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Structural study of the catalytic domain of PKCζ using infrared spectroscopy and two‐dimensional infrared correlation spectroscopy

The secondary structure of the catalytic domain from protein kinase C ζ was studied using IR spectroscopy. In the presence of the substrate MgATP, there was a significant change in the secondary structure. After heating to 80 °C, a 14% decrease in the α‐helix component was observed, accompanied by a 6% decrease in the β‐pleated sheet; no change was observed in the large loops or in 310‐helix plus associated loops. The maximum increase with heating was observed in the aggregated β‐sheet component, with an increase of 14%. In the presence of MgATP, and compared with the sample heated in its absence, there was a substantial decrease in the 310‐helix plus associated loops and an increase in α‐helix. Synchronous 2D‐IR correlation showed that the main changes occurred at 1617 cm−1, which was assigned to changes in the intermolecular aggregated β‐sheet of the denaturated protein. This increase was mainly correlated with the change in α‐helix. In the presence of MgATP, the main correlation was between aggregated β‐sheet and the large loops component. The asynchronous 2D‐correlation spectrum indicated that a number of components are transformed in intermolecularly aggregated β‐sheet, especially the α‐helix and β‐sheet components. It is interesting that changes in 310‐helix plus associated loops and in α‐helix preceded changes in large loops, which suggests that the open loops structure exists as an intermediate state during denaturation. In summary, IR spectroscopy revealed an important effect of MgATP on the secondary structure and on the thermal unfolding process when this was induced, whereas 2D‐IR correlation spectroscopy allowed us to show the establishment of the denaturation pathway of this protein.