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-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<p>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.</p>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-002160nas a2200205   4500008004100000022001400041245011400055210006900169260011100238300001200349490000700361520132800368653002001696653002001716653000801736100002801744700002501772700002501797856013201822       2013                        eng d  a0929-866500aPurification and characterization of a thermolysin like protease from thermoactinomyces thalpophilus MCMB-3800 aPurification and characterization of a thermolysin like protease  aEXECUTIVE STE Y-2, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATESbBENTHAM SCIENCE PUBL LTDcAUG  a918-9250 v203 a<p>The extracellular thermolysin like protease (TLP) was purified and characterized from Thermoactinomyces thalpophilus MCMB-380 (Genbank Accession No. EF397000). The enzyme was purified to homogeneity by successive ultra filtration steps using 50 kDa and 10 kDa membrane filters followed by anion exchange chromatography. The molecular mass and isoelectric point of the enzyme were found to be 34.4 kDa and 9.5, respectively. The proteolytic activity was inhibited by EDTA and the enzyme required Ca2+ to show the full activity as well as thermostability. The T-50 of the enzyme at 80 degrees C was 1 h and the activation energy was estimated to be 11.02 Kcal / mol. Atomic absorption spectrophotometric analysis revealed the presence of Zn2+ ion in the protein core indicating that it is a metalloprotease. This protease has commercial potential in catalyzing the condensation reaction of two amino acids for production of the dipeptide aspartame, an artificial sweetener. The one hour time-frame is significantly faster than that of the enzyme thermolysin from Bacillus thermoproteolyticus. Moreover the TLP was stable at 80 degrees C for one hour which makes it industrially robust. The Zn2+ ion in the T. thalpophilus protease appears to be necessary for maintaining the active conformation of the enzyme molecule.</p>10aMetalloprotease10aThermostability10aTLP1 aMajumder, Devipriya, R.1 aKanekar, Pradnya, P.1 aGaikwad, Sushama, M.  uhttps://library.ncl.res.in/content/purification-and-characterization-thermolysin-protease-thermoactinomyces-thalpophilus-mcmb-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<p>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.</p>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-002566nas a2200253   4500008004100000022001400041245011200055210006900167260000800236300001200244490000700256520172500263653002601988653001502014653001802029653001902047653002002066653002002086100001702106700001802123700002002141700002502161856012602186       2017                        eng d  a0739-110200aFunctional stability and structural transitions of kallikrein: spectroscopic and molecular dynamics studies0 aFunctional stability and structural transitions of kallikrein sp  cMAR  a330-3420 v353 a<p>Kallikrein, a physiologically vital serine protease, was investigated for its functional and conformational transitions during chemical (organic solvents, Gdn-HCl), thermal, and pH induced denaturation using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The enzyme was exceptionally stable in isopropanol and ethanol showing 110% and 75% activity, respectively, after 96h, showed moderate tolerance in acetonitrile (45% activity after 72h) and much lower stability in methanol (40% activity after 24h) (all the solvents [90% v/v]). Far UV CD and fluorescence spectra indicated apparent reduction in compactness of KLKp structure in isopropanol system. MD simulation studies of the enzyme in isopropanol revealed (1) minimal deviation of the structure from native state (2) marginal increase in radius of gyration and solvent accessible surface area (SASA) of the protein and the active site, and (3) loss of density barrier at the active site possibly leading to increased accessibility of substrate to catalytic triad as compared to methanol and acetonitrile. Although kallikrein was structurally stable up to 90 degrees C as indicated by secondary structure monitoring, it was functionally stable only up to 45 degrees C, implicating thermolabile active site geometry. In GdnHCl [1.0M], 75% of the activity of KLKp was retained after incubation for 4h, indicating its denaturant tolerance. A molten globule-like structure of KLKp formed at pH 1.0 was more thermostable and exhibited interesting structural transitions in organic solvents. The above results provide deeper understanding of functional and structural stability of the serine proteases at molecular level.</p>10aisopropanol tolerance10aKallikrein10aMD Simulation10aMolten globule10aSerine protease10aThermostability1 aDalal, Sayli1 aMhashal, Anil1 aKadoo, Narendra1 aGaikwad, Sushama, M.  uhttps://library.ncl.res.in/content/functional-stability-and-structural-transitions-kallikrein-spectroscopic-and-molecular