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000042202 0247_ $$2DOI$$a10.1021/bi0493362
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000042202 084__ $$2WoS$$aBiochemistry & Molecular Biology
000042202 1001_ $$0P:(DE-Juel1)131961$$aFitter, J.$$b0$$uFZJ
000042202 245__ $$aStructural Stability and Unfolding Properties of Thermostable bacterial alpha-amylases: A Comparative Study on Homologous Enzymes
000042202 260__ $$aColumbus, Ohio$$bAmerican Chemical Society$$c2004
000042202 300__ $$a9589 - 9599
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000042202 440_0 $$0798$$aBiochemistry$$v43$$x0006-2960
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000042202 520__ $$aIn a comparative investigation on two thermostable alpha-amylases [Bacillus amyloliquefaciens (BAA), T(m) = 86 degrees C and Bacillus licheniformis (BLA), T(m) = 101 degrees C], we studied thermal and guanidine hydrochloride (GndHCl)-induced unfolding using fluorescence and CD spectroscopy, as well as dynamic light scattering. Depletion of calcium from specific ion-binding sites in the protein structures reduces the melting temperature tremendously for both alpha-amylases. The reduction is nearly the same for both enzymes, namely, in the order of 50 degrees C. Thus, the difference in thermostability between BLA and BAA (DeltaT(m) approximately 15 degrees C) is related to intrinsic properties of the respective protein structures themselves and is not related to the strength of ion binding. The thermal unfolding of both proteins is characterized by a full disappearance of secondary structure elements and by a concurrent expansion of the 3D structure. GndHCl-induced unfolding also yields a fully vanishing secondary structure but with more expanded 3D structures. Both alpha-amylases remain much more compact upon thermal unfolding as compared to the fully unfolded state induced by chemical denaturants. Such rather compact thermal unfolded structures lower the conformational entropy change during the unfolding transition, which principally can contribute to an increased thermal stability. Structural flexibilities of both enzymes, as measured with tryptophan fluorescence quenching, are almost identical for both enzymes in the native states, as well as in the unfolded states. Furthermore, we do not observe any difference in the temperature dependence of the structural flexibilities between BLA and BAA. These results indicate that conformational dynamics on the time scale of our studies seem not to be related to thermal stability or to thermal adaptation.
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000042202 650_2 $$2MeSH$$aBacillus: enzymology
000042202 650_2 $$2MeSH$$aBacterial Proteins: chemistry
000042202 650_2 $$2MeSH$$aEnzyme Stability
000042202 650_2 $$2MeSH$$aGuanidine: chemistry
000042202 650_2 $$2MeSH$$aKinetics
000042202 650_2 $$2MeSH$$aLight
000042202 650_2 $$2MeSH$$aProtein Denaturation
000042202 650_2 $$2MeSH$$aProtein Folding
000042202 650_2 $$2MeSH$$aScattering, Radiation
000042202 650_2 $$2MeSH$$aSequence Homology, Amino Acid
000042202 650_2 $$2MeSH$$aSpectrometry, Fluorescence
000042202 650_2 $$2MeSH$$aTemperature
000042202 650_2 $$2MeSH$$aThermodynamics
000042202 650_2 $$2MeSH$$aalpha-Amylases: chemistry
000042202 650_7 $$00$$2NLM Chemicals$$aBacterial Proteins
000042202 650_7 $$0113-00-8$$2NLM Chemicals$$aGuanidine
000042202 650_7 $$0EC 3.2.1.1$$2NLM Chemicals$$aalpha-Amylases
000042202 650_7 $$2WoSType$$aJ
000042202 7001_ $$0P:(DE-Juel1)VDB12272$$aHaber-Pohlmeier, S.$$b1$$uFZJ
000042202 773__ $$0PERI:(DE-600)1472258-6$$a10.1021/bi0493362$$gVol. 43, p. 9589 - 9599$$p9589 - 9599$$q43<9589 - 9599$$tBiochemistry$$v43$$x0006-2960$$y2004
000042202 8567_ $$uhttp://hdl.handle.net/2128/701$$uhttp://dx.doi.org/10.1021/bi0493362
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