01544nas a2200205 4500008004100000022001400041245009700055210006900152260007300221300001200294490000700306520077500313653002201088653002501110653001301135653001601148100002401164700002501188856012501213 2009 eng d a0141-813000aClass II alpha-mannosidase from aspergillus fischeri: energetics of catalysis and inhibition0 aClass II alphamannosidase from aspergillus fischeri energetics o aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcJAN a112-1150 v443 a

Energetics of the catalysis of Class II alpha-mannosidase (E.C.3.2.1.24) from Aspergillus fischeri was studied. The enzyme showed K(cat)/K(m) for Man (alpha 1-3) Man, Man (alpha 1-2) Man and Man (alpha 1-6) Man as 7488, 5376 and 3690 M(-1) min(-1), respectively. The activation energy, Ea was 15.14, 47.43 and 71.21 kJ/mol for a1-3, alpha 1-2 and alpha 1-6 linked mannobioses, respectively, reflecting the energy barrier in the hydrolysis of latter two substrates. The enzyme showed K(cat)/K(m) as 3.56 x 10(5) and 4.61 x 10(5) M(-1) min(-1) and E(a) as 38.7 and 8.92 kJ/mol, towards pNP alpha Man and 4-MeUmb alpha Man, respectively. Binding of Swainsonine to the enzyme is stronger than that of 1-deoxymannojirimycin. (C) 2008 Elsevier B.V. All rights reserved.

10aalpha-Mannosidase10aEnergy of activation10akinetics10aSwainsonine1 aShashidhara, K., S.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/class-ii-alpha-mannosidase-aspergillus-fischeri-energetics-catalysis-and-inhibition-002108nas a2200277 4500008004100000022001400041245011500055210006900170260007500239300001200314490000700326520110400333653002001437653001701457653001701474653002601491653002401517100002401541700002501565700001901590700001801609700002601627700002001653700002701673856013001700 2009 eng d a1053-050900aComparative studies of two araceous lectins by steady state and time-resolved fluorescence and CD spectroscopy0 aComparative studies of two araceous lectins by steady state and  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScMAR a239-2480 v193 a

Transitions in the tryptophan microenvironment and secondary structure of two monocot lectins from Sauromatum guttatum and Arisaema tortuosum under different denaturing conditions were studied by steady state and time resolved fluorescence and CD spectroscopy. The lectins exist as tetramers with a single tryptophan residue estimated per monomer, present in a polar environment. Quenching with ionic quenchers showed predominantly electropositive environment for tryptophan residues. Acrylamide had maximum quenching effect. A decrease in KI quenching due to lectin denaturation indicated redistribution of charges as a result of possible conformational change. The two values for lifetimes of tryptophanyl population (1.2-1.4 and 6.3-6.4 ns) reduced substantially on quenching or denaturation. Similarly, both the lectins showed a drastic loss of secondary structure in 5 M Gdn-HCl or 6 M Urea or at pH 2.0 and below. For the first time araceous lectins, like legume lectins are shown to bind adenine. The presence of a compact structure at alkaline pH 10.0-12.0 was observed in CD spectra.

10aAraceae lectins10aDenaturation10afluorescence10aLifetime spectroscopy10aSecondary structure1 aDharker, Poorva, N.1 aGaikwad, Sushama, M.1 aSuresh, C., G.1 aDhuna, Vikram1 aKhan, Mohammad, Islam1 aSingh, Jatinder1 aKamboj, Sukhdev, Singh uhttps://library.ncl.res.in/content/comparative-studies-two-araceous-lectins-steady-state-and-time-resolved-fluorescence-and-002426nas a2200277 4500008004100000022001400041245011100055210006900166260009500235300001200330490000800342520139600350653002301746653001601769653001501785653001901800653004801819100002101867700002401888700002601912700002201938700001901960700002501979700002102004856012302025 2009 eng d a0006-291X00aConformational characterization of human eukaryotic initiation factor 2 alpha: a single tryptophan protein0 aConformational characterization of human eukaryotic initiation f a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcDEC a273-2790 v3903 a

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.

10aCircular dichroism10aheIF2 alpha10aK562 cells10aMolten globule10aSteady-state and time-resolved fluorescence1 aSreejith, R., K.1 aYadav, Viveka, Nand1 aVarshney, Nishant, K.1 aBerwal, Sunil, K.1 aSuresh, C., G.1 aGaikwad, Sushama, M.1 aPal, Jayanta, K. uhttps://library.ncl.res.in/content/conformational-characterization-human-eukaryotic-initiation-factor-2-alpha-single-001944nas a2200253 4500008004100000022001400041245010200055210006900157260007500226300001200301490000700313520104400320653001601364653002201380653001601402653002301418653001701441653001101458653001601469653002501485100002401510700002501534856013101559 2010 eng d a1053-050900aConformational and functional transitions in class II alpha-mannosidase from aspergillus fischeri0 aConformational and functional transitions in class II alphamanno a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScJUL a827-8360 v203 a

The conformational transitions in an oligomeric and high molecular weight class II alpha-mannosidase from Aspergillus fischeri were examined using fluorescence and CD spectroscopy under chemical, thermal and acid denaturing conditions. The enzyme lost the activity first and then the overall folded conformation and secondary structure. The midpoint values of GdnHCl mediated changes measured by inactivation; fluorescence and negative ellipticity were 0.48 M, 1.5 M and 1.9 M, respectively. The protein almost completely unfolded in 4.0 M GdnHCl but not at 90 A degrees C. The inactivation and unfolding were irreversible. At pH 2.0, the protein exhibited molten-globule like intermediate with rearranged secondary and tertiary structures and exposed hydrophobic amino acids on the surface. This species showed increased accessibility of Trp to the quenchers and got denatured with GdnHCl in a different manner. The insoluble aggregates of a thermally denatured protein could be detected only in the presence of 0.25-0.75 M GdnHCl.

10aAggregation10aalpha-Mannosidase10aANS binding10aCircular dichroism10afluorescence10aGdnHCl10aHydrobicity10aThermal denaturation1 aShashidhara, K., S.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/conformational-and-functional-transitions-class-ii-alpha-mannosidase-aspergillus-fischeri-002160nas a2200289 4500008004100000022001400041245011900055210006900174260007500243300001200318490000700330520114000337653001201477653002601489653001801515653001901533653002401552653002001576653001401596100002001610700002501630700001901655700001801674700002001692700002701712856013101739 2011 eng d a1053-050900aConformational transitions in ariesaema curvatum lectin: characterization of an acid induced active molten globule0 aConformational transitions in ariesaema curvatum lectin characte a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScMAR a753-7630 v213 a

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.

10aAraceae10aFluorescence lifetime10aHemagglutinin10aMolten globule10aSecondary structure10aThermostability10aUnfolding1 aSharma, Urvashi1 aGaikwad, Sushama, M.1 aSuresh, C., G.1 aDhuna, Vikram1 aSingh, Jatinder1 aKamboj, Sukhdev, Singh uhttps://library.ncl.res.in/content/conformational-transitions-ariesaema-curvatum-lectin-characterization-acid-induced-active-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-2-002087nas a2200229 4500008004100000022001400041245011300055210006900168260011100237300001200348490000700360520113600367653002501503653005401528653002501582653003201607100002201639700002501661700002001686700001901706856013201725 2013 eng d a0929-866500aCrystal structure and fluorescence analysis of alkaline thermostable xylanase from bacillus sp (NCL 87-6-10)0 aCrystal structure and fluorescence analysis of alkaline thermost aEXECUTIVE STE Y-2, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATESbBENTHAM SCIENCE PUBL LTDcFEB a125-1320 v203 a

Structural information deduced from the new crystal form of xylanase from Bacillus sp (NCL 87-6-10) (ATBXYL-C) helped us to identify the active site and interpret the stability of the enzyme. The analysis of the tetragonal crystal structure of ATBXYL-C with a bound and cleaved xylotriose revealed the two glutamic acid residues in the structure that could act as nucleophile (Glu94) and base (Glu184) in the enzyme activity and also the tryptophan residues interacting with the substrate. The cleavage of xylotriose in the crystal showed xylobiose to be the major product. Intrinsic fluorescence of the enzyme showed the presence of tryptophans in partially exposed to the solvent at the active site and surface tryptophans in electropositive environment. The titration experiments with xylobiose and xylotriose revealed slightly enhanced preference for longer chain X3 compared with X2. The crystal structure also account for some of the factors, such as increased number of ionic interactions and additional interactions at the N-terminus, which contributed to increased alkalophilicity and thermostability of the enzyme.

10aactive site residues10aAlkaline thermophile Bacillus xylanase (ATBXYL-C)10afluorescence studies10athree-dimensional structure1 aSatyanarayana, L.1 aGaikwad, Sushama, M.1 aBalkrishnan, H.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/crystal-structure-and-fluorescence-analysis-alkaline-thermostable-xylanase-bacillus-sp-ncl-002079nas a2200229 4500008004100000022001400041245008700055210006900142260007300211300001000284490000700294520124300301653004001544653001701584653001901601653002001620653001401640100002701654700002701681700002501708856011601733 2014 eng d a0141-813000aConformational transitions of cinnamoyl CoA reductase 1 from leucaena leucocephala0 aConformational transitions of cinnamoyl CoA reductase 1 from leu aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcMAR a30-350 v643 a

Conformational transitions of cinnamoyl CoA reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (L1-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 C and starts forming aggregates at 55 C. LI-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of Ll -CCRH1 at pH 2.0 was stable up to 90 C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCI), it got transformed into different structure which was stable in the vicinity of 2 M GdnHCI (as compared to drastic loss of native structure in 2 M GdnHC1) as seen in far UV-CD spectra. The structural transition of LI-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein. (C) 2013 Elsevier B.V. All rights reserved.

10aAggregation Cinnamoyl CoA reductase10aConformation10aMolten globule10aThermostability10aUnfolding1 aSonawane, Prashant, D.1 aKhan, Bashir, Mohammad1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/conformational-transitions-cinnamoyl-coa-reductase-1-leucaena-leucocephala-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

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.

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-002008nas a2200157 4500008004100000245013300041210006900174260000800243300001200251490000700263520138000270100001701650700002801667700002501695856013001720 2017 eng d00aConformational and functional transitions and in silico analysis of a serine protease from conidiobolus brefeldianus (MTCC 5185)0 aConformational and functional transitions and in silico analysis cMAY a387-3970 v983 aThis work describes functional and structural transitions of a novel protease isolated from Conidiobolus brefeldianus MTCC 5185 (Cprot), in detail using biophysical and bioinformatics tools. The commercial importance of Cprot in silk and leather industries made it an interesting candidate for structural investigations. Cprot possesses 8.2%alpha-helix, 31.1% beta-sheet and 23.8% turns. The enzyme was found to be active over a wide pH range and up to 55 degrees C. The protease was also stable in organic solvents up to 50% (v/v) concentration of alcohols and DMSO for >24 h and in 2 M guanidine hydrochloride for >12 h. Cprot was also resistant to trypsin, chymotrypsin, proteinase K and fluorinated alcohols (5-10%). The melting temperatures observed for the native Cprot and for the enzyme treated under various stress conditions correlated well with the corresponding structural and functional transitions obtained. The structural information was supported by the homology model of its closest homologue from C. coronatus; revealing its similarity to PA clan of proteases (Proteases of mixed nucleophile, superfamily A), with His-64, Asp-113 and Ser-208 as putative catalytic triad. Three tryptophan residues in Cprot are surrounded by positively charged residues, as evident from solute quenching studies and homology model. (C) 2017 Elsevier B.V. All rights reserved.1 aShukla, Ekta1 aAgrawal, Sanskruthi, B.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/conformational-and-functional-transitions-and-silico-analysis-serine-protease-conidiobolus02644nas a2200277 4500008004100000022001400041245011100055210006900166260000800235300000800243490000600251520174300257653002102000653002302021653001102044653002402055653001402079100001702093700001802110700002202128700002002150700002102170700002402191700002502215856012602240 2018 eng d a2190-572X00aCloning and characterization of trehalase: a conserved glycosidase from oriental midge, Chironomus ramosus0 aCloning and characterization of trehalase a conserved glycosidas cAUG a3520 v83 a

Insect trehalase is a multiferous enzyme, crucial for normal physiological functions as well as under stress conditions. In this report, we present a fundamental study of the trehalase gene segment (1587 bp) from Chironomus ramosus (CrTre) encoding for 529 amino acids, using appropriate bioinformatics tools. C. ramosus, a tropical midge is an emerging animal model to investigate the consequences of environmental stresses. We observed that CrTre belongs to GH family 37 in the CAZy database and possess 57-92% identity to dipteran trehalases. In silico characterization provided information regarding the structural, functional and evolutionary aspects of midge trehalase. In the phylogenetic tree, CrTre clustered with the soluble dipteran trehalases. Moreover, domain functional characterization of the deduced protein sequence by InterProScan (IPR001661), ProSite (PS00927 and PS00928) and Pfam (PF01204) indicated presence of highly conserved signature motifs which are important for the identification of trehalase superfamily. Furthermore, the instability index of CrTre was predicted to be < 40 suggesting its in vivo stability while, the high aliphatic index indicated towards its thermal stability (index value 71-81). The modelled 3D tertiary structure of CrTre depicts a (alpha/alpha)(6) barrel toroidal core. The catalytic domain of the enzyme comprised Glu424 and Asp226 as the putative active site residues. Interestingly, the conserved motifs were observed to be formed by the flexible loopy regions in the tertiary structure. This study revealed essential sequence features of the midge trehalase and offers better insights into the structural aspects of this enzyme which can be correlated with its function.

10aConserved motifs10aHomology modelling10aMidges10aSecondary structure10atrehalose1 aShukla, Ekta1 aThorat, Leena1 aBendre, Ameya, D.1 aJadhav, Santosh1 aPal, Jayanta, K.1 aNath, Bimalendu, B.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/cloning-and-characterization-trehalase-conserved-glycosidase-oriental-midge-chironomus