Azidotyrosylcalmodulin derivatives. Specific probes for protein-binding domains.
Azidocalmodulin has been shown to be a useful probe for calmodulin-binding proteins in a variety of systems (Andreasen, T. J., Keller, C. H. LaPorte, D. C., Edelman, A. M., and Storm, D. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2782-2785). In previous work, this calmodulin derivative was generated by the modification of lysyl residues. We report here that, on the basis of tryptic peptide mapping by reverse-phase high-pressure liquid chromatography, azidolysylcalmodulin prepared by these procedures is modified at 4 of the 7 lysines. We also report the preparation of azidocalmodulins in which the photolabile moiety is incorporated into a single known residue of the molecule by modifying one or the other of the two tyrosyl side chains. This yields azido-Tyr 99-calmodulin, with the photoaffinity label in Ca2+-binding loop III, and azido-Tyr 138-calmodulin, with the photoaffinity label in Ca2+-binding loop IV. The cross-linking characteristics of these two calmodulin derivatives show that the formation of a covalent adduct upon photolysis of a mixture of azidocalmodulin and a target protein is dependent on the location of the nitrene generated by the irradiation. Azido-Tyr 138-calmodulin shows a significant decrease in cross-linking efficiency to targets such as troponin I, troponin T, and myosin light-chain kinase, relative to azido-Tyr 99-calmodulin and the azidolysyl derivatives.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000494:pharmacology; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins; D002417:Cattle; D004789:Enzyme Activation; D007202:Indicators and Reagents; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010782:Photolysis; D011485:Protein Binding; D013329:Structure-Activity Relationship
D000596:Amino Acids; D001386:Azides; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007202:Indicators and Reagents; C030955:azidocalmodulin; D010749:Phosphoprotein Phosphatases; D010727:Phosphoric Diester Hydrolases