02425nas a2200277 4500008004100000022001400041245011100055210006900166260009500235300001200330490000800342520139600350653002301746653001601769653001501785653001901800653004801819100002101867700002401888700002601912700002201938700001901960700002501979700002102004856012202025 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. uhttp://library.ncl.res.in/content/conformational-characterization-human-eukaryotic-initiation-factor-2-alpha-single-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-002053nas a2200253 4500008004100000022001400041245009800055210006900153260009500222300001400317490000800331520110600339653001001445653002301455653002201478653003901500653002201539100001901561700002201580700002501602700002101627700002001648856013101668 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. uhttp://library.ncl.res.in/content/structural-functional-insights-single-and-multi-domain-capsicum-annuum-protease-inhibitors-0