Structural characterization of the C2 domain of novel protein kinase Cepsilon.
Infrared spectroscopy (IR) and differential scanning calorimetry (DSC) were used to study the biophysical properties of the PKCepsilon-C2 domain, a C2 domain that possess special characteristics as it binds to acidic phospholipids in a Ca2+-independent manner and no structural information about it is available to date. When the secondary structure was determined by IR spectroscopy in H2O and D2O buffers, beta sheet was seen to be the major structural component. Spectroscopic studies of the thermal denaturation in D2O showed a broadening in the amide I' band starting at 45 degrees C. Curve fitting analysis of the spectra demonstrated that two components appear upon thermal denaturation, one at 1623 cm(-1) which was assigned to aggregation and a second one at 1645 cm(-1), which was assigned to unordered or open loop structures. A lipid binding assay has demonstrated that PKCepsilon-C2 domain has preferential affinity for PIP2 although it exhibits maximal binding activity for phosphatidic acid when 100 mol% of this negatively charged phospholipid 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 did not change upon lipid binding and the thermal stability was very high with no significant changes occurring in the secondary structure after heating. DSC experiments demonstrated that when the C2-protein was scanned alone, it showed a Tm of 49 degrees C and a calorimetric denaturation enthalpy of 144.318 kJ x mol(-1). However, when phoshatidic acid vesicles were included in the mixture, the transition disappeared and further IR experiments demonstrated that the protein structure was not modified under these conditions.
García-García
J
J
Departamento de Bioquímica y Biología Molecular 'A', Facultad de Veterinaria, Universidad de Murcia, Spain.
Gómez-Fernández
J C
JC
Corbalán-García
S
S
D000818:Animals; D002152:Calorimetry, Differential Scanning; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011489:Protein Denaturation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051744:Protein Kinase C-epsilon; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D007527:Isoenzymes; D010712:Phosphatidic Acids; D010743:Phospholipids; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon