Calcium Binding and Thermostability of Carbohydrate Binding Module CBM4-2 of Xyn10A from Rhodothermus marinus
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- 作者：Maher Abou-Hachem ; Eva Nordberg Karlsson ; Peter J. Simpson ; Sara Linse ; Peter Sellers ; Michael P. Williamson ; Stuart J. Jamieson ; Harry J. Gilbert ; David N. Bolam ; and Olle Holst
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：May 7, 2002
- 年：2002
- 卷：41
- 期：18
- 页码：5720 - 5729
- 全文大小：353K
- 年卷期：v.41,no.18(May 7, 2002)
- ISSN：1520-4995
文摘
Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) fromRhodothermus 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. Themoderate-affinity site has an association constant of (1.3 ± 0.3) × 105 M-1 and a binding enthalpy 
Haof -9.3 ± 0.4 kJ mol-1, while the high-affinity site has an association constant of approximately 1010M-1 and a binding enthalpy Ha of -40.5 ± 0.5 kJ mol-1. The locations of the binding sites have beenidentified 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, whilethe 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 ina recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (Tm) byapproximately 23 
C at pH 7.5. No correlation between binding affinity and Tm change was noted, aseach of the two calcium ions contributes almost equally to the increase in unfolding temperature.
Haof -9.3 ± 0.4 kJ mol-1, while the high-affinity site has an association constant of approximately 1010M-1 and a binding enthalpy Ha of -40.5 ± 0.5 kJ mol-1. The locations of the binding sites have beenidentified 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, whilethe 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 ina recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (Tm) byapproximately 23 C at pH 7.5. No correlation between binding affinity and Tm change was noted, aseach of the two calcium ions contributes almost equally to the increase in unfolding temperature.