Elsevier

Methods in Enzymology

Volume 345, 2002, Pages 198-206
Methods in Enzymology

[15] - Crystallization of Complex between Soluble Domains of Adenylyl Cyclase and Activated Gsα

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Purification of Hexahistidyl-VC1

The VC1 domain used for these experiments is produced by expression of a cDNA encoding residues 364–580 of canine adenylyl cyclase V. The expression construct is ligated into pQE60-H6 (Qiagen, Chatsworth, CA) at NcoI and HindIII sites. The expressed protein contains a hexahistidine tag at its amino terminus, followed by the residue corresponding to Met-564. Escherichia coli BL21(DE3) is cotransformed with pREP412 and pQE60-H6-VC1 and grown overnight at 30° in 200 ml of Luria–Bertani (LB) medium

Formation of VCl:IIC2:forskolin:Gsα GTPγS complex

VC1 (100 μM), IIC2 (200 μM), Gsα · GTPγS (100 μM), and forskolin (50 μM) are incubated for 30 min on ice in gel-filtration buffer containing 20 mM Na-HEPES (pH 8.0), 2 mM MgCl2, 1 mM EDTA, 2 mM DTT, 100 mM NaCl, 25 μM forskolin, and 10 μM GTPγS. The mixture is then applied to a Superdex 75 (HR 10/30) gel-filtration column in tandem with a Superdex 200 (HR 10/30) column (Amersham Pharmacia Biotech). The column is eluted with gel-filtration buffer and 0.5-ml fractions containing complex are

Ciystallization of VC 1:11C 2: forskolin : Gsα · GTPγS Complex

Crystals of the complex are obtained by vapor diffusion at 20°, either by a sitting drop or hanging drop configuration as described elsewhere in this series.14 For a hanging drop experiment, a 2-to 3-μl drop containing concentrated solution of the complex is deposited on a clean 22-mm siliconized glass coverslip (Hampton Research, Laguna Niguel, CA). Into this drop is injected an equal volume of well solution containing 7.2–7.5% (w/v) polyethylene glycol (PEG) 8000,500 mM NaCl, and 100 mM

Limitations and Outlook

The methods described here provide a route to structural elucidation of Gsα-and forskolin-coactivated catalytic domains of adenylyl cyclase. Crystals described here can be used to investigate the binding modes and conformational effects of active site-based ligands in the presence of Zn2+, Mg2+, and Mn2+.2,4 Although we have done little work in this area, it should also be possible to study a variety of forskolin analogs and mutants of Gsα On the other hand, because VC1 and IIC2 homodimerize in

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    Citation Excerpt :

    Except for IIC2, both VC1 and Gαs constructs contained a hexahistidine tag at their N termini. Gαs was activated by incubation with 10 μm GTPγS and 2 mm MgCl2 at 30 °C for 1 h, and the resulting Gαs·GTPγS complex was further digested by trypsin to a smaller fragment comprising residues 39–387 (27). MANT-GTP, 2′-deoxy-3′-O-(N-methylanthraniloyl)-adenosine 5′-triphosphate, MANT-ATP, and 2′(3′)-O-(N-methylanthraniloyl)-xanthosine 5′-triphosphate were obtained from Molecular Probes (Eugene, OR) or JenaBioscience (Jena, Germany).

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