Supporting Information

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Protein Expression and Purification. The cyanobacterial tandem GAF A/B domains (residues 58-445) were cloned into the NcoI–BamHI sites of Qiagen vector pQE-60. This vector adds a GSRS-His-6 tag to the C terminus. The plasmid was heat-shocked into C41 cells, a derivative of the Escherichia coli strain BL21(DE3). A 2-ml overnight culture in LB plus 100 m g/ml carbenicillin was expanded to 2 liters of LB at 200 rpm at 37°C until an OD₆₀₀ of ~0.7. IPTG was added to 0.6 mM and shaken 18 h at 22°C. The cells were spun down, washed once with 40 ml PBS, spun, and the pellet frozen at –80°C until use. For purification, four buffers were prepared: lysis (50 mM Tris·HCl, pH 8.0/50 mM NaCl/5 mM imidazole/20% glycerol/0.1 mg/ml lysozyme); wash 1 (50 mM Tris·HCl, pH 8.0/15 mM imidazole/20% glycerol/400 mM NaCl/2 mM MgCl₂); wash 2 (same as wash 1, but 10 mM NaCl); and elution (50 mM Tris·HCl, pH 8.0/250 mM imidazole/20% glycerol/10 mM NaCl/2 mM MgCl₂). The cell pellet was thawed in a 37°C water bath for 5 min. Cells were suspended in 5 ml of lysis buffer per g and incubated on ice for 1 h, turning a few times. Cells were sonicated 3 × 30 sec with a 30-sec rest on ice in between. The Virsonic 100 was set at 6-W rms (reading with tip in lysate). The lysate was spun for 30 min at 37,000 ´ g. Ten milliliters of Ni-NTA resin (Invitrogen), washed with lysis buffer, was added to the supernatant and put on the orbital shaker for 30 min. The resin was collected with a low-speed spin. The resin was resuspended and poured into a 15-mm-diameter column then washed with 2 × 1 bed volumes each of wash 1 and wash 2 buffers. Protein was eluted with 2 × 1 bed volumes of elution buffer, and 0.1-ml aliquots were frozen in liquid N₂ and stored at –80°C. The yield was 200 mg/liter cell culture.

Expression of SeMet Protein. A selenomethionine (SeMet)-labeled derivative was produced in M9 minimal media supplemented with SeMet and several amino acids that suppress Met biosynthesis (1). Expression and purification were as for SuMet, except for a 21-h induction.

1. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L. & Clardy, J. (1993) J. Mol. Biol. 229, 105-124.

Fig. 7. Superposition of the GAF-A/B monomers from PDE2A and cyaB2 adenylyl cyclase. The connecting helix between GAF-A and -B superimposes well between the two structures. Linkers attaching GAF-A or -B to the helix have different conformations, causing the GAF domains to be arranged differently between the two structures. The orientations differ by a 90° turn. The figure was prepared with SWISS-PDB VIEWER [Kaplan, W. & Littlejohn, T. G. (2001) Brief Bioinform. 2, 195-197] and rendered with POV-RAY.

Fig. 8. Superposition of the four GAF domains from cyaB2 and PDE2A shows a new conserved Asn. GAF A of cyaB2 (green) was fit individually to GAF B (cyan), and to GAF-A (magenta) and -B (yellow) of PDE2A. The rms deviation for the NKFDE residues (excluding the proposed new Asn addition) is 0.91, 0.98, and 2.05 Å, respectively, for 45 atoms. The Glu clearly shows the most translational and conformational variability. Note that, in PDE2A GAF-A, which does not bind cGMP, the proposed new Asn for the conserved motif is a Gln.