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Expression of a chimeric, cGMP‐sensitive regulatory subunit of the cAMP‐depedent protein kinase type Iα
Author(s) -
Norbert Wild,
Friedrich W. Herberg,
Franz Hofmann,
Wolfgang R. Dostmann
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)01146-6
Subject(s) - protein subunit , protein kinase a , förster resonance energy transfer , cgmp dependent protein kinase , intracellular , biophysics , gi alpha subunit , chemistry , kinase , microbiology and biotechnology , biochemistry , biology , mitogen activated protein kinase kinase , fluorescence , gene , physics , quantum mechanics
To study the fluctuations of cGMP in living cells through changes of energy transfer of dissociable fluorescence labeled subunits, we constructed a cGMP‐sensitive probe by combining the N‐terminus of the type I regulatory subunit of cAMP‐dependent protein kinase (PKA) with the cGMP binding sites of cGMP‐dependent protein kinase Iα (PKG). This chimeric regulatory subunit retained PKA‐like dimerization and PKG‐compatible cGMP binding constants ( K d = 53 nM) for both binding sites. High affinity interaction with the PKA catalytic subunit was verified by Surface Plasmon Resonance ( K d = 3.15 nM). Additionally, the chimera inhibits the formation of wild‐type holoenzyme with an apparent K i of 1.05 nM. Furthermore, cGMP dissociated the mutant holoenzyme with an apparent activation constant of 146 nM. Thus, our construct provides all the requirements needed to investigate changes in intracellular cGMP concentrations.