Binding between calmodulin and the calmodulin-binding peptide of Sema4D (CBPS). (A) A myristoylated peptide corresponding to the calmodulin-binding sequence of Sema4D (Myr-RQCLKFRSALLIGKKKPK) was synthesized and designated as CBPS. A variant peptide in which four conserved amino acids were replaced with Ala (*) is designated as vCBPS (Myr-AQCLAFASALLIGKKAPK). (B) Ten micrograms of CBPS or vCBPS in PBS were incubated with 20 μL of calmodulin-agarose beads at 4°C for 4 hours. After washing extensively, bound peptide was eluted with Laemmli sample buffer and analyzed by gel electrophoresis followed by silver staining. (C) Platelet lysate was incubated with 10 µg of biotinylated CBPS or vCBPS overnight at 4°C. Biotinylated peptide-calmodulin complexes were captured by using avidin-agarose beads, eluted, and subjected to immunoblot analysis by using a calmodulin monoclonal antibody. (D) Fluorescence emission spectra of IAEDANS-labeled calmodulin (I-CaM). (●) I-CaM was added to 3 mL of 10 mM 4-morpholinepropanesulfonic acid (pH 7.4), 0.1 mM CaCl₂, 100 mM NaCl, and 0.1 mg/mL bovine serum albumin to a final concentration of 50 nM, and an emission scan was obtained. (▪) CBPS or (▲) vCBPS was added to a final concentration of 40 μΜ, and the scans was obtained. The excitation wavelength was 340 nm. All spectra were corrected for background reading from the buffer. (E) Binding affinity of CBPS with I-CaM. Association of CBPS to I-CaM was monitored by the change in IAEDANS emission fluorescence at 460 nm. Calmodulin protein was diluted to 50 nM in a 100-μL system and added to the 96-well plate. CBPS (●) or vCBPS (○) polypeptide was added to the calmodulin solution. The buffers were the same as in (D). I-CaM denotes IAEDANS-labeled calmodulin. Data are representative of at least 3 independent experiments.