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Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity
- Source :
- Journal of Biological Inorganic Chemistry, Journal of Biological Inorganic Chemistry, 2015, 20 (5), pp.905-919. ⟨10.1007/s00775-015-1275-1⟩, Journal of Biological Inorganic Chemistry, Springer Verlag, 2015, 20 (5), pp.905-919. ⟨10.1007/s00775-015-1275-1⟩
- Publication Year :
- 2015
-
Abstract
- International audience; Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.
- Subjects :
- Models, Molecular
Calmodulin
[SDV]Life Sciences [q-bio]
030303 biophysics
Molecular Sequence Data
chemistry.chemical_element
Cooperativity
Calcium
Protein Engineering
Biochemistry
Inorganic Chemistry
03 medical and health sciences
Protein structure
Amino Acid Sequence
030304 developmental biology
0303 health sciences
Binding Sites
biology
Mutagenesis
Tryptophan
Cooperative binding
Isothermal titration calorimetry
Protein Structure, Tertiary
Crystallography
chemistry
Biophysics
biology.protein
Mutagenesis, Site-Directed
Thermodynamics
Subjects
Details
- ISSN :
- 14321327 and 09498257
- Volume :
- 20
- Issue :
- 5
- Database :
- OpenAIRE
- Journal :
- Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry
- Accession number :
- edsair.doi.dedup.....741045600838a821717de4262022f792