Structural study of the C2 domains of the classical PKC isoenzymes using infrared spectroscopy and two-dimensional infrared correlation spectroscopy.
The secondary structure of the C2 domains of the classical PKC isoenzymes, alpha, betaII, and gamma, has been studied using infrared spectroscopy. Ca(2+) and phospholipids were used as protein ligands to study their differential effects on the isoenzymes and their influence on thermal protein denaturation. Whereas the structures of the three isoenzymes were similar in the absence of Ca(2+) and phospholipids at 25 degrees C, some differences were found upon heating in their presence, the C2 domain of the gamma-isoenzyme being better preserved from thermal denaturation than the domain from the alpha-isoenzyme and this, in turn, being better than that from the beta-isoenzyme. A two-dimensional correlation study of the denaturation of the three domains also showed differences between them. Synchronous 2D-IR correlation showed changes (increased aggregation of denaturated protein) occurring at 1616-19 cm(-1), and this was found in the three isoenzymes. On the other hand, the asynchronous 2D-IR correlation study of the domains in the absence of Ca(2+) showed that, in all cases, the aggregation of denaturated protein increased after changes in other structural components, an increase perhaps related with the hard-core role of the beta-sandwich in these proteins. The differences observed between the three C2 domains may be related with their physiological specialization and occurrence in different cell compartments and in different cells.
Torrecillas
Alejandro
A
Departamento de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30080-Murcia, Spain.
Corbalán-García
Senena
S
Gómez-Fernández
Juan C
JC
D000595:Amino Acid Sequence; D002122:Calcium Chloride / Q000737:chemistry; D004795:Enzyme Stability; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011493:Protein Kinase C / Q000737:chemistry*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013055:Spectrophotometry, Infrared / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D007527:Isoenzymes; D008024:Ligands; C087857:protein kinase C gamma; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D002122:Calcium Chloride