02138nas a2200217 4500008004100000022001400041245012400055210006900179260007500248300001200323490000700335520131900342653002201661653001701683653001701700653001301717653002101730100002401751700002501775856012001800 2007 eng d a1053-050900aFluorescence quenching and time-resolved fluorescence studies of alpha-Mannosidase from Aspergillus fischeri (NCIM 508)0 aFluorescence quenching and timeresolved fluorescence studies of  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScNOV a599-6050 v173 a

Apart from the vital role in glycoprotein biosynthesis and degradation, alpha-mannosidase is currently an important therapeutic target for the development of anticancer agents. Fluorescence quenching and time-resolved fluorescence of alpha-mannosidase, a multitryptophan protein from Aspergillus fischeri were carried out to investigate the tryptophan environment. The tryptophans were found to be differentially exposed to the solvent and were not fully accessible to the neutral quencher indicating heterogeneity in the environment. Quenching of the fluorescence by acrylamide was collisional. Surface tryptophans were found to have predominantly positively charged amino acids around them and differentially accessible to the ionic quenchers. Denaturation led to more exposure of tryptophans to the solvent and consequently in the significant increase in quenching with all the quenchers. The native enzyme showed two different lifetimes, tau (1) (1.51 ns) and tau (2) (5.99 ns). The average lifetime of the native protein (tau) (3.187 ns) was not affected much after denaturation (tau) (3.219 ns), while average lifetime of the quenched protein samples was drastically reduced (1.995 ns for acrylamide and 1.537 ns for iodide). This is an attempt towards the conformational studies of alpha-mannosidase.

10aalpha-Mannosidase10aDenaturation10afluorescence10alifetime10aSolute quenching1 aShashidhara, K., S.1 aGaikwad, Sushama, M. uhttp://library.ncl.res.in/content/fluorescence-quenching-and-time-resolved-fluorescence-studies-alpha-mannosidase-002045nas a2200265 4500008004100000022001400041245009700055210006900152260007500221300001200296490000700308520112300315653001601438653001701454653001801471653001301489653002101502653002301523653002101546100001901567700001901586700002601605700002501631856012301656 2008 eng d a1053-050900aSteady state and time-resolved fluorescence studies of a hemagglutinin from Moringa oleifera0 aSteady state and timeresolved fluorescence studies of a hemagglu a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScMAR a479-4850 v183 a

The saccharide binding and conformational characterization of a hemagglutinin, a low molecular weight protein from the seeds of Moringa oleifera was studied using steady state and time resolved fluorescence. The lectin binds sugars LacNAc (K-a = 1380 M-1) and fructose (K-a = 975 M-1), as determined by the fluorescence spectroscopy. It has a single tryptophan per monomer which is exposed on the surface and is in a strong electropositive environment as revealed by quenching with iodide. Quenching of the fluorescence by acrylamide involved both static (K-s = 0.216 M-1) and collisional (K-sv= 8.19 M-1) components. The native protein showed two different lifetimes, tau(1) (1.6 ns) and tau(2) (4.36 ns) which decrease and get converted into a single one, (2.21 ns) after quenching with 0.15 M acrylamide. The bimolecular quenching constant, k(q) was 7.55 x 10(11) M-1 s(-1). ANS binding studies showed that the native protein has exposed hydrophobic patches which get further exposed at extreme acidic or alkaline pH. However, they get buried in the interior of the protein in presence of 1 M GdnHCl or urea.

10aANS binding10afluorescence10aHemagglutinin10alifetime10aMoringa oleifera10asaccharide binding10aSolute quenching1 aKatre, Uma, V.1 aSuresh, C., G.1 aKhan, Mohammad, Islam1 aGaikwad, Sushama, M. uhttp://library.ncl.res.in/content/steady-state-and-time-resolved-fluorescence-studies-hemagglutinin-moringa-oleifera-002107nas a2200277 4500008004100000022001400041245011500055210006900170260007500239300001200314490000700326520110400333653002001437653001701457653001701474653002601491653002401517100002401541700002501565700001901590700001801609700002601627700002001653700002701673856012901700 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 uhttp://library.ncl.res.in/content/comparative-studies-two-araceous-lectins-steady-state-and-time-resolved-fluorescence-and-001943nas a2200253 4500008004100000022001400041245010200055210006900157260007500226300001200301490000700313520104400320653001601364653002201380653001601402653002301418653001701441653001101458653001601469653002501485100002401510700002501534856013001559 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. uhttp://library.ncl.res.in/content/conformational-and-functional-transitions-class-ii-alpha-mannosidase-aspergillus-fischeri-002647nas a2200241 4500008004100000022001400041245013000055210006900185260009500254300001200349490000800361520173000369653002202099653002302121653001702144653002802161653001902189653002002208653001402228100001902242700002502261856011902286 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. uhttp://library.ncl.res.in/content/multistate-unfolding-alpha-mannosidase-canavalia-ensiformis-jack-bean-evidence-002754nas a2200253 4500008004100000022001400041245012600055210006900181260007500250300001200325490000700337520179300344653002402137653002802161653001702189653001902206653002102225100002302246700003202269700001802301700002502319700002702344856012902371 2014 eng d a1053-050900aSteady state fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1) and its active site mutants0 aSteady state fluorescence studies of wild type recombinant cinna a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScMAY a665-6730 v243 a

Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA (K (a) = 3.72 x 10(5) M-1) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy. Quenching of the fluorescence by acrylamide for wild type and active site mutants was collisional with almost 100 % of the tryptophan fluorescence accessible under native condition and remained same after denaturation of protein with 6 M GdnHCl. In wild type Ll-CCRH1, the extent of quenching achieved with iodide (f (a) = 1.0) was significantly higher than cesium ions (f (a) = 0.33) suggesting more density of positive charge around surface of trp conformers under native conditions. Denaturation of wild type protein with 6 M GdnHCl led to significant increase in the quenching with cesium (f (a) = 0.54), whereas quenching with iodide ion was decreased (f (a) = 0.78), indicating reorientation of charge density around trp from positive to negative and heterogeneity in trp environment. The Stern-Volmer plots for wild type and mutants Ll-CCRH1 under native and denatured conditions, with cesium ion yielded biphasic quenching profiles. The extent of quenching for cesium and iodide ions under native and denatured conditions observed in active site mutants was significantly different from wild type Ll-CCRH1 under the same conditions. Thus, single substitution type mutations of active site residues showed heterogeneity in tryptophan microenvironment and differential degree of conformation of protein under native or denatured conditions.

10aActive site mutants10aCinnamoyl CoA reductase10afluorescence10aLigand binding10aSolute quenching1 aSonawane, Prashant1 aVishwakarma, Rishi, Kishore1 aSingh, Somesh1 aGaikwad, Sushama, M.1 aKhan, Bashir, Mohammad uhttp://library.ncl.res.in/content/steady-state-fluorescence-studies-wild-type-recombinant-cinnamoyl-coa-reductase-ll-ccrh1-0