Mapping distances in HCN2 bound to cAMP with transition metal ion FRET. (a) X-ray crystal structure of a single subunit of the cysteineless HCN2 carboxyl-terminal fragment (Clinker and CNBD) bound to cAMP. (b) Organization of the tetrameric complex viewed parallel to the membrane. Cartoon of transmembrane domain based on the structure of Kv1.2 is shown in blue. (c,d) Plots of Ni²⁺-dependent quenching of HCN2 constructs labeled with fluorescein-5-maleimide at N520C (c) or K570C (d) with Ni²⁺-binding motifs in the C-helix. Control contains no introduced histidines. Fluorescence was measured in solution containing 250 μM cAMP, normalized to the intensity before the addition of Ni²⁺, and fit by a single-site binding model (solid lines). Error bars are s.e.m. (n = 4–6). (e) Structural model for positions of fluorescein-5-maleimide conjugated to N520C (cyan) and K570C (red) in relation to the 4 Ni²⁺-binding motifs. Point sources used for calculations are shown as mesh spheres. (f) Plot of distances determined from FRET (experimental) for each position compared to distances determined from the crystal structure (model). Error bars are s.e.m. (n = 4–6). Colors match c with open circles corresponding to distances to K570C_F5M and closed circles distances to N520C_F5M.

Structural rearrangements in HCN2 determined with transition metal ion FRET. Ni²⁺-dependent quenching of apo state HCN2 labeled with fluorescein-5-maleimide at N520C (a) and K570C (b) with Ni²⁺-binding motifs in the C-helix. Control contains no introduced histidines. Fluorescence was normalized to the intensity before the addition of Ni²⁺ and fit by a single-site binding model (solid line). (c) Plot of FRET changes (right) and corresponding changes in distance (left) between the apo and holo states. Error bars are s.e.m. (n = 4–6).

Measuring distances with transition metal ion FRET. (a) Spectral overlap between fluorescein emission (grey) and the absorbance of Ni²⁺ (green line) and Cu²⁺ (blue line) bound to the peptide ACAAKAAAKHAAAHKA. (b) Cartoon of FRET between a fluorescein-modified cysteine and a Ni²⁺-binding motif in an α-helix. A helical wheel projection of the position of the cysteine (blue) and histidine (green) residues in the model peptides. (c) Quenching of fluorescein by increasing concentrations of Ni²⁺ in the model peptide AC_F5MHAKHAAKAAAAAKA. (d) Average quenching of model peptides AC_F5MAAKAAAKAAAAAKA (black), AC_F5MHAKHAAKAAAAAKA (purple), AC_F5MAAKHAAKHAAAAKA (green), and AC_F5MAAKAAAKHAAAHKA (orange). Fluorescence was normalized to the intensity before the addition of Ni²⁺. Data were fit to a single-site binding model (solid line). (e) Average quenching of fluorescein by Cu²⁺ in the same peptides as c. Error bars are s.e.m. (n = 4–6). (f) Comparison of modeled distances to experimental FRET values in all peptides with Ni²⁺ and Cu²⁺.

Crystal structure of the mouse wild-type HCN2 carboxyl-terminal fragment (C-linker and CNBD) in the absence of cAMP. Ribbon diagram of a single subunit (a) and tetrameric organization (b) of the apo state. (c) Overlay of the apo (blue) and holo (grey) state HCN2 crystal structures. (d) Expanded view of the structural differences in the F′- and C-helices that occur as a result of cAMP-binding. Dotted line indicates the end of the C-helix in the apo state. Residues used for FRET measurements are labeled yellow.

The C-helix is stabilized during cAMP-binding. (a,b) Overlay of apo (grey) and holo (red) Ni²⁺-quenching curves for (a) N520C_F5M constructs and (b) K570C_F5M constructs. (c) Current working hypothesis for the conformational rearrangements (red) that occur during cAMP-binding. Asterisk indicates the position of C-helix in the apo state.