Figure 6.
CaM/segment-C interaction studies by photoaffinity labeling and surface plasmon resonance. Amino acid sequences of segment-C variants are given in Table 3. (A,B) PAL-based competition assay of segment-C-Bpa with segment-C and its variants V709D (green), I714D (blue), and F723D (orange). (A) SDS-PAGE analysis of photoreactions; (□) CaM, (∆) CaM/segment-C-Bpa 1:1 complex. (B) quantification of photoadduct yield (CaM/segment-C-Bpa signal); solid lines, quantification by SDS-PAGE analysis; dotted lines, quantification by MALDI-TOF-MS. The plot shows the mean of three independent experiments with standard error of mean (SEM) as error bars. A representative set of SDS gels and corresponding mass spectra is shown in Figure S11. (C) SPR measurements of wild-type (WT) and mutated segment-C variants V709D, I714D and F723D. Upper panel: sensograms of all variants for one concentration, lower panel: sensograms of one segment-C variant at different concentrations. KD values are given; n.d.: not determined.