Crystallizationh of PKA regulatory subunit from Saccharomyces cerevisiae

J.J.RINALDI, J.YANG, S.ROSSI, S.GANAPATHY, S.MORENO AND S.TAYLOR

San Diego, USA

Congreso; ASBMB Annual Meeting; 2008

ASBMB

In mammals, the PKA holoenzyme exists as a complex of two catalytic subunits and a regulatory (R) subunit dimer. R subunits have a dimerization and docking domain at the N terminus; at the C terminus, two tandem cAMP-binding domains and in between a flexible hinge region, including a substrate-like inhibitor sequence that docks to the active site cleft of the C subunit. Much effort has been put in studying structure-function relationships in mammalian PKAs. The aim of this work is to solve the structure of the R subunit from S. cerevisiae in order to learn which of the features of mammalian R are general and which are specific to this system. We over-expressed and purified different deletion mutants of the protein: D85, and D167. The over-expression and stability of the proteins was assessed both in yeast and in bacterial expression systems. D167 was crystallized at 21ºC in sitting or hanging drop in 15-18% w/v PEG 3350, 0.1M Tris-HCl pH 8.5. These crystals were transferred to a cryoprtectant solution Images of each crystal trial were manually taken at 0, 2, 7, 14, 21 and 28 days after setup. X-ray diffraction data of single crystals were collected at Stanford Synchrotron Radiaton Laboratory (SSRL).