caa a22 4500 605507651 CHVBK 20210128100633.0 cr unu---uuuuu 210128e20150701xx s 000 0 eng 10.1007/s00775-015-1275-1 doi (NATIONALLICENCE)springer-10.1007/s00775-015-1275-1 Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity [Elektronische Daten] [Maria Beccia, Sandrine Sauge-Merle, David Lemaire, Nicolas Brémond, Romain Pardoux, Stéphanie Blangy, Philippe Guilbaud, Catherine Berthomieu] 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. SBIC, 2015 Calmodulin nationallicence Calcium nationallicence Microscopic binding constants nationallicence Thermodynamics nationallicence Cooperativity nationallicence Beccia Maria CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut Sauge-Merle Sandrine CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut Lemaire David CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut Brémond Nicolas CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut Pardoux Romain CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut Blangy Stéphanie Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques, 13284, Marseille Cedex 09, France aut Guilbaud Philippe CEA, DRCPC, SMCS, LILA, Bagnols Sur Cèze, France aut Berthomieu Catherine CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/5(2015-07-01), 905-919 0949-8257 20:5<905 2015 20 775 https://doi.org/10.1007/s00775-015-1275-1 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00775-015-1275-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Beccia Maria CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut NATIONALLICENCE 700 1- Sauge-Merle Sandrine CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut NATIONALLICENCE 700 1- Lemaire David CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut NATIONALLICENCE 700 1- Brémond Nicolas CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut NATIONALLICENCE 700 1- Pardoux Romain CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut NATIONALLICENCE 700 1- Blangy Stéphanie Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques, 13284, Marseille Cedex 09, France aut NATIONALLICENCE 700 1- Guilbaud Philippe CEA, DRCPC, SMCS, LILA, Bagnols Sur Cèze, France aut NATIONALLICENCE 700 1- Berthomieu Catherine CEA, DSV, IBEB, Laboratoire des Interactions Protéine Métal, Saint-Paul-Lez-Durance, France aut NATIONALLICENCE 773 0- JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/5(2015-07-01), 905-919 0949-8257 20:5<905 2015 20 775