Fig. 3. Protein kinase A regulatory subunit binding to neurobeachin measured by surface plasmon resonance (SPR). A, RIIα binds to regions B of neurobeachin and DAKAP550 but not BGL (analyte concentrations, 1.5 μm each). B, SPR tracings of a concentration series of neurobeachin region B binding to RIIα performed for Kd determination.C, Neurobeachin region B binds RIIα and RIIβ but not RIα or RIβ (analyte concentration, 1.5 μm).D, The RII binding peptide from AKAP Ht31 competes with neurobeachin region B for RIIα binding (neurobeachin region B concentration, 1 μm). E, Dissection of neurobeachin region B to delineate the RII binding sequence. Region B comprises amino acids 951–1311. Constructs C1–C3 reach from amino acid 951 to 1225, 1133, and 1041, respectively. Constructs D1–D3 extend from amino acids 1005, 1088, and 1177, respectively, to 1311.Kd values were determined for these constructs by measuring concentration series as in part B and are indicated beside the constructs. As described in Materials and Methods, construct C2 (asterisk) displayed high-affinity binding only if prepared under nondenaturing conditions. D2 and D3 binding were very weak, with approximate Kd values of 5 and 15 μm, respectively, and no binding was detectable with C3 (detection limit, 20 μm). An open frame indicates the sequence region in which the high-affinity RII binding property resides (amino acids 1022–1107). Because known RII binding sites are ∼20 amino acids long, we assume that if the binding site in region B should overlap with the end of a nonbinding construct, C3 or D2, it will do so for a maximum of 20 residues. The candidate core RII binding sequence (amino acids 1081–1099) with high α-helix potential is indicated by a hatched frame in the overview and an open box in the sequence at the bottom. F, Helical representation of amino acids 1081–1099 illustrating the highly amphiphilic nature of this structure. Filled circles symbolize hydrophobic residues, and open circles indicate hydrophilic amino acids.