Abstract
Calmodulin (CaM) is a crucial sensor protein for cellular calcium level. CaM has four highly conserved E-F hand domains for calcium binding. Upon calcium binding, CaM undergoes conformational exchange and recruits the suitable down-stream target molecules. The highly flexible central linker of CaM allows the protein to adapt different conformation when binding to different targets. Recently, many natively unfolded proteins have been reported. These proteins are highly flexible with no average structure in solution. MARCKS (myristoylated alanine-rich protein kinase C substrate) is one of such protein and it is capable of binding to CaM. The CaM-binding domain of MARCKS is a 25-amino acid region called “effector domain” (ED). Although crystal structure of CaM/MARCKS ED complex has been determined, the detail information about the conformational changes upon binding is still not available. This information is critical for understanding the relationship between structure and function. In this research, we inveatigate different conformational states using NMR spectroscopy. The recombinant gene of CaM was expressed in E.coli, and purified with ion-excahnge and phenyl columns. The 15N-labeled CaM titrated with synthetic peptide of MARCKS effector domain was monitored by 1H-15N HSQC spectrum. The corresponding peaks in NMR spectra were traced and assigned. Our data show that not only the residues in the central region but also some residues in other regions changed. The changes in the HSQC spectra provide information for us to understand conformational states in CaM.