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

Abou-Hachem, Maher LU ; Nordberg Karlsson, Eva LU ; Simpson, Peter J; Linse, Sara LU ; Sellers, Peter LU ; Williamson, Michael P; Jamieson, Stuart J; Gilbert, Harry J; Bolam, David N and Holst, Olle LU (2002) In Biochemistry 41(18). p.5720-5729
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... (More)
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. (Less)
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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
in
Biochemistry
volume
41
issue
18
pages
5720 - 5729
publisher
The American Chemical Society
external identifiers
  • wos:000175365300004
  • pmid:11980476
  • scopus:0037035542
ISSN
0006-2960
DOI
10.1021/bi012094a
language
English
LU publication?
yes
id
ca06fc6b-8ee0-4fc2-b88e-d69b2ac4598d (old id 107891)
alternative location
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11980476&dopt=Abstract
date added to LUP
2007-06-26 08:27:28
date last changed
2017-07-23 04:53:49
@article{ca06fc6b-8ee0-4fc2-b88e-d69b2ac4598d,
  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.},
  author       = {Abou-Hachem, Maher and Nordberg Karlsson, Eva and Simpson, Peter J and Linse, Sara and Sellers, Peter and Williamson, Michael P and Jamieson, Stuart J and Gilbert, Harry J and Bolam, David N and Holst, Olle},
  issn         = {0006-2960},
  keyword      = {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},
  language     = {eng},
  number       = {18},
  pages        = {5720--5729},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.},
  url          = {http://dx.doi.org/10.1021/bi012094a},
  volume       = {41},
  year         = {2002},
}