Biochemical characterization of the talin 1 R35E mutation to block binding to Rap1b. (A) Surface electrostatic potential of talin 1 F0 (left panel) and Rap1b (right panel) binding interface as open book view. The charge complementarity between the 2 proteins’ binding interface is represented by the dashed line. (B-E) 2D NMR ¹H,¹⁵N-sfHMQC spectra (298K, 600 MHz) of 100 μM ¹⁵N-labeled talin 1 F0. (B) Free talin 1 F0 wild-type (blue) and R35E (red). Almost all peaks are in the same position, suggesting that THD(R35E) is well folded and has a fold similar to wild-type protein. (C) Talin 1 F0 wild-type (blue); with sevenfold excess Rap1b (red), specific chemical shift changes are observed and indicated by arrows. (D) Close-up view of the 10-step Rap1b titration for residues G11 and A41. (E) Talin 1 F0(R35E) (blue); in the presence of sevenfold excess Rap1b (red), minimal chemical shift changes are observed, showing that THD(R35E) drastically reduced interaction. (F) Titration curves for the interaction of talin 1 F0 wild-type (blue) or R35E (red) (100 μM) with increasing amounts of Rap1b (0-700 μM). Wild-type dissociation constants were measured for multiple residues, and binding curves are shown for G11 and G42 talin 1 F0 wild-type and mutant proteins. The F0(R35E) affinity for Rap1b was dramatically reduced (at 700 μM Rap1b, no binding was detected).