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Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity

Maria Rosa Beccia 1 Sandrine Sauge-Merle 2 David Lemaire 2 Nicolas Brémond 2 Romain Pardoux 3, 4, 5, 2 Stéphanie Blangy 6, 7, 3, 8 Philippe Guilbaud 9 C. Berthomieu 7, 2, 10
1 Instituto de Química
2 BVME - Biologie végétale et microbiologie environnementale - UMR7265
3 AMU - Aix Marseille Université
4 DRF (CEA) - Direction de Recherche Fondamentale (CEA)
5 CNRS - Centre National de la Recherche Scientifique
6 CEA Cadarache
7 BIAM - Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB)
8 EBM - Bioénergie et Microalgues
BIAM - Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) : DRF/BIAM
9 DRCP - Département RadioChimie et Procédés
10 IPM - Interactions Protéine Métal
BIAM - Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) : DRF/BIAM
Abstract : 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.
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https://hal-amu.archives-ouvertes.fr/hal-01561163
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Submitted on : Wednesday, July 12, 2017 - 2:16:52 PM
Last modification on : Friday, January 21, 2022 - 10:40:01 AM

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UNIV-AMU | CNRS | CEA | DEN | CEA-DRF | CEA-CAD | BIAM | DEN-MARCO

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Maria Rosa Beccia, Sandrine Sauge-Merle, David Lemaire, Nicolas Brémond, Romain Pardoux, et al.. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity. Journal of Biological Inorganic Chemistry, Springer Verlag, 2015, 20 (5), pp.905 - 919. ⟨10.1007/s00775-015-1275-1⟩. ⟨hal-01561163⟩

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