Plant calmodulin-like proteins: calcium binding and target interactions

Calcium (Ca2+) is an ubiquitous key second messenger in all living organisms, from bacteria to differentiated neuronal cells and is involved in a large variety of biological processes such as apoptosis, cell differentiation and proliferation, fertilization, transcription factors activation and many others. Plants in particular are known to be capable of exploiting Ca2+ signalling in a more sophisticated way than other organisms as demonstrated by the larger set of Ca2+-binding proteins (CBPs). Although the study of CBP has been an open and highly studied field for over a century, a biochemical and structural characterization of many CBPs from different organisms is still missing. Here, we provided a deep characterisation of two plant (Arabidopsis thaliana) Ca2+ sensors, a specific class of CBPs, to understand the differences in their ability to respond to Ca2+ and the regulation of downstream targets. Thus we analysed the metal and target binding properties of Calmodulin like protein 7 and 19 (CML7 and CML19). By exploiting spectroscopic techniques (NMR and CD) we evaluated the conformational changes induced by metal binding and assigned the specific affinities for each binding site by a combination of calorimetric and spectroscopic techniques (ITC and NMR). To further understand the Ca2+ sensing properties and the ability to interact with target proteins, we analysed the interaction of CML7 and CML19 with synthetic peptides, representing the binding region on physiological or model targets. Because the majority of CBPs are characterised by multiple metal binding sites, clustered affinities and cooperativity events, the determination of Ca2+/CBPs stoichiometry requires time and sample consuming procedures and sophisticated instrumentations (i.e. ITC and NMR). Thus, we report the optimisation of a new method, based on X-ray fluorescence, to overcome the most common limitations of the currently used techniques, allowing the quick estimation of the Ca2+/protein stoichiometry using low amounts of sample. Finally, initial studies on the recombinant production and characterisation of a neuronal family of CBPs are reported. These protein are called NECABs and are characterised by a peculiar domain composition and might be involved in the development of neurodegenerative diseases.