Torrecillas Alejandro, Corbalán-García Senena, Gómez-Fernández Juan C
Departamento de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia. Apartado de Correos 4021, E-30080-Murcia, Spain.
Biochemistry. 2004 Mar 2;43(8):2332-44. doi: 10.1021/bi035128i.
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.
在H₂O和D₂O缓冲液存在的情况下，利用红外光谱研究了蛋白激酶Cα（PKCα）的二级结构。在20℃且不存在配体时，结果表明β-折叠是主要成分，约占总结构的44%，而α-螺旋占22%。在20℃添加Ca²⁺后，二级结构仅发生微小变化，β-折叠增加至48%。另一方面，其他配体，如佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）、ATP和磷脂，并未产生任何显著变化。当将PKCα加热至75℃后研究其热解折叠时，配体的存在影响了解折叠过程。在Ca²⁺存在的情况下，PKCα能更好地抵御热变性，1618 cm⁻¹处的聚集β-折叠从不存在该配体时的19%降至存在时的13%。PMA或磷脂的存在也提供了保护作用。对PKCα在这些不同配体存在下的变性进行二维相关研究也显示出它们之间的差异。同步二维红外相关显示主要变化发生在1616 - 1619 cm⁻¹处，该成分被归属于变性蛋白的分子间聚集β-折叠。这种增加在所有情况下主要与α-螺旋成分的变化相关，除了存在包括Ca²⁺、ATP、PMA和磷脂的配体混合物时，此时β-折叠的分子间聚集与β-折叠成分的变化相关。此外，PKCα的异步二维红外相关研究表明，在其他成分发生变化后，聚集β-折叠增加。有趣的是，在对照实验和存在Ca²⁺时，α-螺旋在β-折叠之前发生变化，而添加PMA时变化顺序则相反。
