Fig. 3.

Characterization of the PDZ3:CRIPT interaction from extant species with a type 1 PBM in CRIPT. (A–D) Examples of ITC experiments for native PDZ3:CRIPT interactions for four extant species: Homo sapiens, Saccoglossus kowalevskii, Loa loa, and Octopus bimaculoides. (E–L) Binding kinetics from stopped-flow experiments for the same native interactions as in panel (A–D), and also including experiments with a reconstructed CRIPT from the ancestor of all eukaryotes (CRIPT_Eukaryota). (E–H) Observed rate constants were obtained from kinetic-binding traces (examples shown in panels I–L) and plotted as function of CRIPT concentration at a constant concentration of PDZ3 (1 µM) to estimate the association rate constant k_on. (I–L) Examples of kinetic-binding traces from stopped-flow experiments using 4 µM CRIPT and 1 µM PDZ3. The fitted line (red) is a single exponential from which the observed rate constant k_obs was obtained. k_off was determined in a separate experiment as explained in Materials and Methods section, and K_d calculated as k_off/k_on. Experiments were performed at 25 °C for ITC and at 10 °C for stopped-flow.