Structural characterization of the C2 domain of novel protein kinase C epsilon

Infrared spectroscopy (IR) and differential scanning calorimetry (DSC) were used to study the biophysical properties of the PKC epsilon -C2 domain, a C2 domain that possess special characteristics as it binds to acidic phosph olipids in a Ca2+-independent manner and no structural information about it is available to date. When the secondary structure was determined by IR sp ectroscopy in H2O and D2O buffers, beta sheet was seen to be the major stru ctural component. Spectroscopic studies of the thermal denaturation in D2O showed a broadening in the amide I' band starting at 45 degreesC. Curve fit ting analysis of the spectra demonstrated that two components appear upon t hermal denaturation, one at 1623 cm(-1) which was assigned to aggregation a nd a second one at 1645 cm(-1) which was assigned to unordered or open loop structures. A lipid binding assay has demonstrated that PKC epsilon -C2 do main has preferencial affinity for PIP2 although it exhibits maximal bindin g activity for phosphatidic acid when 100 mol% of this negatively charged p hospholipid was used. Thus, phosphatidic acid containing vesicles were used to characterize the effect of lipid binding on the secondary structure and thermal stability. These experiments showed that the secondary structure d id not change upon lipid binding and the thermal stability was very high wi th no significant changes occurring in the secondary structure after heatin g. DSC experiments demonstrated that when the C2-protein was scanned alone, it showed a T-m of 49 degreesC and a calorimetric denaturation enthalpy of 144.318 kJ.mol(-1). However, when phoshatidic acid vesicles were included in the mixture, the transition disappeared and further IR experiments demon strated that the protein structure was not modified under these conditions.