Membrane-specific and calcium-dependent binding of the Arabidopsis C2 domain protein CaLB revealed by ATR-FTIR spectroscopy.

C2 domain-containing proteins bind to cellular membranes and mediate diverse cellular processes. Although many of these membrane-interacting proteins have been identified, the molecular mechanisms of protein-membrane interactions and conformational dynamics are often poorly understood and remain to be investigated with appropriate methods. Here, we used attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and biomimetic membrane systems to analyse CalB, a yet uncharacterized Arabidopsis C2 domain protein. We studied membrane binding, lipid specificity and calcium dependency with solid-supported lipid membranes (SSLB) and small unilamellar lipid vesicles (SUVs). Membranes were composed of pure POPC lipids or of POPC/PI(3)P lipid mixtures. A significantly increased protein binding affinity was observed with membranes containing 1% PI(3)P indicating the high binding specificity of CaLB for PI(3)P. Furthermore, membrane binding occurs in a calcium-dependent manner with a higher calcium concentration increasing the binding of CaLB to the POPC/PI(3)P membrane. Secondary structure analysis of IR-spectra reveals that only minor conformational changes take place upon binding with a slight increase in the helical and disordered regions of CaLB.