Calcium (Ca2+) is one of the most important second messengers in eukaryotes. Ca2+ binding proteins can be subdivided into two categories: “Ca2+ buffers” that modulate Ca2+ ion concentrations in cells, and “Ca2+ sensors” that decode Ca2+ signals in a wide array ofphysiological processes in response to external stimuli. Calmodulin (CaM) is the prototypicalexample of Ca2+ sensor proteins in both animals and plants. In addition to conserved CaM,plants possess a unique family of 50 CaM-like proteins (CMLs). Many of these CMLs still remainuncharacterized and the investigation of their biochemical and biophysical properties willprovide insight into Ca2+ signalling in plants. Herein, a detailed characterization of Arabidopsisthaliana CML14 is reported. CML14 is a protein of 148 amino acids with a theoretical molecularweight of 16,579 Da and 50% amino acid sequence identity with AtCaM2. CML14 is predictedto have one functional Ca2+ binding site despite the presence of three EF-hand motifs(Prosite). We overexpressed CML14 in E. coli and analyzed its biochemical and biophysicalcharacteristics, i.e. calcium affinity and stoichiometry and eventual changes in conformation,thermal stability and proteolytic susceptibility upon Ca2+ binding. Isothermal titrationcalorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy identified one Ca2+binding site in CML14 and showed that Ca2+ and Mg2+ compete for the same binding site. TheKd values determined by ITC established that CML14 has higher affinity for Ca2+ than forMg2+. Our data were consistent with the sequence based prediction of one functional calciumbinding site. Differential scanning calorimetry (DSC) showed that Ca2+ and Mg2+ have thesame stabilizing effects on protein folding. Apo-CML14 undergoes two thermal unfoldingtransitions, but in the presence of Ca2+ or Mg2+ only one unfolding event at an intermediatetemperature occurs. Limited proteolysis experiments showed that Ca2+ binding affordsprotection against CML14 digestion by trypsin. Surprisingly, CML14 exhibits very fewconformational changes upon calcium binding, which were evaluated by ANS fluorescence andStokes radius measurements in the apo- and Ca2+ bound-forms. These results suggest thatCML14 does not show the characteristics of a classical Ca2+ sensor protein. To betterunderstand the physiological role of CML14 in plants, in vivo analysis will be performed.

Biophysical and biochemical characterization of Arabidopsis thaliana Calmodulin-like protein CML14

Vallone, Rosario;LA VERDE, Valentina;D'ONOFRIO, Mariapina;ASTEGNO, Alessandra;DOMINICI, Paola
2015

Abstract

Calcium (Ca2+) is one of the most important second messengers in eukaryotes. Ca2+ binding proteins can be subdivided into two categories: “Ca2+ buffers” that modulate Ca2+ ion concentrations in cells, and “Ca2+ sensors” that decode Ca2+ signals in a wide array ofphysiological processes in response to external stimuli. Calmodulin (CaM) is the prototypicalexample of Ca2+ sensor proteins in both animals and plants. In addition to conserved CaM,plants possess a unique family of 50 CaM-like proteins (CMLs). Many of these CMLs still remainuncharacterized and the investigation of their biochemical and biophysical properties willprovide insight into Ca2+ signalling in plants. Herein, a detailed characterization of Arabidopsisthaliana CML14 is reported. CML14 is a protein of 148 amino acids with a theoretical molecularweight of 16,579 Da and 50% amino acid sequence identity with AtCaM2. CML14 is predictedto have one functional Ca2+ binding site despite the presence of three EF-hand motifs(Prosite). We overexpressed CML14 in E. coli and analyzed its biochemical and biophysicalcharacteristics, i.e. calcium affinity and stoichiometry and eventual changes in conformation,thermal stability and proteolytic susceptibility upon Ca2+ binding. Isothermal titrationcalorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy identified one Ca2+binding site in CML14 and showed that Ca2+ and Mg2+ compete for the same binding site. TheKd values determined by ITC established that CML14 has higher affinity for Ca2+ than forMg2+. Our data were consistent with the sequence based prediction of one functional calciumbinding site. Differential scanning calorimetry (DSC) showed that Ca2+ and Mg2+ have thesame stabilizing effects on protein folding. Apo-CML14 undergoes two thermal unfoldingtransitions, but in the presence of Ca2+ or Mg2+ only one unfolding event at an intermediatetemperature occurs. Limited proteolysis experiments showed that Ca2+ binding affordsprotection against CML14 digestion by trypsin. Surprisingly, CML14 exhibits very fewconformational changes upon calcium binding, which were evaluated by ANS fluorescence andStokes radius measurements in the apo- and Ca2+ bound-forms. These results suggest thatCML14 does not show the characteristics of a classical Ca2+ sensor protein. To betterunderstand the physiological role of CML14 in plants, in vivo analysis will be performed.
EF-hand, calmodulin-like protein, Arabidopsis thaliana, metal binding; conformational change, protein folding.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/930434
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