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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 orcid ; 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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Contribution to journal
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published
subject
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 (ACS)
external identifiers
  • wos:000175365300004
  • pmid:11980476
  • scopus:0037035542
ISSN
0006-2960
DOI
10.1021/bi012094a
language
English
LU publication?
yes
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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
2016-04-04 08:32:56
date last changed
2022-01-29 03:32:14
@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}},
  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}},
  language     = {{eng}},
  number       = {{18}},
  pages        = {{5720--5729}},
  publisher    = {{The American Chemical Society (ACS)}},
  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}},
  doi          = {{10.1021/bi012094a}},
  volume       = {{41}},
  year         = {{2002}},
}