Premium
Crystallization of PKA regulatory subunit from Saccharomyces cerevisiae
Author(s) -
Rinaldi Jimena Julieta,
Yang Jie,
Rossi Silvia,
Ganapathy Sarma,
Moreno Silvia,
Taylor Susan
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1050.13
Subject(s) - protein subunit , dimer , saccharomyces cerevisiae , crystallography , chemistry , yeast , docking (animal) , crystallization , mutant , stereochemistry , biochemistry , biophysics , biology , gene , organic chemistry , medicine , nursing
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: Δ85, and Δ167. The over‐expression and stability of the proteins was assessed both in yeast and in bacterial expression systems. Δ167 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).