Circular dichroism analysis of ligand-induced conformational changes in protein kinase C. Mechanism of translocation of the enzyme from the cytosol to the membranes and its implications.
The structural changes following the binding to protein kinase C (PKC) of activators that promote its translocation to lipid environments were studied by far-u.v. c.d. and intrinsic fluorescence measurements of the protein. In the absence of activators, PKC contained 40% alpha-helix, with an average size of 13 amino acids per alpha-helix segment, and 12% beta-structure as deduced from c.d. spectral analysis while fitting a set of model proteins of known structure. Ligands that promote translocation and activation of the enzyme, such as Ca2+ ions and phorbol esters, produced drastic changes in the c.d. spectra which may be interpreted as a reduction in the average number of consecutive amino acids in the alpha-helix. Most of the total alpha-helix structure was conserved and an increase in beta-structure was produced by active phorbol esters. These activators differentially affected the fluorescence of PKC: phorbol esters shifted the emission maximum to the red, whereas Ca2+ produced a marked decrease in the intensity of the fluorescence emission, suggesting in both cases that tryptophan residues were exposed to increased polar environments after binding of the ligands.
Boscá
L
L
Instituto de Bioquímica, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Morán
F
F
D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology*; D002942:Circular Dichroism; D003600:Cytosol / Q000201:enzymology*; D004533:Egtazic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011487:Protein Conformation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D015240:Phorbol 12,13-Dibutyrate; D004533:Egtazic Acid; D011493:Protein Kinase C; D002118:Calcium