02107nas a2200241 4500008004100000022001400041245014100055210006900196260008600265300001200351490000700363520118300370653001601553653002401569653001901593653001401612100001801626700001901644700002501663700002001688700002501708856013201733 2007 eng d a1521-654300aBile salt hydrolase, the member of Ntn-hydrolase family: differential modes of structural and functional transitions during denaturation0 aBile salt hydrolase the member of Ntnhydrolase family differenti a325 CHESTNUT ST, SUITE 800, PHILADELPHIA, PA 19106 USAbTAYLOR & FRANCIS INCcFEB a118-1250 v593 a

Conformational transitions and functional stability of the bile salt hydrolase (BSH; cholylglycine EC: 3.5.1.24) from Bifidobacterium longum (BlBSH) cloned and expressed in E. coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and CD spectroscopy. Thermal and Gdn-HCl-mediated denaturation of BlBSH is a multistep process of inactivation and unfolding. The inactivation and unfolding of the enzyme was found to be irreversible. Enzyme activity seems sensitive to even minor conformational changes at the active site. Thermal denaturation as such did not result in any insoluble protein aggregates. However, on treating with 0.25-1 M Gdn-HCl the enzyme showed increasing aggregation at temperatures of 40-55 degrees C indicating more complex structural changes taking place in the presence of chemical denaturants. The enzyme secondary structure was still intact at acidic pH (pH 1-3). The perturbation in the tertiary structure at the acidic pH was detected through freshly formed solvent exposed hydrophobic patches on the enzyme. These changes could be due to the formation of an acid-induced molten globule-like state.

10aAggregation10aBile salt hydrolase10amolten-globule10aUnfolding1 aKumar, Suresh1 aSuresh, C., G.1 aBrannigan, James, A.1 aDodson, Guy, G.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/bile-salt-hydrolase-member-ntn-hydrolase-family-differential-modes-structural-and-functional02648nas 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-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-001792nas a2200265 4500008004100000022001400041245008800055210006900143260007300212300001200285490000700297520083600304653001601140653003501156653001701191653001901208653004801227653001401275100002801289700002901317700002301346700002201369700002501391856011001416 2013 eng d a0141-813000aSolution and in silico studies on the recombinant lectin from Cicer arietinum seeds0 aSolution and in silico studies on the recombinant lectin from Ci aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcMAY a149-1550 v563 a

The Cicer arietinum seed lectin was cloned and expressed in Escherichia coli and purified in active form. Conformational characterization of the recombinant lectin (rCAL) was performed using biophysical and bioinformatics tools. Thermal denaturation of rCAL caused rapid secondary structural rearrangements above 50 degrees C and transient exposure of hydrophobic residues at 55 degrees C, leading to aggregation. Treatment of rCAL with GdnHCl resulted in unfolding followed by dissociation of the dimer. The single tryptophan in rCAL present on the surface of the protein is surrounded by hydrophobic and acidic amino acids and exists as different conformers. The experimental observations correlated well with the structural information revealed from the homology model of rCAL. (C) 2013 Elsevier B.V. All rights reserved.

10aAggregation10aBL21-CodonPlus (DE3)-RIL cells10aCicer lectin10aHomology model10aSteady-state and time resolved fluorescence10aUnfolding1 aWakankar, Madhurima, S.1 aKrishnasastry, Musti, V.1 aJaokar, Tulika, M.1 aPatel, Krunal, A.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/solution-and-silico-studies-recombinant-lectin-cicer-arietinum-seeds-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-002418nas a2200253 4500008004100000022001400041245010700055210006900162260007300231300001200304490000700316520147800323653001601801653002001817653002701837653004101864653001401905100002101919700001801940700002701958700002701985700002502012856012702037 2016 eng d a0141-813000aFunctional and conformational transitions of mevalonate diphosphate decarboxylase from Bacopa monniera0 aFunctional and conformational transitions of mevalonate diphosph aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcFEB a160-1700 v833 a

Functional and conformational transitions of mevalonate diphosphate decarboxylase (MDD), a key enzyme of mevalonate pathway in isoprenoid biosynthesis, from Bacopa monniera (BmMDD), cloned and overexpressed in Escherichia coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and Circular dichroism spectroscopy. Native BmMDD is a helix dominant structure with 45% helix and 11% sheets and possesses seven tryptophan residues with two residues exposed on surface, three residues partially exposed and two situated in the interior of the protein. Thermal denaturation of BmMDD causes rapid structural transitions at and above 40 degrees C and transient exposure of hydrophobic residues at 50 degrees C, leading to aggregation of the protein. An acid induced molten globule like structure was observed at pH 4, exhibiting altered but compact secondary structure, distorted tertiary structure and exposed hydrophobic residues. The molten globule displayed different response at higher temperature and similar response to chemical denaturation as compared to the native protein. The surface tryptophans have predominantly positively charged amino acids around them, as indicated by higher K-SV for IC as compared to that for CsCl. The native enzyme displayed two different lifetimes, vi (1.203 +/- 0.036 ns) and tau 2 (3.473 +/- 0.12 ns) indicating two populations of tryptophan. (C) 2015 Elsevier B.V. All rights reserved.

10aAggregation10aCD spectroscopy10aFluorescence quenching10aMevalonate diphosphate decarboxylase10aUnfolding1 aShakeel, Abbassi1 aPatel, Krunal1 aKhan, Bashir, Mohammad1 aBhosale, Siddharth, H.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/functional-and-conformational-transitions-mevalonate-diphosphate-decarboxylase-bacopa-0