Folding and activity of cAMP-dependent protein kinase mutants.
The catalytic subunit of cAMP-dependent protein kinase (PKA) can easily be expressed in Escherichia coli and is catalytically active. Four phosphorylation sites are known in PKA (S10, S139, T197 and S338), and the isolated recombinant protein is a mixture of different phosphorylated forms. Obtaining uniformly phosphorylated protein requires separation of the protein preparation leading to significant loss in protein yield. It is found that the mutant S10A/S139D/S338D has similar properties as the wild-type protein, whereas additional replacement of T197 with either E or D reduces protein expression yield as well as folding propensity of the protein. Due to its high sequence homology to Akt/PKB, which cannot easily be expressed in E. coli, PKA has been used as a surrogate kinase for drug design. Several mutations within the ATP binding site have been described to make PKA even more similar to Akt/PKB. Two proteins with Akt/PKB-like mutations in the ATP binding site were made (PKAB6 and PKAB8), and in addition S10, S139 and S338 phosphorylation sites have been removed. These proteins can be expressed in high yields but have reduced activity compared to the wild-type. Proper folding of all proteins was analyzed by 2D 1H, 15N-TROSY NMR experiments.
Langer
Thomas
T
Johann Wolfgang Goethe-University Frankfurt, Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Marie Curie Strasse 11, D-60439 Frankfurt am Main, Germany. t.langer@nmr.uni-frankfurt.de
Sreeramulu
Sridhar
S
Vogtherr
Martin
M
Elshorst
Bettina
B
Betz
Marco
M
Schieborr
Ulrich
U
Saxena
Krishna
K
Schwalbe
Harald
H
D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases