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Differential scanning calorimetric, circular dichroism, and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans.
Proteins. 2003 Feb 01; 50(2):341-54.P

Abstract

The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64 degrees C and an exothermic event centered near 75 degrees C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications.

Authors+Show Affiliations

Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Laval, Quebec, Canada.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

12486727

Citation

Roberge, Martin, et al. "Differential Scanning Calorimetric, Circular Dichroism, and Fourier Transform Infrared Spectroscopic Characterization of the Thermal Unfolding of Xylanase a From Streptomyces Lividans." Proteins, vol. 50, no. 2, 2003, pp. 341-54.
Roberge M, Lewis RN, Shareck F, et al. Differential scanning calorimetric, circular dichroism, and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans. Proteins. 2003;50(2):341-54.
Roberge, M., Lewis, R. N., Shareck, F., Morosoli, R., Kluepfel, D., Dupont, C., & McElhaney, R. N. (2003). Differential scanning calorimetric, circular dichroism, and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans. Proteins, 50(2), 341-54.
Roberge M, et al. Differential Scanning Calorimetric, Circular Dichroism, and Fourier Transform Infrared Spectroscopic Characterization of the Thermal Unfolding of Xylanase a From Streptomyces Lividans. Proteins. 2003 Feb 1;50(2):341-54. PubMed PMID: 12486727.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Differential scanning calorimetric, circular dichroism, and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans. AU - Roberge,Martin, AU - Lewis,Ruthven N A H, AU - Shareck,François, AU - Morosoli,Rolf, AU - Kluepfel,Dieter, AU - Dupont,Claude, AU - McElhaney,Ronald N, PY - 2002/12/18/pubmed PY - 2003/5/28/medline PY - 2002/12/18/entrez SP - 341 EP - 54 JF - Proteins JO - Proteins VL - 50 IS - 2 N2 - The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64 degrees C and an exothermic event centered near 75 degrees C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications. SN - 1097-0134 UR - https://www.unboundmedicine.com/medline/citation/12486727/Differential_scanning_calorimetric_circular_dichroism_and_Fourier_transform_infrared_spectroscopic_characterization_of_the_thermal_unfolding_of_xylanase_A_from_Streptomyces_lividans_ L2 - https://doi.org/10.1002/prot.10262 DB - PRIME DP - Unbound Medicine ER -