Biophysical characterization of the DARPin-LmrCD complexes.
(A, B) Stoichiometry analysis as exemplified by the LmrCD/α-LmrCD#2 complex. (A) LmrCD and the LmrCD/α-LmrCD#2 complex were separated by SEC (Superdex 200 PC3.2/30, GE Healthcare) with a void volume V0 = 0.85 ml and a total volume Vt = 2.4 ml. A fraction corresponding to heterodimeric LmrCD in complex with α-LmrCD#2 complex (red bar) was subjected to protein chip analysis (lane 3, inset). LmrCD and the DARPin α-LmrCD#2 were also analyzed (lanes 1 and 2, inset). The peak at a retention volume of 1.2 ml corresponds to aggregated LmrCD. (B) The peak area of the protein chip chromatogram corresponding to LmrCD and α-LmrCD#2 of lane 3 in (A) were calibrated with dilution series of LmrCD and DARPin of known protein concentrations (not shown) and were used to determine the stoichiometry of the LmrCD-DARPin complexes (Table 1). (C) Affinities of the DARPins to LmrCD were determined by surface plasmon resonance as shown for α-LmrCD#3. The colored lines correspond to the measured traces at different DARPin concentrations, the fitted curves (1∶1 binding model) are shown as black lines. (D) The steady state DARPin binding signals achieved at the end of the association phase shown in (C) were plotted against the DARPin concentration and fitted using an equilibrium binding equation equivalent to the Michaelis-Menten equation. In this analysis, equilibrium dissociation constants (KD, eq.) were generated.