Structural study of the catalytic domain of PKCfusinginfrared spectroscopy and two-dimensional infraredcorrelation spectroscopy

Abstract

The secondary structure of the catalytic domain from protein kinase Cfwas studied using IR spectroscopy. In the presence of the substrateMgATP, there was a significant change in the secondary structure. Afterheating to 80°C, a 14% decrease in thea-helix component was observed,accompanied by a 6% decrease in theb-pleated sheet; no change wasobserved in the large loops or in 310-helix plus associated loops. The maxi-mum increase with heating was observed in the aggregatedb-sheet compo-nent, with an increase of 14%. In the presence of MgATP, and comparedwith the sample heated in its absence, there was a substantial decrease inthe 310-helix plus associated loops and an increase ina-helix. Synchronous2D-IR correlation showed that the main changes occurred at 1617 cm)1,which was assigned to changes in the intermolecular aggregatedb-sheet ofthe denaturated protein. This increase was mainly correlated with thechange ina-helix. In the presence of MgATP, the main correlation wasbetween aggregatedb-sheet and the large loops component. The asynchro-nous 2D-correlation spectrum indicated that a number of components aretransformed in intermolecularly aggregatedb-sheet, especially thea-helixandb-sheet components. It is interesting that changes in 310-helix plusassociated loops and ina-helix preceded changes in large loops, which sug-gests that the open loops structure exists as an intermediate state duringdenaturation. In summary, IR spectroscopy revealed an important effect ofMgATP on the secondary structure and on the thermal unfolding processwhen this was induced, whereas 2D-IR correlation spectroscopy allowed usto show the establishment of the denaturation pathway of this protein