02046nas a2200265 4500008004100000022001400041245009700055210006900152260007500221300001200296490000700308520112300315653001601438653001701454653001801471653001301489653002101502653002301523653002101546100001901567700001901586700002601605700002501631856012401656 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. uhttps://library.ncl.res.in/content/steady-state-and-time-resolved-fluorescence-studies-hemagglutinin-moringa-oleifera-001652nas a2200217 4500008004100000022001400041245008300055210006900138260007300207300001200280490000700292520085400299653002301153653003501176653002401211100001901235700001901254700002601273700002501299856011001324 2008 eng d a0141-813000aStructure-activity relationship of a hemagglutinin from Moringa oleifera seeds0 aStructureactivity relationship of a hemagglutinin from Moringa o aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcMAR a203-2070 v423 a

The hemagglutinin from the seeds of Moringa oleifera (MoL) agglutinates human as well as rabbit erythrocytes; the affinity for the latter is almost 250 times more than that for the former. MoL was inhibited by glycoproteins namely thyroglobulin, fetuin and holotransferin indicating the complex sugar specificity of the lectin. The protein is a homodimer with molecular mass of 14 kDa, subunits (7.1 kDa) linked by the disulfide bond(s). The secondary structure elements of MoL area-helix, 28%; beta-sheet, 23%; turn 20% and unordered 28%. While the activity and secondary structure were not affected at extreme pH and high temperature, they were drastically affected in presence of dithiothreitol at and above pH 7.0, indicating that disulfide linkages hold the active conformation of the protein. (C) 2007 Elsevier B.V. All rights reserved.

10adisulfide linkages10aMoringa oleifera hemagglutinin10aSecondary structure1 aKatre, Uma, V.1 aSuresh, C., G.1 aKhan, Mohammad, Islam1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/structure-activity-relationship-hemagglutinin-moringa-oleifera-seeds-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-002054nas a2200253 4500008004100000022001400041245009800055210006900153260009500222300001400317490000800331520110600339653001001445653002301455653002201478653003901500653002201539100001901561700002201580700002501602700002101627700002001648856013201668 2013 eng d a0006-291X00aStructural-functional insights of single and multi-domain capsicum annuum protease inhibitors0 aStructuralfunctional insights of single and multidomain capsicum a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcJAN a1060-10650 v4303 a

Pin-II protease inhibitors (PIs) are the focus of research interest because of their large structural-functional diversity and relevance in plant defense. Two representative Capsicum annuum PI genes (CanPI-15 and -7) comprising one and four inhibitory repeat domains, respectively, were expressed and recombinant proteins were characterized. beta-Sheet and unordered structure was found predominant in CanPI-15 while -7 also displayed the signatures of polyproline fold, as revealed by circular dichroism studies. Inhibition kinetics against bovine typsin indicated three times higher potency of CanPI-7 (K-i similar to 57 mu M) than -15 (similar to 184 mu M). Activity and structural stability of these CanPIs were revealed under various conditions of pH, temperature and denaturing agent. Structure prediction, docking studies with proteases and mass spectroscopy revealed the organization of multiple reactive site loops of multi domain PIs in space as well as the steric hindrances imposed while binding to proteases due to their close proximity. (C) 2012 Elsevier Inc. All rights reserved.

10aCanPI10aCircular dichroism10aDisulfide bonding10aPotato type-II protease inhibitors10aProtein stability1 aMishra, Manasi1 aJoshi, Rakesh, S.1 aGaikwad, Sushama, M.1 aGupta, Vidya, S.1 aGiri, Ashok, P. uhttps://library.ncl.res.in/content/structural-functional-insights-single-and-multi-domain-capsicum-annuum-protease-inhibitors-002755nas a2200253 4500008004100000022001400041245012600055210006900181260007500250300001200325490000700337520179300344653002402137653002802161653001702189653001902206653002102225100002302246700003202269700001802301700002502319700002702344856013002371 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 uhttps://library.ncl.res.in/content/steady-state-fluorescence-studies-wild-type-recombinant-cinnamoyl-coa-reductase-ll-ccrh1-002772nas a2200241 4500008004100000022001400041245012800055210006900183260007300252300001200325490000800337520181200345653003102157653002202188653003702210653003102247653002402278100002402302700002502326700002302351700002602374856013002400 2014 eng d a0378-517300aStructure function attributes of gold nanoparticle vaccine association: effect of particle size and association temperature0 aStructure function attributes of gold nanoparticle vaccine assoc aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcAUG a439-4480 v4713 a

Many biotherapeutic applications of gold nanoparticles make use of conjugated or adsorbed protein moieties. Physical parameters of association such as particle size, morphology, surface chemistry and temperature influences the protein-nanoparticle association and thereby their interaction with the biological environment. In present study, effect of size of chitosan reduced gold nanoparticles (CsAuNPs) and association temperature on structure and function of tetanus toxoid (TT) vaccine has been investigated. CsAuNPs were synthesized in the sizes of 20 + 3, 40 + 5 and 80 + 7 nm followed by loading of TT. Binding process of CsAuNPs with TT was investigated at their predetermined micro molar concentrations. Upon binding of TT onto CsAuNPs, particle surface was characterized using X-ray photoelectron spectroscopy. CD spectroscopic evaluation of TT bound 20 nm CsAuNPs led to 75% reduction in secondary structure of TT and thereby compromised immune function. Binding of TT with 40 and 80 nm sized CsAuNPs did not cause significant modifications in secondary structure or function of TT. Thermodynamic studies using temperature dependent fluorescence spectroscopy revealed an increase in association constants with the temperature. Based on thermodynamic data three phases in CsAuNPs and TT association process were traced. Samples from these distinct phases were also investigated for immunological recognition. Ex-vivo interaction of TT-CsAuNPs with TT positive and negative sera followed by relative change in particle size and zeta potential was studied. The findings here suggests prominent role of particle size and association temperature on adsorbed TT structure and function. Such studies may help in engineering functional nanotherapeutics. (C) 2014 Elsevier B.V. All rights reserved.

10aAssociation thermodynamics10aGold nanoparticle10aNanoparticle protein interaction10aStructure function studies10aVaccine formulation1 aBarhate, Ganesh, A.1 aGaikwad, Sushama, M.1 aJadhav, Suresh, S.1 aPokharkar, Varsha, B. uhttps://library.ncl.res.in/content/structure-function-attributes-gold-nanoparticle-vaccine-association-effect-particle-size-002593nas a2200277 4500008004100000022001400041245009900055210006900154260005700223300001200280490000700292520162200299653001401921653002601935653003101961653002001992653001402012653002502026653002802051100002102079700002102100700001802121700002502139700002502164856012602189 2014 eng d a0175-757100aSubtilase from Beauveria sp.: conformational and functional investigation of unusual stability0 aSubtilase from Beauveria sp conformational and functional invest a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcSEP a393-4030 v433 a

Retention of total activity of the subtilisin-like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50 % methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the protein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chromatography in the presence of GdnHCl (1.0-3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentration of denaturant (1.0-4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k (unf) of native protein to be 5.407 x 10(-5) s(-1) and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resistant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interesting structural transitions.

10aBeauveria10aChemical denaturation10aConformational transitions10aSerine protease10aSubtilase10aThermal denaturation10aThermodynamic stability1 aDalal, Sayli, A.1 aMore, Snehal, V.1 aShankar, Shiv1 aLaxman, Ryali, Seeta1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/subtilase-beauveria-sp-conformational-and-functional-investigation-unusual-stability-0