Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/107891
- author
- 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
- organization
- publishing date
- 2002
- type
- Contribution to journal
- publication status
- 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
- 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
- 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}}, }