Structural study of the catalytic domain of PKCzeta using infrared spectroscopy and two-dimensional infrared correlation spectroscopy.
The secondary structure of the catalytic domain from protein kinase C zeta 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 degrees C, a 14% decrease in the alpha-helix component was observed, accompanied by a 6% decrease in the beta-pleated sheet; no change was observed in the large loops or in 3(10)-helix plus associated loops. The maximum increase with heating was observed in the aggregated beta-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 3(10)-helix plus associated loops and an increase in alpha-helix. Synchronous 2D-IR correlation showed that the main changes occurred at 1617 cm(-1), which was assigned to changes in the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in alpha-helix. In the presence of MgATP, the main correlation was between aggregated beta-sheet and the large loops component. The asynchronous 2D-correlation spectrum indicated that a number of components are transformed in intermolecularly aggregated beta-sheet, especially the alpha-helix and beta-sheet components. It is interesting that changes in 3(10)-helix plus associated loops and in alpha-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.
Sánchez-Bautista
Sonia
S
Departamento de Bioquímica y Biología Molecular, Universidad de Murcia, Spain.
Kazaks
Andris
A
Beaulande
Melanie
M
Torrecillas
Alejandro
A
Corbalán-García
Senena
S
Gómez-Fernández
Juan C
JC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002021:Buffers; D020134:Catalytic Domain; D017666:Deuterium Oxide; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011493:Protein Kinase C / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry; D000099221:Protein Kinase C zeta
D000255:Adenosine Triphosphate; D002021:Buffers; D017666:Deuterium Oxide; D007527:Isoenzymes; D011493:Protein Kinase C; D014867:Water; D000099221:Protein Kinase C zeta