Type II beta regulatory subunit of cAMP-dependent protein kinase: purification strategies to optimize crystallization.
To elucidate the structural basis for important differences between types I and II regulatory subunit isoforms (RI and RII) of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase, the full-length RII beta isoform and five RII beta deletion mutants were constructed, expressed, purified, and screened for crystallization. Only one of these six proteins yielded diffraction quality crystals. Crystals were grown of the RII beta deletion mutant (delta 1-111) monomer potentially in complex with two cAMP molecules. X-ray diffraction quality data were obtained only after significant modification to existing purification procedures. Modifications required a Sepharose, not agarose, support for cAMP affinity chromatography followed by rapid, quantitative removal of free cAMP by size-exclusion chromatography under reducing conditions. Data to 2.4 A resolution were collected at 29 degrees C using synchrotron radiation on a single crystal measuring 0.2 x 0.3 x 1.2 mm(3). Data were 99% complete. The hexagonal crystal belonged to space group P6((1)) or P6((5)) with unit cell dimensions a = b = 161.62 A and c = 39.66 A.
Diller
T C
TC
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0654, USA.
Xuong
N H
NH
Taylor
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; C517839:Prkar2b protein, rat; D011994:Recombinant Proteins; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases