Structural Characterization of the C2 Domains of Classical Isozymes of Protein Kinase C and Novel Protein Kinase Cε by using Infrared Spectroscopy

Abstract

Abstract. The amide I regions in the original infrared spectra of PKCα-C2 in the Ca 2+ -free and Ca 2+ -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 Ca 2+ , the thermal stability of the protein increased considerably. Phosphatidic acid binding to the PKCα-C2 domain was characterized, and the lipid-protein binding becoming Ca 2+ -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.

Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy Discriminates the Elderly with a Low and High Percentage of Pathogenic CD4+ T Cells

“…Spectral peaks at 1629 and 1626 cm −1 were assigned to β-sheet amide I region structure. The last bands at 1555/8 consisted of ring base vibration [51,[62][63][64][65]. In general, the spectral peaks associated with the LP and HP discrimination from exosome spectra were associated with differences in protein, lipid, and nucleic acid biomolecules.…”

“…Prominent ATR-FTIR exosome spectral bands for discrimination of the LP and HP groups using PCA and PLS-DA[51,[62][63][64][65].…”

An Infrared Spectroscopic Study of the Secondary Structure of Protein Kinase Cα and Its Thermal Denaturation

“…The asynchronous spectrum in the presence of Ca 2+ was also different from that in the presence of EGTA, and the main correlation was between the aggregated β-sheet and the α-helix component, confirming our previous conclusion from the FT-IR 1D spectroscopic studies that Ca 2+ somehow protects the β-sheet. It should be pointed out that Ca 2+ is known to bind to the C2 domain, the structure of which is based on a β-sandwich (), and we have already shown that Ca 2+ protects this type of structure in the isolated C2 domain of PKCα from thermal denaturation ( , ). Therefore, it seems likely that the effect that we are observing in the whole enzyme is due to the C2 domain.…”