CORDIS provides links to public deliverables and publications of HORIZON projects.
Links to deliverables and publications from FP7 projects, as well as links to some specific result types such as dataset and software, are dynamically retrieved from OpenAIRE .
Deliverables
Calmodulin (CaM) has been particularly studied as the prototypical Ca2+-binding protein by using recombinant proteins in order to study the structure/activity relationships. Obviously, a better understanding of the Ca2+-binding mechanism of CaM must allow to effectively design drugs able to interfere with the calcium signals and, ultimately, to modify cellular events such as apoptosis or cellular differentiation.
Our aim was to evaluate the importance of electrostatic potential in the calcium binding mechanism of calmodulin. Therefore, we studied SynCaM, a synthetic hybrid of mammalian and plant CaM, which is able to activate all the Ca2+CaM-dependent proteins, and three charge reversal mutants. Using differential scanning calorimetry and CD, we evidenced the major role of the electrostatic potential in the stability and flexibility of CaM and in the influence of calcium binding lobes on each other.
Mass spectrometry, flow dialysis and isothermal microcalorimetry allowed the thermodynamic parameters associated with the binding of calcium to SynCaM mutants to be characterized. The parameters follow an enthalpy-entropy compensation for the conformational changes induced by Ca2+ binding. This result suggests that the different mutants explore different subspaces of conformation and, therefore, shed some light on the specificity of CaM responce. Each calmodulin binding structure may be associated to a given subset of calmodulin conformations.
We have shown that denaturation transitions of the N- and C-terminal lobes of Nereis sarcoplasmic calcium binding protein (NSCP) are very sensitive to Ca2+-binding and to the mutations altering the affinity of different cites and the structure of the central helix. The addition of calcium causes the significant increase of NSCP thermostability so as to form one single cooperative folding unit instead of two. The two lobes in NSCP are not independant and the influence of the lobes on each other is much stronger in Ca2+-saturated proteins. The high affinity of NSCP for Ca2+ is due to allosteric interaction between the two halves, rather than to the main structure.
Comparison of Ca2+-binding mechanism of calmodulin and NSCP allowed us to start to understand how the evolution using a common structure has been able to design through selection different thermodynamic and kinetic molecular mechanisms in order to decipher the calcium signal of a given cell in a given organism.
We then focused on the interplay between a cellular stress response involving general chaperones and the calcium signal. Indeed, molecular chaperones such as heat shock protein hsp90 seem to interact with CaM. This led us to study hsp90 thermal denaturation by DSC, CD, fluorescence, and Nature/PAGE electrophoresis. Our results evidenced the dramatic loss of stability induced by magnesium. The binding of hsp90 to CaM and to tubulin is under investigation.
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