Figure 3. Calmodulin binds two AQP0^CBD peptides in a step-wise manner.

A, Overlay of ¹⁵N-calmodulin HSQC spectra of unbound (yellow), singly bound (green) and doubly bound (blue) species in the presence of Ca²⁺. B, Combined ¹H/¹⁵N chemical shift changes (Δδ) for several amino acids from the N- and C-lobes of calmodulin. Green and blue bars correspond to Δδ values from the 1:1 and 1:2 complexes, respectively. Red bars correspond to the overall Δδ values from the S235-P titration. Horizontal lines indicate the mean and standard deviation of the overall Δδ values from the AQP0^CBD titration. C, Close-up view of the boxed area from panel A, for AQP0^CBD. Residue assignments are identified in the figure. Arrows indicate different points in the titration experiment, corresponding to singly bound (Δδ₁) and doubly bound (Δδ₂) ¹⁵N-calmodulin species. D, Titration curves of normalized Δδ values for several ¹⁵N-calmodulin residues versus mole ratio of AQP0^CBD peptide (colored and labeled). The flat portion of the trace “Δδ₁” indicates slow-exchange. Theoretical fits to the fast exchange Δδ₂ used to obtain binding constants are overlaid in black hatch lines. E and F, are the same as C and D, but were obtained for the S235-P modified AQP0^CBD. In E, arrows indicate S235-P titration points that resulted in fast-exchange. In F, the titration data for both binding events were fit to a two-state binding isotherm.