Effect of calcium and phosphatidic acid binding on the C2 domain of PKC alpha as studied by Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural and thermal denaturation of the C2 domain of PKC alpha (PKC-C2) and its complexes with Ca2+ and phosphatidic acid vesicles. The amide I reg ions in the original spectra of PKC-C2 in the Ca2+-free and Ca2+-bound stat es are both consistent with a predominantly beta-sheet secondary structure below the denaturation temperatures. Spectroscopic studies of the thermal d enaturation revealed that for the PKC-C2 domain alone the secondary structu re abruptly changed at 50 degrees C. While in the presence of 2 and 12.5 mM Ca2+, the thermal stability of the protein increased to 60 and 70 degrees C, respectively. Further studies using a mutant lacking two important amino acids involved in Ca2+ binding (PKC-C2D246/248N) demonstrated that these m utations were inherently more stable to thermal denaturation than the wild- type protein. Phosphatidic acid binding to the PKC-C2 domain was characteri zed, and the lipid-protein binding became Ca2+-independent when 100 mol % p hosphatidic acid vesicles were used. The mutant lacking two Ca2+ binding si tes was also able to bind to phosphatidic acid vesicles. The effect of lipi d binding on secondary structure and thermal stability was also studied. be ta-Sheet was the predominant structure observed in the lipid-bound state, a lthough the percentage represented by this structure in the total area of t he amide I band significantly decreased from 60% in the lipid-free state to 47% in the lipid-bound state. This decrease in the beta-sheet component of the lipid-bound complex correlates well with the significant increase obse rved in the 1644 cm(-1) band which can be assigned to loops and disordered structure. Thermal stability after lipid binding was very high, and no sign of thermal denaturation was observed in the presence of lipids under the c onditions that were studied.