Fig. 1

Description of the SPR-based screening method. a Isolated PAS and C-linker/CNB(CNBH) domains of HCN and KCNH channels were purified with affinity and size-exclusion chromatography. b The purified domains were immobilized on the NTA sensor chip. The NTA chip contained 20 spots for protein immobilization, five spots (S1-S5) per each of the four flow cells (FCs). For each FC, S3 was used as a control spot for non-specific binding, S1 and S2 were used to immobilize the same protein at high density (HD) and low density (LD), respectively, and similarly, S4 and S5 were used to immobilize the same protein at LD density for S4 and HD for S5. Only spots with HD of the immobilized protein are depicted in the figure. For FC1 only spots S4 and S5 had immobilized protein. c The Spectrum Library compounds at 50 μM concentration distributed into 384-well plates were injected to the seven immobilized proteins simultaneously. In addition to the library compounds cAMP was also injected at the beginning and end of each plate screening. Binding of cAMP to the C-linker/CNB of hHCN4 channels was used as a positive control for the screen. The pdb IDs of the PAS and C-linker/CNB(CNBH) domain structures are: 3OTF (HCN4 CNB), 4HOI (EAG PAS), 4F8A (EAG CNBH), 1BYW (ERG PAS), 2N7G (ERG CNBH), 4HP4 (ELK PAS) and 3UKN (ELK CNBH)