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 Ca(2+) and phosphatidic acid vesicles. The amide I regions in the original spectra of PKC-C2 in the Ca(2+)-free and Ca(2+)-bound states are both consistent with a predominantly beta-sheet secondary structure below the denaturation temperatures. Spectroscopic studies of the thermal denaturation revealed that for the PKC-C2 domain alone the secondary structure abruptly changed at 50 degrees C. While in the presence of 2 and 12.5 mM Ca(2+), 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 Ca(2+) binding (PKC-C2D246/248N) demonstrated that these mutations were inherently more stable to thermal denaturation than the wild-type protein. Phosphatidic acid binding to the PKC-C2 domain was characterized, and the lipid-protein binding became Ca(2+)-independent when 100 mol% phosphatidic acid vesicles were used. The mutant lacking two Ca(2+) binding sites was also able to bind to phosphatidic acid vesicles. The effect of lipid binding on secondary structure and thermal stability was also studied. Beta-sheet was the predominant structure observed in the lipid-bound state, although the percentage represented by this structure in the total area of the 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 observed 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 conditions that were studied.
García-García
J
J
Departamento de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Spain.
Corbalán-García
S
S
Gómez-Fernández
J C
JC
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D017666:Deuterium Oxide; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017433:Protein Structure, Secondary; D051381:Rats; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared
D007527:Isoenzymes; D008024:Ligands; D010446:Peptide Fragments; D010712:Phosphatidic Acids; D010743:Phospholipids; D012996:Solutions; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D017666:Deuterium Oxide; D002118:Calcium