Crystallization of PKA regulatory subunit from Saccharomyces cerevisiae

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

San Diego, California, USA

Congreso; Experimental Biology 2008-ASBMB; 2008

American Society of Biochemistry and Molecular Biology

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 a  the N terminus; at the C terminus, two tandem cAMP-binding domains and in between a flexible hinge region, including a substratelike 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: 85, and 167. The overexpression and stability of the proteins was assessed both in yeast and in bacterial expression systems. 167 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).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: 85, and 167. The overexpression and stability of the proteins was assessed both in yeast and in bacterial expression systems. 167 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).