/spermidine acetyl transferase. Our study provides insights into the intrinsic self-renewing ability of damaged lesional tissue to restore epidermal functionality in vitiligo.1 aSingh, Archana1 aGotherwal, Vishvabandhu1 aJunni, Paivi1 aVijayan, Vinaya1 aTiwari, Manisha1 aGanju, Parul1 aKumar, Avinash1 aSharma, Pankaj1 aFatima, Tanveer1 aGupta, Aayush1 aHolla, Ananthaprasad1 aKar, Hemanta, K.1 aKhanna, Sangeeta1 aThukral, Lipi1 aMalik, Garima1 aNatarajan, Krishnamurthy1 aGadgil, Chetan, J.1 aLahesmaa, Riitta1 aNatarajan, Vivek, T.1 aRani, Rajni1 aGokhale, Rajesh, S. uhttps://library.ncl.res.in/content/mapping-architectural-and-transcriptional-alterations-non-lesional-and-lesional-epidermis