Structural characterization of the C2 domains of classical isozymes of protein kinase C and novel protein kinase Cε by using infrared spectroscopy

Issue title: First International Conference on Biomedical Spectroscopy: From molecules to men, Cardiff, UK, 7–10 July 2002, Part II

Article type: Research Article

Authors: Corbalán‐García, Senena | García‐García, Josefa | Sánchez‐Carrillo, M. Susana | Gómez‐Fernández, Juan C.

Affiliations: Departamento de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E‐30080 Murcia, Spain

Note: [] Corresponding author. Tel.: +34 968 364766; Fax: +34 968 364766; E‐mail: jcgomez@um.es.

Abstract: The amide I regions in the original infrared spectra of PKCα‐C2 in the Ca2+‐free and Ca2+‐bound states are both consistent with a predominantly β‐sheet secondary structure. Spectroscopic studies of the thermal denaturation revealed that for the PKCα‐C2 domain alone the secondary structure abruptly changed at 50°C. While in the presence of Ca2+, the thermal stability of the protein increased considerably. Phosphatidic acid binding to the PKCα‐C2 domain was characterized, and the lipid–protein binding becoming Ca2+‐independent when 100 mol% phosphatidic acid vesicles was used. The effect of lipid binding on secondary structure and thermal stability was also studied. In addition, the secondary structure of the C2 domain from the novel PKCε was also determined by IR spectroscopy and β‐sheet was seen to be the major structural component. Spectroscopic studies of the thermal denaturation in D2O showed a broadening in the amide I′ band starting at 45°C. Phosphatidic acid containing vesicles were used to characterize the effect of lipid binding on the secondary structure. It was observed through thermal stability experiments that the secondary structure did not change upon lipid binding and the protein stability was very high with no significant changes occurring in the secondary structure after heating.

Journal: Spectroscopy, vol. 17, no. 2-3, pp. 399-416, 2003