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Lookup NU author(s): Emeritus Professor Harry Gilbert, Dr David Bolam
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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 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 mol(-1). The locations of the binding sites hake 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 curbonyls 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 degreesC at pH 7.5. No correlation between binding affinity and T-m , contributes almost equally to the increase ill unfolding temperature.
Author(s): Abou-Hachem M, Karlsson EN, Simpson PJ, Linse S, Sellers P, Williamson MP, Jamieson SJ, Gilbert HJ, Bolam DN, Holst O
Publication type: Article
Publication status: Published
Journal: Biochemistry
Year: 2002
Volume: 41
Issue: 18
Pages: 5720-5729
ISSN (print): 0006-2960
ISSN (electronic): 1520-4995
Publisher: American Chemical Society
URL: http://dx.doi.org/10.1021/bi012094a
DOI: 10.1021/bi012094a
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