Abstract:
:The secondary structure of protein kinase C alpha (PKC alpha) has been studied using infrared spectroscopy in the presence of both H(2)O and D(2)O buffers. In the absence of ligands at 20 degrees C, it was shown that beta-sheet is the major component, representing about 44% of the total structure, whereas the alpha-helix amounts to 22%. The addition of Ca(2+) produced only small changes in the secondary structure at 20 degrees C with the beta-sheet increasing up to 48%. On the other hand, the other ligands, such as phorbol 12-myristate 13-acetate (PMA), ATP, and phospholipids, did not produce any significant change. When the thermal unfolding of PKC alpha was studied after heating to 75 degrees C, the presence of the ligands affected the unfolding process. PKC alpha was better preserved from thermal denaturation in the presence of Ca(2+), the aggregated beta-sheet at 1618 cm(-1) decreasing from 19% in the absence of this ligand to 13% in its presence. Protection was also afforded by the presence of PMA or phospholipids. A two-dimensional correlation study of the denaturation of PKC alpha in the presence of these different ligands also showed differences among them. Synchronous 2D-IR correlation showed that the main change occurred at 1616-1619 cm(-1), this component being assigned to the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in the alpha-helix component in all cases except in the presence of a mixture of ligands including Ca(2+), ATP, PMA, and phospholipids, when the intermolecular aggregation of beta-sheet was correlated with the change in the beta-sheet component. In addition, the asynchronous 2D-IR correlation study of PKC alpha showed that the aggregated beta-sheet increased after changes in other components. It was interesting that alpha-helix changed before the beta-sheet in the control experiment and in the presence of Ca(2+), while the order of change was reversed when PMA was added.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Torrecillas A,Corbalán-García S,Gómez-Fernández JCdoi
10.1021/bi035128isubject
Has Abstractpub_date
2004-03-02 00:00:00pages
2332-44issue
8eissn
0006-2960issn
1520-4995journal_volume
43pub_type
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