Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.

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Abstract

Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.

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Subject classification (UKÄ) – MANDATORY

  • Biochemistry and Molecular Biology

Keywords

  • Rhodobacter : genetics, Rhodobacter : enzymology, Protein Denaturation, Protein Conformation, Protein Binding, Biomolecular, Nuclear Magnetic Resonance, Site-Directed, Mutagenesis, Molecular, Models, Hydrogen-Ion Concentration, Enzyme Stability, Cloning, Carbohydrates : metabolism, Calorimetry, Calcium : metabolism, Binding Sites, Xylosidases : chemistry, Xylosidases : genetics, Xylosidases : metabolism, Structure-Activity Relationship, Support, Non-U.S. Gov't, Temperature, Thermodynamics
Original languageEnglish
Pages (from-to)5720-5729
JournalBiochemistry
Volume41
Issue number18
Publication statusPublished - 2002
Publication categoryResearch
Peer-reviewedYes