New Aspects of Calmodulin–Calmodulin Binding Domains Recognition

Abstract

Understanding the role of calmodulin (CaM) in calcium signal transduction implies to describe the -calcium-dependent molecular mechanism of interaction of CaM with the various CaM-binding domains (CBD). In order to fulfill this aim, we have developed a new strategy and the afferent techniques to quantify the interaction of CaM with any CBD as a function of calcium concentration. Excel software has been used to deconvolute the experimental data and to obtain the macroscopic constants characterizing the system. We are illustrating our approach on six different CaM/CBD. This strategy may be used to analyze the interaction between any calcium-binding protein and its targets.

The adaptors Grb10 and Grb14 are calmodulin‐binding proteins

“…To exclude that the adaptor proteins were piggy‐back bound to other CaM‐BPs present in the preparations, we decided to estimate the binding and affinity of peptides corresponding to the identified CaM‐BDs of Grb7, Grb10 and Grb14 to CaM. We used a new technique based on fluorescence polarization to describe the binding affinity of CaM for its target, as well as the minimum number of Ca 2+ bound to CaM to achieve efficient binding . Peptides corresponding to the CaM‐BD of Grb7, and the homologous sequences of Grb10 and Grb14 tagged with the fluorescent probe TAMRA (Fig.…”

“…A) were assayed by fluorescent polarization of the CaM/peptide target interaction. We used a titration matrix in which both Ca 2+ and CaM concentrations were changed to determine the minimum number of Ca 2+ bound to CaM that was required for the interaction to occur with efficiency, as well as the apparent association and dissociation constants (K' a and K' d ) . Figure B shows the apparent dissociation constants (K' d ) of the fluorescent target peptides corresponding to the CaM‐BD of Grb7, Grb10 and Grb14 when they bind to CaM as a function of the Ca 2+ concentration.…”

“…The binding of the tagged peptides to CaM was followed by fluorescence anisotropy in a medium of known changing free Ca 2+ concentrations. Exhaustive description of the methodology used was previously described .…”