3.3.1. Protein Construct Design

Two cysteine mutations are introduced at the residue positions that do not disrupt the properties, binding affinity, or activity of the protein (see Note 3). Any intrinsic cysteines that are solvent exposed need to be mutated to avoid unspecific double-stranded DNA handle attachment (see Note 3). In the regulatory subunit of PKA, the mutations C345A and C360A are introduced in the CNB-B domain to prevent undesired reactions with the thiol-modified dsDNA handles. To manipulate each individual CNB domain (termed Type-I constructs) we introduce the mutations S110C/M243C and M243C/S376C for the isolated CNB-A and CNB-B domains, respectively. To selectively manipulate either the CNB-A domain or the CNB-B domain (termed Type-II constructs) we introduce into the regulatory subunit the mutations S110C/M243C and M243C/S376C, respectively.

All the protein constructs are expressed in BL21(DE3) (NEB) and purified as described previously [22, 28, 31]. Briefly, the protein is expressed in BL21(DE3) competent cells overnight at 18 °C with 1 mM IPTG. The cells are lysed in lysis buffer (20 mM MES, 100 mM NaCl, 2 mM EGTA, 2 mM EDTA, 5 mM DTT, pH 6.5) and the spun-down supernatant is precipitated with 40% ammonium sulfate before binding to a homemade cAMP-coupled agarose resin. The protein is eluted from the cAMP-coupled resin with cGMP (20 mM cGMP in lysis buffer) and run on a size exclusion column to remove the excess cGMP and aggregated proteins. The protein is stored in gel filtration buffer (50 mM MES, 200 mM NaCl, 2 mM EGTA, 2 mM EDTA 5 mM DTT, pH 5.8) before use.