title: Coexpression, copurification, crystallization and preliminary X-ray analysis of a complex of ARL2-GTP and PDE delta.
abstract: The small GTPase ARL2 (from Mus musculus) and an effector protein, the delta subunit of human cGMP phosphodiesterase (hPDE delta), were coexpressed and copurified from Escherichia coli as a stable complex. Coexpression significantly increased the otherwise low yield of PDE delta production in E. coli. The complex, which contains ARL2 in the activated GTP-bound form, was crystallized in two forms. The first belongs to the monoclinic space group P2(1), with unit-cell parameters a = 48.1, b = 45.7, c = 74.7 A, beta = 94.0 degrees and one complex (39 kDa) in the asymmetric unit. Cryocooled crystals diffract to 2.3 A using synchrotron radiation. The micro-focused X-ray beam at beamline ID13 (ESRF) allowed the use of very small crystals, which helped to overcome twinning and enabled the identification of a molecular-replacement solution. The second form recrystallized from the first one after several months. These crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.5, b = 65.4, c = 104.4 A and one complex in the asymmetric unit. They diffracted to 1.8 A using synchrotron radiation.
authors.affiliation: Max-Planck-Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Renault
authors.forename: M
authors.initials: M
authors.lastname: Hanzal-Bayer
authors.forename: R C
authors.initials: RC
authors.lastname: Hillig
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004926:Escherichia coli; D005136:Eye Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006801:Humans; D051379:Mice; D011487:Protein Conformation
chemical_list: D005136:Eye Proteins; D006160:Guanosine Triphosphate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C517431:PDE6B protein, human; C517438:Pde6b protein, mouse; C410130:ARL2 protein, human; C485819:Arl2 protein, mouse; D019204:GTP-Binding Proteins