Coexpression, copurification, crystallization and preliminary X-ray analysis of a complex of ARL2-GTP and PDE delta.
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.
Renault
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Max-Planck-Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Hanzal-Bayer
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Hillig
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RC
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
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