Differential scanning calorimetric, circular dichroism, and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans.


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. Study holds ProTherm entries: 16161, 16162, 16163, 16164, 16165, 16166, 16167, 16168, 16169, 16170, 16171, 16172, 16173, 16174, 16175, 16176, 16177, 16178, 16179, 16180, 16181, 16182, 16183 Extra Details: Catalytic domain; 1mM NaN3 was added in the experiment xylanase A; Streptomyces lividans; thermal unfolding; protein denaturation; domain interactions; differential scanning calorimetry; circular dichroism; infrared spectroscopy

Submission Details

ID: nZvB8kpz3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Roberge M;Lewis RN;Shareck F;Morosoli R;Kluepfel D;Dupont C;McElhaney RN,Proteins (2003) Differential scanning calorimetric, circular dichroism, and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans. PMID:12486727
Additional Information

Study Summary

Number of data points 58
Proteins Endo-1,4-beta-xylanase A ; Endo-1,4-beta-xylanase A
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Tm prot_conc:0.5 mg/mL, buffers:boric acid-NaOH: 50 mM, details:Additives , pH:9.02 ; Experimental Assay: dHvH prot_conc:0.5 mg/mL, buffers:boric acid-NaOH: 50 mM, details:Additives , pH:9.02 ; Experimental Assay: Tm pH:8.57, prot_conc:0.5 mg/mL, buffers:boric acid-NaOH: 50 mM, details:Additives ; Experimental Assay: dHvH pH:8.57, prot_conc:0.5 mg/mL, buffers:boric acid-NaOH: 50 mM, details:Additives ; Experimental Assay: Tm prot_conc:0.5 mg/mL, buffers:boric acid-NaOH: 50 mM, details:Additives , pH:8.01 ; Experimental Assay: dHvH prot_conc:0.5 mg/mL, buffers:boric acid-NaOH: 50 mM, details:Additives , pH:8.01 ; Experimental Assay: Tm prot_conc:0.5 mg/mL, pH:7.46, details:Additives , buffers:sodium phosphate: 50 mM ; Experimental Assay: dHvH prot_conc:0.5 mg/mL, pH:7.46, details:Additives , buffers:sodium phosphate: 50 mM ; Experimental Assay: Tm prot_conc:0.5 mg/mL, pH:7.0, details:Additives , buffers:sodium phosphate: 50 mM ; Experimental Assay: dHvH prot_conc:0.5 mg/mL, pH:7.0, details:Additives , buffers:sodium phosphate: 50 mM ; Experimental Assay: Tm prot_conc:0.5 mg/mL, pH:6.49, details:Additives , buffers:sodium phosphate: 50 mM ; Experimental Assay: dHvH prot_conc:0.5 mg/mL, pH:6.49, details:Additives , buffers:sodium phosphate: 50 mM ; Experimental Assay: Tm pH:6.0, prot_conc:0.5 mg/mL, details:Additives , buffers:sodium phosphate: 50 mM ; Experimental Assay: dHvH pH:6.0, prot_conc:0.5 mg/mL, details:Additives , buffers:sodium phosphate: 50 mM ; Experimental Assay: Tm prot_conc:0.5 mg/mL, pH:5.5, details:Additives , buffers:citric acid-sodium phosphate: 50 mM ; Experimental Assay: dHvH prot_conc:0.5 mg/mL, pH:5.5, details:Additives , buffers:citric acid-sodium phosphate: 50 mM ; Experimental Assay: Tm prot_conc:0.5 mg/mL, details:Additives , buffers:citric acid-sodium phosphate: 50 mM, pH:5.0 ; Experimental Assay: dHvH prot_conc:0.5 mg/mL, details:Additives , buffers:citric acid-sodium phosphate: 50 mM, pH:5.0 ; Experimental Assay: Tm prot_conc:0.5 mg/mL, details:Additives , pH:4.49, buffers:citric acid-sodium phosphate: 50 mM ; Experimental Assay: dHvH prot_conc:0.5 mg/mL, details:Additives , pH:4.49, buffers:citric acid-sodium phosphate: 50 mM ; Experimental Assay: Tm prot_conc:0.5 mg/mL, details:Additives , buffers:citric acid-sodium phosphate: 50 mM, pH:3.98 ; Experimental Assay: dHvH prot_conc:0.5 mg/mL, details:Additives , buffers:citric acid-sodium phosphate: 50 mM, pH:3.98 ; Experimental Assay: dHcal pH:7.0, buffers:NaH2PO4, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: Tm details:Additives NaN3 (1mM),, pH:7.0, buffers:NaH2PO4, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: dHvH details:Additives NaN3 (1mM),, pH:7.0, buffers:NaH2PO4, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: dHcal pH:6.5, buffers:NaH2PO4, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: Tm details:Additives NaN3 (1mM),, pH:6.5, buffers:NaH2PO4, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: dHvH details:Additives NaN3 (1mM),, pH:6.5, buffers:NaH2PO4, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: dHcal pH:6.0, buffers:NaH2PO4, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: Tm pH:6.0, details:Additives NaN3 (1mM),, buffers:NaH2PO4, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: dHvH pH:6.0, details:Additives NaN3 (1mM),, buffers:NaH2PO4, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: dHcal pH:5.5, buffers:citric acid, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: Tm buffers:citric acid, Na2HPO4: 50 mM, 50 mM, pH:5.5, details:Additives NaN3 (1mM), ; Experimental Assay: dHvH buffers:citric acid, Na2HPO4: 50 mM, 50 mM, pH:5.5, details:Additives NaN3 (1mM), ; Experimental Assay: dHcal pH:5.0, buffers:citric acid, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: Tm pH:5.0, details:Additives NaN3 (1mM),, buffers:citric acid, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: dHvH pH:5.0, details:Additives NaN3 (1mM),, buffers:citric acid, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: dHcal pH:4.5, buffers:citric acid, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: Tm pH:4.5, details:Additives NaN3 (1mM),, buffers:citric acid, Na2HPO4: 50 mM, 50 mM ; Experimental Assay: dHvH pH:4.5, details:Additives NaN3 (1mM),, buffers:citric acid, Na2HPO4: 50 mM, 50 mM
Libraries Mutations for sequence AESTLGAAAAQSGRYFGTAIASGRLSDSTYTSIAGREFNMVTAENEMKIDATEPQRGQFNFSSADRVYNWAVQNGKQVRGHTLAWHSQQPGWMQSLSGRPLRQAMIDHINGVMAHYKGKIVQWDVVNEAFADGSSGARRDSNLQRSGNDWIEVAFRTARAADPSAKLCYNDYNVENWTWAKTQAMYNMVRDFKQRGVPIDCVGFQSHFNSGSPYNSNFRTTLQNFAALGVDVAITELDIQGAPASTYANVTNDCLAVSRCLGITVWGVRDSDSWRSEQTPLLFNNDGSKKAAYTAVLDA

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1V0L 2004-08-16 0.98 Xylanase Xyn10A from Streptomyces lividans in complex with xylobio-isofagomine at pH 5.8
1V0K 2004-08-16 1.03 Xylanase Xyn10A from Streptomyces lividans in complex with xylobio-deoxynojirimycin at pH 5.8
1OD8 2003-04-08 1.05 Xylanase Xyn10A from Streptomyces lividans in complex with xylobio-isofagomine lactam
1V0M 2004-08-16 1.07 Xylanase Xyn10a from Streptomyces lividans in complex with xylobio-deoxynojirimycin at pH 7.5
1V0N 2004-08-16 1.1 Xylanase Xyn10a from Streptomyces lividans in complex with xylobio-isofagomine at pH 7.5
1E0W 2001-04-05 1.2 Xylanase 10A from Sreptomyces lividans. native structure at 1.2 angstrom resolution
1KNM 2002-06-19 1.2 Streptomyces lividans Xylan Binding Domain cbm13 in Complex with Lactose
1KNL 2002-06-19 1.2 Streptomyces lividans Xylan Binding Domain cbm13
1E0X 2001-04-05 1.65 XYLANASE 10A FROM SREPTOMYCES LIVIDANS. XYLOBIOSYL-ENZYME INTERMEDIATE AT 1.65 A
1E0V 2001-04-05 1.7 Xylanase 10A from Sreptomyces lividans. cellobiosyl-enzyme intermediate at 1.7 A
1MC9 2002-09-11 1.7 STREPROMYCES LIVIDANS XYLAN BINDING DOMAIN CBM13 IN COMPLEX WITH XYLOPENTAOSE
1XAS 1995-05-31 2.6 CRYSTAL STRUCTURE, AT 2.6 ANGSTROMS RESOLUTION, OF THE STREPTOMYCES LIVIDANS XYLANASE A, A MEMBER OF THE F FAMILY OF BETA-1,4-D-GLYCANSES

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Endo-1,4-beta-xylanase A P26514 XYNA_STRLI