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<p>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.</p>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-002224nas a2200205   4500008004100000245011400041210006900155260000800224300001200232490000700244520150600251100001701757700001801774700002101792700002201813700001601835700001701851700002501868856012501893       2016                        eng d00aMolecular cloning and in silico studies of physiologically significant trehalase from drosophila melanogaster0 aMolecular cloning and in silico studies of physiologically signi  cNOV  a282-2920 v923 aTrehalase, a physiologically important glycosidase is known for its crucial role in insect glycometabolism and stress recovery. The present study describes the molecular cloning of a gene fragment, encoding the catalytically active trehalase from Drosophila melanogaster (DmTre) and its heterologous expression in Escherichia coli. The 1275 bp gene was overexpressed in two different vectors viz., pET28a and pCOLD TF and investigated for variable soluble expression, purification and activity of the recombinant enzyme with optimum pH and temperature of enzyme as 6 and 55 degrees C, respectively. The sequence was characterized in silico by subjecting it to homology search, multiple sequence alignment and phylogenetic tree construction revealing its identity to other trehalases which belong to glycoside hydrolase family 37. The deduced amino acid sequence and modeled 3D structure of DmTre possessed all features of trehalase superfamily, including signature motifs and catalytic domain. The active site pocket of recombinant DmTre was compared with the crystal structure of E. coli trehalase identifying Glu424 and Asp226 as the putative catalytic residues. Additionally, enzyme-substrate docking suggests possible involvement of other residues in the catalysis along with Asp226. The present study holds significance in understanding the structural aspects of Drosophila trehalase in spite of unavailabilty of eukaryotic trehalase crystal structure. (C) 2016 Elsevier B.V. All rights reserved.1 aShukla, Ekta1 aThorat, Leena1 aBhavnani, Varsha1 aBendre, Ameya, D.1 aPal, J., K.1 aNath, B., B.1 aGaikwad, Sushama, M.  uhttps://library.ncl.res.in/content/molecular-cloning-and-silico-studies-physiologically-significant-trehalase-drosophila